RESUMEN
Bovine papillomavirus (BPV) types 1 and 2 play an important role in the pathogenesis of equine sarcoids (ES), the most common cutaneous tumour affecting horses. MicroRNAs (miRNAs), small non-coding RNAs that regulate essential biological and cellular processes, have been found dysregulated in a wide range of tumours. The aim of this study was to identify miRNAs associated with ES. Differential expression of miRNAs was assessed in control equine fibroblasts (EqPalFs) and EqPalFs transformed with the BPV-1 genome (S6-2 cells). Using a commercially available miRNA microarray, 492 mature miRNAs were interrogated. In total, 206 mature miRNAs were differentially expressed in EqPalFs compared with S6-2 cells. Aberrant expression of these miRNAs in S6-2 cells can be attributed to the presence of BPV-1 genomes. Furthermore, we confirm the presence of 124 miRNAs previously computationally predicted in the horse. Our data supports the involvement of miRNAs in the pathogenesis of ES.
Asunto(s)
Papillomavirus Bovino 1 , MicroARNs/metabolismo , Infecciones por Papillomavirus/veterinaria , Animales , Transformación Celular Viral , Fibroblastos/metabolismo , Fibroblastos/virología , Enfermedades de los Caballos/metabolismo , Caballos , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/veterinaria , Neoplasias Cutáneas/virologíaRESUMEN
Samples of faeces were taken from 183 healthy pet dogs in a census-based, cross-sectional study in Cheshire; culture methods were used to detect any Campylobacter species and a direct PCR was used to detect Campylobacter upsaliensis. Forty-six of the dogs were positive for C upsaliensis by either culture or direct PCR, giving a prevalence of 25.1 per cent (95 per cent confidence interval [CI] 19.0 to 32.1 per cent). One sample was positive by culture for Campylobacter jejuni (95 per cent CI 0.0 to 3.0 per cent) and one for Campylobacter lari. Multivariable logistic regression identified risk factors for the carriage of C upsaliensis by a dog as: living with another dog that also carried C upsaliensis; being small rather than medium-sized; being less than three years old; living in a household that kept fish; being fed commercial dog treats; and being fed human food titbits, particularly in the dog's bowl.
Asunto(s)
Infecciones por Campylobacter/veterinaria , Campylobacter upsaliensis/aislamiento & purificación , Enfermedades de los Perros/microbiología , Animales , Infecciones por Campylobacter/epidemiología , Infecciones por Campylobacter/microbiología , Portador Sano , Enfermedades de los Perros/epidemiología , Perros , Inglaterra/epidemiología , Heces/microbiología , Análisis Multivariante , Factores de RiesgoRESUMEN
Bovine papillomavirus type 1 (BPV-1) and, less commonly, BPV-2 are associated with the pathogenesis of common equine skin tumors termed sarcoids. In an attempt to understand the mechanisms by which BPV-1 induces sarcoids, we used gene expression profiling as a screening tool to identify candidate genes implicated in disease pathogenesis. Gene expression profiles of equine fibroblasts transformed by BPV-1 experimentally or from explanted tumors were compared with those of control equine fibroblasts to identify genes associated with expression of BPV-1. Analysis of the microarray data identified 81 probe sets that were significantly (P < 0.01) differentially expressed between the BPV-1-transformed and control cell lines. Expression of several deregulated genes, including MMP-1, CXCL5, FRA-1, NKG7, TLR4, and the gene encoding the major histocompatibility complex class I (MHC-I) protein, was confirmed using other BPV-1-transformed cell lines. Furthermore, expression of these genes was examined using a panel of 10 sarcoids. Increased expression of MMP-1, CXCL5, FRA-1, and NKG7 was detected in a subset of tumors, and TLR4 and MHC I showed robust down-regulation in all tumors. Deregulated expression was confirmed at the protein level for MMP-1 and MHC-I. The present report identifies genes modulated by BPV-1 transformation and will help identify the molecular mechanisms involved in disease pathogenesis.
Asunto(s)
Papillomavirus Bovino 1 , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Enfermedades de los Caballos/virología , Infecciones por Papillomavirus/metabolismo , Animales , Línea Celular Transformada , Cartilla de ADN/genética , Fibroblastos/virología , Citometría de Flujo , Perfilación de la Expresión Génica , Caballos , Complejo Mayor de Histocompatibilidad/genética , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Interleukin 18 (IL-18) is a cytokine capable of induction of IFNgamma, granulocyte monocyte-colony stimulating factor (GM-CSF), TNFalpha and IL-1 in immunocompetent cells. Equine and feline plasmid vectors expressing pro-IL-18, mature IL-18 and IL-18 fused to a synthetic signal sequence from human IL-1beta receptor antagonist protein (ILRAP), ILRAP-IL-18, have been generated. In vitro protein expression of these constructs was compared by Western blot analysis. These data demonstrated that ILRAP-IL-18 protein was secreted readily from transfected chinese hamster ovary (CHO) cells. A simple bioassay for human IL-18 was recently described using human myelomonocytic KG-1 cells, which produce human IFNgamma in response to human IL-18 in a dose dependent manner (Konishi et al., 1997). We demonstrated bioactivity of equine and feline IL-18 protein in transfection products of CHO cells using this assay. Bioactivity of ILRAP-IL-18 protein was demonstrated in the culture medium of transfected CHO cells. These data imply that the ILRAP-IL-18 construct shows potential for use in vivo, where cell secretion of protein is crucial.
Asunto(s)
Gatos/inmunología , Regulación de la Expresión Génica/inmunología , Caballos/inmunología , Interleucina-18/biosíntesis , Animales , Bioensayo , Células CHO , Cricetinae , Cricetulus , Técnicas In Vitro , Interferón gamma/biosíntesis , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-18/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Sialoglicoproteínas/genéticaRESUMEN
To examine the mode of natural transmission and persistence of feline coronavirus (FCoV), FCoV strains shed by domestic cats were investigated over periods of up to 7 years. An RT-PCR that amplified part of the 3' end of the viral spike (S) gene was devised to distinguish FCoV types I and II. All but 1 of 28 strains of FCoV from 43 cats were type I. Nucleotide identities of the amplified 320 bp product from 49 type I FCoVs ranged from 79 to 100 %. The consensus partial S sequence of isolates recovered from persistently infected cats at time intervals spanning years was generally conserved. While most cats were infected with a single strain, a few may have been infected by more than one strain. Cats that were transiently infected and ceased shedding could be re-infected with either the same, or a different, strain. In most cases, whether a cat became persistently or transiently infected was independent of the virus strain. However, one strain was unusual in that it infected the majority of cats in the household simultaneously and was still being shed 18 months later. Factors that influence whether FCoV establishes lifelong infection in some cats and not others are determined mainly by the host response to infection.
Asunto(s)
Portador Sano/veterinaria , Enfermedades de los Gatos/transmisión , Infecciones por Coronavirus/veterinaria , Coronavirus Felino/clasificación , Animales , Portador Sano/virología , Enfermedades de los Gatos/virología , Gatos , Infecciones por Coronavirus/transmisión , Infecciones por Coronavirus/virología , Coronavirus Felino/genética , Coronavirus Felino/patogenicidad , Glicoproteínas de Membrana/genética , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/genética , Esparcimiento de VirusAsunto(s)
Cardiomiopatía Dilatada/veterinaria , Enfermedades de los Bovinos/virología , Infecciones por Enterovirus/veterinaria , Enterovirus Bovino/genética , Animales , Cardiomiopatía Dilatada/virología , Bovinos , ADN Viral/química , ADN Viral/genética , Enterovirus Humano B/genética , Infecciones por Enterovirus/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinariaRESUMEN
The expectation that cell-mediated immunity is important in the control of feline leukemia virus (FeLV) infection led us to test a DNA vaccine administered alone or with cytokines that favored the development of a Th1 immune response. The vaccine consisted of two plasmids, one expressing the gag/pol genes and the other expressing the env gene of FeLV-A/Glasgow-1. The genetic adjuvants were plasmids encoding the feline cytokines interleukin-12 (IL-12), IL-18, or gamma interferon (IFN-gamma). Kittens were immunized by three intramuscular inoculations of the FeLV DNA vaccine alone or in combination with plasmids expressing IFN-gamma, IL-12, or both IL-12 and IL-18. Control kittens were inoculated with empty plasmid. Following immunization, anti-FeLV antibodies were not detected in any kitten. Three weeks after the final immunization, the kittens were challenged by the intraperitoneal inoculation of FeLV-A/Glasgow-1 and were then monitored for a further 15 weeks for the presence of virus in plasma and, at the end of the trial, for latent virus in bone marrow. The vaccine consisting of FeLV DNA with the IL-12 and IL-18 genes conferred significant immunity, protecting completely against transient and persistent viremia, and in five of six kittens protecting against latent infection. None of the other vaccines provided significant protection.
Asunto(s)
ADN Viral/inmunología , Proteínas de Fusión gag-pol/inmunología , Interleucina-12/inmunología , Interleucina-18/inmunología , Virus de la Leucemia Felina/inmunología , Infecciones por Retroviridae/prevención & control , Infecciones Tumorales por Virus/prevención & control , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos , Animales , Gatos , Línea Celular , Proteínas de Fusión gag-pol/genética , Expresión Génica , Vectores Genéticos , Humanos , Interleucina-12/administración & dosificación , Interleucina-12/genética , Interleucina-18/administración & dosificación , Interleucina-18/genética , Virus de la Leucemia Felina/genética , Recombinación Genética , Vacunación , Vacunas de ADN/administración & dosificación , Vacunas Virales/administración & dosificación , Virión/fisiología , Ensamble de Virus/fisiología , Latencia del VirusRESUMEN
Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-gamma production in cells derived from equine lymph nodes. Preincubation of IFN-gamma inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-gamma induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-gamma production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species.
Asunto(s)
Caballos/inmunología , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Baculoviridae , Línea Celular , ADN Complementario/genética , Regulación de la Expresión Génica , Vectores Genéticos , Enfermedades de los Caballos/inmunología , Enfermedades de los Caballos/terapia , Caballos/genética , Inmunidad Celular , Interleucina-12/química , Interleucina-12/genética , Interleucina-12/inmunología , Subunidad p35 de la Interleucina-12 , Subunidad p40 de la Interleucina-12 , Datos de Secuencia Molecular , Subunidades de Proteína , SpodopteraRESUMEN
Caspases are cysteine proteases which have important roles in the activation of cytokines and in apoptosis. The ICE subfamily of caspases comprise peptides closely related to caspase-1, or interleukin-1beta (IL-1beta) converting enzyme (ICE), which promotes maturation of interleukin IL-1beta and interleukin-18 (IL-18) by proteolytic cleavage of precursor forms to generate biologically active peptides. Other members of this subfamily include caspase-4, -5, -13 and isoforms of these proteins. We report the cloning and sequencing of two feline and canine ICE-related cDNAs amplified by RT-PCR. The predicted proteins are 410 and 404 amino acids in length respectively and are most closely related to caspase-1 sequences across the N-terminal 115 amino acids and to human caspase-13 across the C-terminal sequence.
Asunto(s)
Caspasa 1/genética , Caspasas/genética , ADN Complementario/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Gatos , Clonación Molecular , ADN Complementario/química , Perros , Datos de Secuencia Molecular , Precursores de Proteínas/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Caspases are a family of cysteine proteases which have important roles in activation of cytokines and in apoptosis. Caspase-1, or interleukin-1 beta converting enzyme (ICE), promotes maturation of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) by proteolytic cleavage of precursor forms to generate biologically active peptides. We report the cloning and sequencing of equine caspase-1 cDNA. Equine caspase-1 is 405 amino acids in length and has 72% and 63% identity to human and mouse caspase-1, respectively, at the amino acid level. Sites of proteolytic cleavage and catalytic activity as identified in human caspase-1, are conserved.
Asunto(s)
Caspasa 1/genética , Equidae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
Interleukin-18, originally termed interferon-gamma inducing factor, is a recently described cytokine which is intimately involved in the generation of the immune response. The human and mouse sequences have been reported and studies have demonstrated the potent biological functions of this protein including the induction of interferon-gamma and the enhancement of NK cytotoxicity. This paper describes the cloning, sequencing, and characterization of the dog homologue of this gene. The coding sequence for dog IL-18 was obtained using reverse transcription-polymerase chain reaction from mRNA harvested from PMA-stimulated alveolar macrophages. Sequence analysis of the dog gene has demonstrated an open reading frame of 582 base pairs coding for a 193 amino acid precursor protein. The dog coding sequence shares 84% and 74% similarity to the human and mouse equivalents, respectively, at the nucleotide level. Based upon sequence analysis, we propose that this new gene is the dog equivalent of human IL-18.
Asunto(s)
Perros/genética , Interleucina-18/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Humanos , Macrófagos Alveolares/citología , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The complete DNA sequence of a field strain of canine adenovirus type 1 was determined by sequencing random fragments of viral DNA cloned into pBluescript. The virus has a genome of 30536 bp flanked by two identical 161 bp inverted terminal repeats. Thirty ORFs have been identified, based on genomic location or sequence identity with published adenoviruses. These are arranged into similar discrete regions found in the human adenoviruses. ORFs in the late region show greatest identity with published human adenovirus sequences, whereas the E3 and E4 ORFs show little or none.
Asunto(s)
Adenoviridae/genética , ADN Viral/genética , Genoma Viral , Análisis de Secuencia de ADN , Animales , Perros , Humanos , Datos de Secuencia MolecularRESUMEN
Transforming growth factor beta (TGF-beta) belongs to a family of peptide growth factors which control critical stages of cell proliferation and differentiation. We report the cloning and sequencing of the cDNA for TGF-beta type 1 isoform of the horse. The predicted mature equine TGF beta-1 peptide is 112 amino acids in length and exhibits 99% identity to mature human TGF beta-1.
Asunto(s)
Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Caballos , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Canine herpesvirus 1 (CHV-1), a member of the alphaherpesvirus sub-family, is known to cause fatal infections in litters of puppies and may also be involved in infertility, abortion, and stillbirths in adult dogs. The purpose of this study was to determine the presence of CHV-1 DNA using the polymerase chain reaction (PCR) in twelve key sites that have been associated with latency for the other herpesviruses. A 605 base pair portion of the viral glycoprotein B (gB) gene was amplified using degenerate primers, cloned, and sequenced. Conventional 20 mer primers were designed using this sequence information to amplify a 120 bp fragment of gB situated between the original degenerate primers. The specificity of amplification was confirmed by Southern Blot hybridisation using an internal oligonucleotide probe. DNA was extracted from tissue samples taken from twelve dogs at post mortem and from twenty-four blood samples. Nine out of twelve dogs showed evidence of infection with CHV-1; the tissues most commonly affected were lumbo-sacral ganglia (5/12 dogs), tonsil (5/12), parotid salivary gland (4/9), and liver (4/9). No positive results were detected within the twenty-four blood samples. These results indicate that exposure to CHV-1 may be much more common than previously suggested.
Asunto(s)
Enfermedades de los Perros , Infecciones por Herpesviridae/veterinaria , Herpesvirus Cánido 1/aislamiento & purificación , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Southern Blotting , Cartilla de ADN , ADN Viral/análisis , Perros , Femenino , Ganglios Espinales/virología , Infecciones por Herpesviridae/diagnóstico , Hígado/virología , Sondas de Oligonucleótidos , Especificidad de Órganos , Tonsila Palatina/virología , Glándula Parótida/virología , Placenta/virología , Reacción en Cadena de la Polimerasa/métodos , Embarazo , Ovinos , Proteínas del Envoltorio Viral/genéticaRESUMEN
We have modified the commercial cosmid vector, triple helix vector (THV), such that I-Sce-I restriction endonuclease sites flank the cloning site. I-Sce-I is a rare-cutting endonuclease which recognizes an 18-bp sequence. It does not restrict the genome of either of the equine herpesvirus 1 or 4 (EHV-1 and EHV-4) strains we have cosmid cloned. Thus, cosmid-cloned EHV fragments can be excised intact from the vector by I-Sce-I digestion, facilitating production of large overlapping EHV fragments for use in transfections to produce recombinant virus.
Asunto(s)
Clonación Molecular/métodos , Cósmidos , ADN Viral/genética , Vectores Genéticos , Herpesvirus Équido 1/genética , Varicellovirus/genética , Secuencia de Bases , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Mapeo RestrictivoRESUMEN
Interferon gamma, a cytokine produced by T-lymphocytes and natural killer cells, plays a central role in the modulation of the immune response, and its antiviral and antitumourigenic properties have made it a potential candidate for use in immunoprophylactic and therapeutic regimes. We have cloned the equine IFN gamma cDNA to facilitate production of this cytokine for clinical evaluation in the horse. The predicted equine IFN gamma amino acid sequence is 67% identical to that of the human equivalent and 78% to the bovine equivalent.
Asunto(s)
Caballos/genética , Interferón gamma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Complementario , Humanos , Leucocitos Mononucleares/química , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Proteínas RecombinantesRESUMEN
The polymerase chain reaction (PCR) is a sensitive technique used to detect DNA of viral pathogens. We have applied the technique to the detection of Equid herpesviruses-1 and -4 (EHV-1 and EHV-4) DNA within nasopharyngeal swab samples from horses. Ninety-eight samples from suspected field cases and in-contact horses were analysed. The assays were conducted blind and later decoded and compared with virus isolation data. Our results indicate that PCR is a sensitive and rapid technique for the diagnosis of EHV-1 and EHV-4 infection.
Asunto(s)
ADN Viral/análisis , Infecciones por Herpesviridae/veterinaria , Herpesviridae/genética , Herpesvirus Équido 1/genética , Enfermedades de los Caballos/diagnóstico , Animales , Secuencia de Bases , Herpesviridae/aislamiento & purificación , Infecciones por Herpesviridae/diagnóstico , Herpesvirus Équido 1/aislamiento & purificación , Caballos , Datos de Secuencia Molecular , Nasofaringe/microbiología , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Hamsters were immunized with either an affinity-purified preparation of equid herpesvirus 1 (EHV-1) glycoprotein 13 (gp13) or synthetic peptides representing three sequences within the homologous glycoprotein of EHV-4, resulting in the production of anti-peptide (in the case of peptide-immunized animals) or antivirus antibodies. The sera from gp13-immunized hamsters contained antibodies which showed virus-neutralizing activity and complement-mediated antibody lysis of EHV-1-infected target cells. These hamsters were protected from EHV-1 challenge. The characteristics of a panel of anti-gp13 monoclonal antibodies (P28, P17, 14H7, 16E4 and 16H9) were assessed both in vivo and in vitro. 16E4 and P28 showed high levels of complement-mediated neutralization of virus, complement-mediated lysis of virus-infected target cells and passive protection of hamsters. Furthermore, epitope mapping studies demonstrated that this glycoprotein contains a neutralizing epitope recognized by EHV-1-immune horse serum. The data imply that gp13 has potential as a candidate antigen for a molecular vaccine.
Asunto(s)
Herpesvirus Équido 1/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Formación de Anticuerpos , Cromatografía de Afinidad , Proteínas del Sistema Complemento , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Herpesvirus Équido 1/aislamiento & purificación , Inmunización Pasiva , Hígado/microbiología , Pulmón/microbiología , Mesocricetus , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos/síntesis química , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
The genomic position of an equine herpesvirus 4 (EHV-4) gene homologue of the herpes simplex virus 1 (HSV-1) gC gene was determined by Southern analysis and DNA sequencing. The gene lies within a 2-kbp Bg/II-EcoRI fragment mapping between 0.15 and 0.17 within the long unique component of the EHV-4 genome and is transcribed from right to left. Putative promoter elements were identified upstream of the 1455-bp open reading frame which encodes a 485-amino-acid protein of unglycosylated molecular weight 52,513. Computer-assisted analysis of the primary sequence predicts the protein possesses a domain structure characteristic of a type 1 integral membrane glycoprotein. Four domains were distinguished--(i) an N-terminal signal sequence, (ii) a large extracellular domain containing 11 putative N-linked glycosylation sites, (iii) a hydrophobic transmembrane domain, and (iv) a C-terminal charged domain. Comparison of the predicted amino acid sequence to that of other herpesvirus glycoproteins indicated identities of between 22 and 29% with HSV-1 gC, HSV-2 gC, VZV gpV, PRV gIII, BHV-1 gIII, and MDV A antigen and of 79% with EHV-1 gp13. A gene with no apparent homologue in HSV-1 or VZV maps immediately downstream of the EHV-4 gC gene homologue.