RESUMEN
RNA transcripts of the B7 family molecule (CD80) are diminished in blood leukocytes from animals clinically affected with Visna/Maedi virus (VMV) infection. This work investigates whether the use of B7 genes enhances immune responses and protection in immunization-challenge approaches. Sheep were primed by particle-mediated epidermal bombardment with VMV gag and env gene recombinant plasmids together with plasmids encoding both CD80 and CD86 or CD80 alone, boosted with gag and env gene recombinant modified vaccinia Ankara virus and challenged intratracheally with VMV. Immunization in the presence of one or both of the B7 genes resulted in CD4+ T cell activation and antibody production (before and after challenge, respectively), but only immunization with CD80 and CD86 genes together, and not CD80 alone, resulted in a reduced number of infected animals and increased early transient cytotoxic T lymphocytes (CTL) responses. Post-mortem analysis showed an immune activation of lymphoid tissue in challenge-target organs in those animals that had received B7 genes compared to unvaccinated animals. Thus, the inclusion of B7 genes helped to enhance early cellular responses and protection (diminished proportion of infected animals) against VMV infection.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígeno B7-1/administración & dosificación , Neumonía Intersticial Progresiva de los Ovinos/prevención & control , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Virus Visna-Maedi/inmunología , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Antivirales/sangre , Antígeno B7-1/genética , Antígeno B7-1/farmacología , Antígeno B7-2/administración & dosificación , Antígeno B7-2/genética , Antígeno B7-2/farmacología , Linfocitos T CD4-Positivos/inmunología , Productos del Gen env/administración & dosificación , Productos del Gen env/genética , Productos del Gen gag/administración & dosificación , Productos del Gen gag/genética , Vectores Genéticos , Inmunización Secundaria/métodos , Masculino , Ovinos , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/genética , Virus Visna-Maedi/genéticaRESUMEN
To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.
Asunto(s)
Biolística , Genes env/genética , Genes gag/genética , Neumonía Intersticial Progresiva de los Ovinos/prevención & control , Vacunas de ADN , Virus Vaccinia/genética , Animales , Anticuerpos Antivirales/sangre , Epidermis/virología , Femenino , Genes env/inmunología , Genes gag/inmunología , Inmunización , Masculino , Neumonía Intersticial Progresiva de los Ovinos/virología , Provirus/aislamiento & purificación , Ovinos , Linfocitos T/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Virus Vaccinia/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/inmunología , Virión/genética , Virión/inmunología , Virus Visna-MaediRESUMEN
Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.
Asunto(s)
Productos del Gen env/inmunología , Productos del Gen pol/inmunología , Neumonía Intersticial Progresiva de los Ovinos/prevención & control , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Virus Visna-Maedi/inmunología , Animales , Anticuerpos Antivirales/sangre , Proliferación Celular , Pruebas Inmunológicas de Citotoxicidad , Femenino , Productos del Gen env/genética , Productos del Gen pol/genética , Vectores Genéticos , Leucocitos Mononucleares/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Masculino , Nasofaringe/inmunología , Provirus/aislamiento & purificación , Índice de Severidad de la Enfermedad , Ovinos , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/administración & dosificación , Virus Vaccinia/genética , Carga Viral , Vacunas Virales/administración & dosificaciónRESUMEN
The long terminal repeats (LTR) sequence divergence among Maedi Visna virus (MVV) isolates leads to LTRs with distinct transcriptional activities, which may result in distinct biological behaviours. The genetic heterogeneity, as well as basal and Tat-induced transcriptional activity of the LTRs from P1OLV and WLC-1 MVV viruses, slow/low and rapid/high isolates, respectively, have been examined and compared with LTRs from other strains of small ruminant lentiviruses (SRLV). Transfection assays using a reporter construct containing the LTR fused to a luciferase gene demonstrated that the LTR from P1OLV virus had the weakest promoter activity, suggesting a correlation between the level of promoter activity and the viral replication rate. To confirm this hypothesis, the promoter of P1OLV was cloned into infectious molecular clone KV1772kv72/67 and the resulting chimeric virus was tested for growth in various cell types. Compared to the parental KV1772, the LTR-chimeric virus KV1772/P1OLV exhibited a drastic reduction in replication rate in sheep choroid plexus (SCP) and lung cells, while in ovine macrophages and goat synovial membrane cells (GSM), chimeric virus showed a growth rate similar to that of parental virus. These observations suggest that the LTR is responsible for the slow/low in vitro phenotype presented by P1OLV in SCP and lung cells.
Asunto(s)
Secuencias Repetidas Terminales/fisiología , Virus Visna-Maedi/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Cabras , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Virus Reordenados , Alineación de Secuencia , Ovinos , Especificidad de la Especie , Secuencias Repetidas Terminales/genética , Replicación Viral , Virus Visna-Maedi/genética , Virus Visna-Maedi/crecimiento & desarrolloRESUMEN
Infectious molecular clones have been isolated from two maedi-visna virus (MVV) strains, one of which (KV1772kv72/67) is an antigenic escape mutant of the other (LV1-1KS1). To map the type-specific neutralization epitope, we constructed viruses containing chimeric envelope genes by using KV1772kv72/67 as a backbone and replacing various parts of the envelope gene with equivalent sequences from LV1-1KS1. The neutralization phenotype was found to map to a region in the envelope gene containing two deletions and four amino acid changes within 39 amino acids (positions 559 to 597 of Env). Serum obtained from a lamb infected with a chimeric virus, VR1, containing only the 39 amino acids from LV1-1KS1 in the KV1772kv72/67 backbone neutralized LV1-1KS1 but not KV1772kv72/67. The region in the envelope gene that we had thus shown to be involved in escape from neutralization was cloned into pGEX-3X expression vectors, and the resulting fusion peptides from both molecular clones were tested in immunoblots for reactivity with the KV1772kv72/67 and VR1 type-specific antisera. The type-specific KV1772kv72/67 antiserum reacted only with the fusion peptide from KV1772kv72/67 and not with that from LV1-1KS1, and the type-specific VR1 antiserum reacted only with the fusion peptide from LV1-1KS1 and not with that from KV1772kv72/67. Pepscan analysis showed that the region contained two linear epitopes, one of which was specific to each of the molecularly cloned viruses. This linear epitope was not bound by all type-specific neutralizing antisera, however, which indicates that it is not by itself the neutralization epitope but may be a part of it. These findings show that mutations within amino acids 559 to 597 in the envelope gene of MVV virus result in escape from neutralization. Furthermore, the region contains one or more parts of a discontinuous neutralization epitope.
Asunto(s)
Mutación Puntual , Proteínas del Envoltorio Viral/genética , Virus Visna-Maedi/genética , Secuencia de Aminoácidos , Aminoácidos/genética , Animales , Datos de Secuencia Molecular , Fenotipo , Alineación de Secuencia , Virus Visna-Maedi/clasificación , Virus Visna-Maedi/aislamiento & purificaciónRESUMEN
Expression of major histocompatibility complex (MHC) antigens was studied in the brains of 10 healthy sheep 2 months to 5 years old and 13 sheep infected with visna virus by intracerebral inoculation and killed one and 6 months post infection (p.i.). In healthy sheep there was prominent expression of class I, mainly on endothelial cells but also detected on ependyma, choroid plexus and in the leptomeninges. Class II expression was sparse. It was observed on perivascular cells, in choroid plexus, leptomeninges and on microglial cells in the white matter. No definite increase with age in the constitutive expression of class I and II was observed, confirming that we are dealing with a true constitutive expression. In visna-infected sheep a considerable induction of MHC antigens on microglia was observed, which correlated with severity of lesions and was mainly found in or adjacent to inflammatory infiltrates of the white matter. Increase in class II antigen expression was detected in all sheep but class I only in sheep with the most severe lesions 6 months p.i., an indication of a higher threshold for induction of class I than class II antigens on microglia. Few cells expressed viral antigens, indicating that direct immune-mediated destruction of infected cells plays a minor role in evolution of lesions. Since the preferential induction of MHC antigens on microglia in the white matter correlated with the lesion pattern, activated microglia may play a considerable role in the pathogenesis of lesions.
Asunto(s)
Encéfalo/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Antígenos de Histocompatibilidad Clase I/análisis , Virus Visna-Maedi/fisiología , Visna/inmunología , Visna/patología , Animales , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunohistoquímica , Ovinos , Visna/virologíaRESUMEN
During the epidemic caused by maedi-visna virus (MVV) of sheep in Iceland, the pulmonary affection, maedi, was the predominant clinical manifestation. In some flocks, however, a central nervous system (CNS) affection, visna, was the main cause of morbidity and mortality. As there is only one breed of sheep in the country, host factors did apparently not play an important role in the different clinical manifestations. To obtain some information on possible viral genetic determinants of neurotropism and neurovirulence we studied both phenotypic and genotypic properties of two maedi-visna virus strains; a strain that was originally isolated from the brain of sheep with encephalitis (visna), and another strain isolated from the lungs of a sheep suffering from pneumonia (maedi). The brain isolate was found to grow faster in sheep choroid plexus cells than the lung isolate, whereas the growth rate in macrophages was similar for the maedi and visna virus strains. Intracerebral inoculation indicated that the visna virus isolate induced more severe brain lesions than the maedi isolate. In addition, a pathogenic molecular clone derived from a visna strain (KV1772kv72/67) was tested for growth in sheep choroid plexus cells and macrophages. The molecularly cloned virus retained the fast growth rate in choroid plexus cells. The nucleotide sequence of the env gene and the U3 of the LTR was determined for the maedi strain and compared to that of the visna strains. There was an 11.7% difference in deduced amino acid sequence in the Env protein and a 6% difference in the LTR. The molecular clone KV1772kv72/67 will be a useful reagent for characterization of viral determinants of cell tropism in vitro and possibly neurovirulence in vivo.
Asunto(s)
Encéfalo/virología , Pulmón/virología , Neumonía Intersticial Progresiva de los Ovinos/virología , Virus Visna-Maedi/genética , Visna/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/patología , Línea Celular , ADN Viral , Femenino , Genes env , Inyecciones , Pulmón/patología , Macrófagos/virología , Datos de Secuencia Molecular , Neumonía Intersticial Progresiva de los Ovinos/patología , Secuencias Repetitivas de Ácidos Nucleicos , Ovinos , Visna/patología , Virus Visna-Maedi/crecimiento & desarrolloRESUMEN
The major part of the dUTPase-encoding region of the visna virus genome was deleted. Intracerebral injection of the mutant virus resulted in a somewhat reduced viral load compared to that resulting from injection of the wild type, especially in the lungs, but the neuropathogenic effects were comparable. The dUTPase gene is dispensable for induction of lesions in the brain.
Asunto(s)
Sistema Nervioso/virología , Pirofosfatasas/genética , Virus Visna-Maedi/genética , Visna/virología , Animales , Eliminación de Gen , Sistema Nervioso/patología , Ovinos , Virulencia/genética , Virus Visna-Maedi/patogenicidadRESUMEN
The behavior of two genetically different molecular clones of visna virus KV1772-kv72/67 and LV1-1KS1 was compared in vivo and in vitro. On intracerebral inoculation, clone KV1772-kv72/67 induced a similar response in five sheep as has already been reported with neurovirulent derivates of visna virus. Virus was frequently isolated from blood, cerebrospinal fluid (CSF), and lymphoid organs and induced characteristic central nervous system (CNS) lesions. A strong humoral immune response was detected by ELISA, immunoblotting, and neutralization. Six sheep infected with clone LV1-1KS1 showed a completely different picture. No virus could be isolated from blood or CSF during 6 months of infection. At sacrifice all organs were virus-negative except the CNS of one sheep. None of the six sheep developed significant neutralizing antibodies and only low titer antibodies were detected by ELISA and immunoblotting. Minimal CNS lesions were present in one sheep. The molecular clones were also tested in sheep choroid plexus cells (SCP) and macrophages. In macrophages LV1-1KS1 replicated to a significantly lower titer but induced much more cell fusion than KV1772-kv72/67. The clones replicated equally well in SCP cells. Thus, these molecular clones of visna virus, which differ only by 1% in nucleotide sequence, showed a profound difference in replication and pathogenicity both in vitro and in vivo. These results can be used to map viral genetic determinants important for host-lentivirus interactions.
Asunto(s)
Macrófagos/virología , Virus Visna-Maedi/patogenicidad , Visna/virología , Animales , Anticuerpos Antivirales/sangre , Línea Celular , Macrófagos/citología , Ovinos , Visna/inmunología , Visna/patología , Virus Visna-Maedi/inmunología , Virus Visna-Maedi/aislamiento & purificaciónRESUMEN
An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue.
Asunto(s)
Enfermedades de los Peces/microbiología , Bacterias Grampositivas/aislamiento & purificación , Infecciones por Bacterias Grampositivas/veterinaria , Enfermedades Renales/veterinaria , Reacción en Cadena de la Polimerasa/métodos , Salmón/microbiología , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Genes Bacterianos/genética , Bacterias Grampositivas/genética , Infecciones por Bacterias Grampositivas/microbiología , Riñón/microbiología , Enfermedades Renales/microbiología , Datos de Secuencia Molecular , Ovario/microbiología , ARN Ribosómico 16S/genética , ADN Polimerasa Dirigida por ARN , Sensibilidad y EspecificidadRESUMEN
Intracerebral serial passage of visna virus KV1514 through three Icelandic sheep was used to select for strains with increased neurovirulence. A strain (KV1772) with increased neuropathogenicity was obtained. We isolated several proviral molecular clones from a plaque-purified biological clone of KV1772 that induced typical visna virus pathology in young sheep. One of the clones (kv72) was infectious, while others contained mutations or were permuted and required gene recombination with other proviral clones to generate infectious virus after transfection. Stable plasmids containing functional, full-length, visna virus KV1772 genomes were constructed from the proviral molecular clones. The in vitro cytopathic effects of virus derived from these clones varied depending upon the tissue origin of the infected cells. A goat cell line became persistently infected with molecularly cloned KV1772 virus; these cells resisted the cell-killing effects and continuously shed high levels of infectious virus. We determined the complete nucleotide sequence of a KV1772 provirus; it contains open reading frames for all structural and accessory genes previously identified in the visna virus genome and is highly homologous to other published visna virus sequences. Progeny of molecularly cloned KV1772 virus rapidly induced both a pronounced neuropathology and an unexpected, strong, neutralizing antibody response in experimentally infected young Icelandic sheep. The availability of stable plasmids of replication-competent and pathogenic proviral molecular clones of visna virus should now enable the study of the genetic determinants of neurovirulence and their interaction with the host immune system in visna virus pathogenesis.
Asunto(s)
ADN Viral/genética , Provirus/genética , Virus Visna-Maedi/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Cabras , Datos de Secuencia Molecular , Mutación/genética , Provirus/patogenicidad , Ovinos , Transfección/genética , Virulencia/genética , Virus Visna-Maedi/patogenicidadRESUMEN
Visna, a lingering meningo-encephalitis in sheep, was one of the diseases on which B. Sigurdsson based his theory of a special group of disorders called slow infections. The cause of the disease, a retrovirus, was isolated in 1957. Visna is now classified in a subgroup of retroviridae, lentivirinae, together with virus types in animals and the human immune deficiency virus HIV. Notwithstanding that Visna has been eradicated among Icelandic sheep for 25 years research into this virus continues since it bears many similarities to HIV and also MS.
Asunto(s)
VIH/clasificación , Virus Visna-Maedi/clasificación , Variación Antigénica , VIH/genética , VIH/inmunología , Proteínas Virales , Cultivo de Virus , Virus Visna-Maedi/genética , Virus Visna-Maedi/inmunologíaRESUMEN
Maedi-visna virus (MVV) of sheep was the first lentivirus to be isolated. The genomic organization of MVV is very similar to that of human immunodeficiency virus (HIV) with several genes regulating the expression of the viral genome. Viral replication is severely restricted in the host and some cells apparently contain the genetic information in a DNA provirus form with little or no expression of viral antigens. This seems to be a major factor in causing the "slowness" of lentiviral infections and the persistence of the virus in the host since the immune system may not recognize the provirus-containing cells. The target cells for HIV and MVV are similar although T4 lymphocytes are not specifically destroyed in maedi-visna. There are also certain similarities in the pathological changes in both diseases, both in the central nervous system, the lungs and the lymphatic system. Although the severe final immunodeficiency state characteristic of AIDS has not been observed in maedi-visna, the basic biological features of the MVV and its interaction with host cells are so similar to HIV infection, that we consider ovine maedi-visna useful animal model for the human lentivirus infections.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , VIH , Neumonía Intersticial Progresiva de los Ovinos/microbiología , Virus Visna-Maedi , Visna/microbiología , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/microbiología , Animales , Anticuerpos Antivirales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Modelos Animales de Enfermedad , VIH/genética , VIH/fisiología , Humanos , Infecciones Oportunistas , Neumonía Intersticial Progresiva de los Ovinos/complicaciones , Neumonía Intersticial Progresiva de los Ovinos/inmunología , Ovinos , Visna/complicaciones , Visna/inmunología , Virus Visna-Maedi/genética , Virus Visna-Maedi/inmunología , Virus Visna-Maedi/aislamiento & purificación , Virus Visna-Maedi/fisiología , Virus Visna-Maedi/ultraestructuraRESUMEN
The effect of the glucose analogue 5-thio-D-glucose (5TG) on the yeast Saccharomyces cerevisiae was studied. Derepression of mitochondrial respiratory chain cytochromes, alcohol dehydrogenase (isoenzyme II), NADH dehydrogenase and maltase was inhibited by 0.5-2 mM-5TG. Growth rate was only slightly affected. Ethanol was efficiently produced with 2 mM-5TG in medium initially containing 0.25% glucose. Mutants resistant to the growth inhibitory effects of 5TG on glycerol medium showed resistance to the catabolite repressing effects of glucose. Other mutants, known to be catabolite repression resistant, showed resistance to 5TG. The analogue seems to inhibit derepression of glucose repressible enzymes with greater potency than glucose itself.
Asunto(s)
Glucosa/análogos & derivados , Saccharomyces cerevisiae/efectos de los fármacos , Citocromos/biosíntesis , Represión Enzimática , Glucosa/farmacología , Mutación , NADH Deshidrogenasa/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , alfa-Glucosidasas/biosíntesisRESUMEN
The "start" cell division control genes CDC36 and CDC28 have been reported to contain a certain sequence homology to tissue oncogenes (ets and some protein kinase encoding oncogenes respectively). Here we report that temperature sensitive mutations in these genes are suppressed in cytoplasmic "petite" mutants and catabolite repression resistant mutants.
Asunto(s)
Genes Fúngicos , Oncogenes , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido Nucleico , Supresión Genética , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMEN
Subcellular distribution of hexokinase (HK) isoenzymes in 22 human breast cancers (21 primary cancers and 1 axillary metastatic growth) and 7 non-pathological human mammary gland tissue samples was studied with starch gel electrophoresis on isolated cell fractions obtained by differential centrifugation. Fractions used were cytosol, mitochondria and microsomes. A comparison of two methods for detecting HK activity was made, yielding different results regarding HK II and HK III. A method based on the ability of NADPH to fluoresce in UV gave a constant pattern of HK isoenzymes. In non-pathological breast tissue, only HK I was seen, i.e. in the cytosol and the microsomal fractions. HK I was also seen in all fractions of the cancers, but another more anodal band of HK I, as well as HK II and HK III, consistently appeared in the cytosol fractions. The more commonly used staining technique with tetrazolium dye revealed HK II in 45% and HK III in 50% of the samples. The pattern of HK isoenzymes in the cancers was the same irrespective of estrogen and progesterone cytosolic receptor contents and the histology of the tumors. The fluorescence method is, therefore, much more sensitive than the tetrazolium technique for detecting HK activity after electrophoresis and could explain difference in results obtained by various laboratories.