RESUMEN
Previously, we demonstrated that intracerebral (IC) inoculation of a murine coronavirus, MHV-JHM, into two species of primates can result in acute encephalomyelitis (Murray et al., 1992a). Infectious virus isolated from acutely infected animals, designated JHM-OMp1, was inoculated IC into a second group of monkeys. In this report we describe observations on the acutely infected animals and those surviving the acute infection were sacrificed at later times post-infection. Results from dual in situ hybridization/immunohistochemistry screening of tissues show that astrocytes are target cells in white matter lesions during acute infection. In animals sacrificed 150 days post-infection, areas of demyelinated gliotic lesions, prominent in the spinal cord, were seen throughout the neuraxis. No virus products were detected in these late-infection lesions.
Asunto(s)
Aotidae/virología , Astrocitos/virología , Infecciones por Coronavirus/virología , Encefalomielitis/virología , Virus de la Hepatitis Murina/patogenicidad , Enfermedad Aguda , Animales , Sistema Nervioso Central/patología , Sistema Nervioso Central/virología , Líquido Cefalorraquídeo/virología , Convalecencia , Infecciones por Coronavirus/líquido cefalorraquídeo , Infecciones por Coronavirus/patología , Encefalomielitis/líquido cefalorraquídeo , Encefalomielitis/patología , Gliosis/patología , Gliosis/virología , Virus de la Hepatitis Murina/aislamiento & purificación , ARN Viral/análisis , Especificidad de la EspecieRESUMEN
Coronaviruses (CV) are pleomorphic enveloped RNA viruses that are ubiquitous in nature, causing a variety of diseases in both man and domestic animals. In man, CV are generally associated with upper respiratory tract infections. The two prototype strains that are the best studied human CV isolates and which are thought to be responsible for most of the respiratory infections caused by CV are called 229E and OC43. Humoral responses consisting of neutralizing antibodies to CV are present in most individuals by six years of age. Although the cellular immune response to CV in man has not been characterized at all, it is known that the spike (S) and nucleocapsid (N) proteins elicit the major cell mediated immune responses in the mouse. This report describes the production and characterization of eleven independently isolated T cell clones that are specific for the human CV(HCV) 229E. The T cell clones are CD4+ and presumably recognize a processed viral peptide presented by class II molecules on the surface of antigen presenting cells. Of six 229E-specific T cell clones tested against purified viral proteins, three recognize the 180 kD spike glycoprotein while the other three recognize the 55 kD nucleocapsid phosphoprotein. Analysis of the human T cell mediated response to HCV will provide information regarding which viral proteins elicit the immunodominant response, what the fine specificity of these T cell clones are (immuno-dominant peptides), and what the T cell receptor (TCR) and cytokine usage is of these virus specific clones.
Asunto(s)
Coronavirus Humano 229E , Coronavirus Humano OC43 , Coronavirus/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Linfocitos T/virología , Cápside/biosíntesis , Cápside/aislamiento & purificación , Línea Celular , Línea Celular Transformada , Células Clonales , Coronavirus/fisiología , Electroforesis en Gel de Poliacrilamida , Hemaglutininas Virales/biosíntesis , Hemaglutininas Virales/aislamiento & purificación , Herpesvirus Humano 4 , Humanos , Pulmón , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus , Proteínas del Núcleo Viral/biosíntesis , Proteínas del Núcleo Viral/aislamiento & purificación , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
Primary human and primate brain microvascular endothelial cells were tested for permissiveness to coronaviruses JHM and 229E. While sub-genomic viral RNAs could be detected up to 72 hours post-infection, primate cells were abortively infected and neither virus caused cytopathology. Human cells were non-permissive for JHM but permissive for 229E replication; peak production of progeny 229E and observable cytopathic effects occurred approximately 22 and 32 hour post-infection, respectively. Using the criterion of cytopathology induction in infected endothelial cells, 229E was compared to other human RNA and DNA viruses. In addition, virus induced modulation of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and HLA I was monitored by immunostaining of infected cells.
Asunto(s)
Encéfalo/irrigación sanguínea , Coronavirus Humano 229E , Coronavirus/fisiología , Coronavirus/patogenicidad , Endotelio Vascular/virología , Animales , Anticuerpos Monoclonales , Antígenos Virales/análisis , Antígenos Virales/biosíntesis , Línea Celular , Chlorocebus aethiops , Coronavirus/genética , Expresión Génica , Humanos , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/biosíntesis , Cinética , Microcirculación , Primates , ARN Viral/análisis , ARN Viral/biosíntesis , Especificidad de la Especie , Factores de Tiempo , Transcripción Genética , Molécula 1 de Adhesión Celular Vascular/análisis , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Células Vero , Replicación ViralRESUMEN
A previous report demonstrated that intracerebrally inoculated coronavirus produced CNS disease in two species of primates (Murray RS, Cai G-Y, Hoel K, et al., Virol 1992; 188: 274-84). We were therefore interested in testing the potential of coronaviruses to infect primate CNS tissue following peripheral inoculation. Four Owl monkeys (Aotus trivirgatus) were inoculated intranasally and ocularly and four were inoculated intravenously with coronavirus JHM OMp1 (Murray RS, Cai G-Y, Hoel K, et al., Virol 1992; 188: 274-84). Two intranasally and two intravenously inoculated animals received a second intravenous inoculum at 153 days post-infection. The animals were sacrificed 16, 35, 194, and 215 days post-infection. Tissue sections from brain and spinal cord were screened for viral products by in sity hybridization and immunostaining. Virus RNA and/or antigen was detected in the brains of all animals and the distribution corresponded to areas of inflammation and edema. Viral products were predominantly found in blood vessels and perivascular regions, suggesting hematogenous spread with entry into the central nervous system through endothelium.
Asunto(s)
Aotus trivirgatus/virología , Sistema Nervioso Central/virología , Virus de la Hepatitis Murina/fisiología , Administración Intranasal , Animales , Antígenos Virales/análisis , Encéfalo/virología , Susceptibilidad a Enfermedades , Encefalomielitis/virología , Inyecciones Intravenosas , Instilación de Medicamentos , Ratones , Virus de la Hepatitis Murina/aislamiento & purificación , Virus de la Hepatitis Murina/patogenicidad , ARN Viral/análisis , Especificidad de la Especie , Células Tumorales Cultivadas , Cultivo de VirusRESUMEN
Two separate studies are described in this report. First, 5 Owl monkeys were inoculated intracerebrally (IC) with coronavirus JHM OMP1; this virus isolate was cultured from the brain of an animal inoculated with uncloned MHV JHM. Two of the animals became neurological impaired and were sacrificed; these animals had developed severe encephalomyelitis as previously described. Two of the remaining 3 healthy animals were inoculated IC again at 90 days post-inoculation (DPI) and all 3 were sacrificed approximately 5 months after the first virus inoculation. Despite the lack of detectable infectious virus, viral RNA and antigen, all 3 animals had significant white matter inflammation and areas of demyelination in the spinal cord. In the second study 4 Owl monkeys were inoculated intranasally (IN) and ocularly and 4 inoculated intravenously (i.v.) with JHM OMP1. The animals were sacrificed between 16 and 215 DPI with 2 IN and 2 i.v. animals receiving a second i.v. inoculum at 152 DPI. Viral RNA and/or antigen was detected in the brains of all animals and the distribution corresponded to areas of inflammation and edema. One of the animals that received the second inoculum developed neurological impairment and subsequent analysis of tissues showed viral antigen in both brain and spinal cord. Viral products were predominantly found in blood vessels suggesting hematogenous spread with entry into the central nervous system (CNS) through endothelium.
Asunto(s)
Aotidae/microbiología , Infecciones por Coronavirus/etiología , Coronavirus/patogenicidad , Enfermedades Desmielinizantes/microbiología , Encefalomielitis/microbiología , Administración Intranasal , Animales , Antígenos Virales/análisis , Astrocitos/microbiología , Encéfalo , Edema Encefálico/microbiología , Edema Encefálico/patología , Córnea , Coronavirus/aislamiento & purificación , Coronavirus/fisiología , Infecciones por Coronavirus/microbiología , Infecciones por Coronavirus/patología , Enfermedades Desmielinizantes/patología , Encefalomielitis/patología , Gliosis/microbiología , Gliosis/patología , Inyecciones , Inyecciones Intravenosas , Meningitis Viral/microbiología , Meningitis Viral/patología , ARN Viral/análisis , Médula Espinal/microbiología , Médula Espinal/patología , Viremia/microbiologíaAsunto(s)
Encéfalo/microbiología , Coronavirus/aislamiento & purificación , Esclerosis Múltiple/microbiología , ARN Viral/aislamiento & purificación , Médula Espinal/microbiología , Animales , Astrocitos/microbiología , Astrocitoma , Secuencia de Bases , Células Cultivadas , Coronavirus/patogenicidad , Humanos , Ratones , Datos de Secuencia Molecular , Neuronas/microbiología , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
Epidemiological studies of patients with multiple sclerosis (MS) and animal model data support the hypothesis that viruses initiate the immunopathogenic events leading to demyelination in MS. There have been no reports, however, of consistent detection of viruses in MS central nervous system tissue. We probed MS and control brain with cDNA probes specific for human, murine, porcine, and bovine coronaviruses. We report the in situ hybridization detection of coronavirus RNA in 12 of 22 MS brain samples using cloned coronavirus cDNA probes. In addition, tissue was screened for coronavirus antigen by immunohistochemical methods; antigen was detected in two patients with rapidly progressive MS. Significant amounts of coronavirus antigen and RNA were observed in active demyelinating plaques from these two patients. These findings show that coronaviruses can infect the human central nervous system and raise the possibility that these viruses may contribute to the pathogenesis of MS in some patients.
Asunto(s)
Antígenos Virales/análisis , Encéfalo/microbiología , Coronaviridae/genética , Esclerosis Múltiple/microbiología , ARN Viral/análisis , Compuestos Azo , Encéfalo/patología , Colorantes , Coronaviridae/inmunología , Humanos , Esclerosis Múltiple/patologíaRESUMEN
Two species of primates, Owl and African green monkeys, were inoculated intracerebrally with either the neurotropic mouse hepatitis virus JHM or the putative multiple sclerosis brain coronavirus isolate SD. These viruses caused an acute to subacute panencephalitis and/or demyelination in the infected animals. The course of pathogenesis and sites of detected viral RNA and antigen was dependent both on animal species and virus strain but the results clearly showed that these viruses replicated and disseminated in the central nervous system (CNS) of these primates. This study suggests that human CNS may be susceptible to coronavirus infection.
Asunto(s)
Enfermedades del Sistema Nervioso Central/microbiología , Infecciones por Coronaviridae/patología , Enfermedades Desmielinizantes/microbiología , Animales , Aotidae , Secuencia de Bases , Northern Blotting , Western Blotting , Enfermedades del Sistema Nervioso Central/patología , Chlorocebus aethiops , Coronaviridae/patogenicidad , Coronaviridae/fisiología , Infecciones por Coronaviridae/microbiología , ADN Viral , Enfermedades Desmielinizantes/patología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Replicación ViralRESUMEN
Plasmids containing the varicella zoster virus (VZV) open reading frames (ORFs) 61 and 62 were used in a transient co-transfection assay to test for trans-activation of the VZV and herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) promoters. The trans-activating potential of the polypeptides encoded by these VZV ORFs, designated p51 and p140, was compared to that of their HSV-1 homologs ICP0 and ICP4, respectively. VZV p51 was functionally inactive in this system while p140 appeared to be a much stronger transcriptional activator than ICP4. Co-transfection of plasmids encoding VZV p140 and HSV-1 ICP0 resulted in a synergistic activation of the reporter gene as has been shown for the combination of ICP4 and ICP0.
Asunto(s)
Herpesvirus Humano 3/genética , Regiones Promotoras Genéticas , Transactivadores/genética , Luciferasas/genética , Plásmidos , Timidina Quinasa/genética , Transcripción Genética , Transfección , Proteínas Virales/genéticaRESUMEN
Malignant rabbit fibroma virus (MV) is a recombinant poxvirus derived from Shope fibroma virus (SFV) and rabbit myxoma virus (D. S. Strayer, E. Skaletsky, G. F. Cabirac, P. A. Sharp, L. B. Corbeil, S. Sell, and J. L. Leibowitz, 1983a, J. Immunol. 130, 399-404; W. Block, C. Upton, and G. McFadden, 1984, Virology 140, 113-124). We report here the transcriptional mapping of early RNAs transcribed from the SFV sequences within MV and from the corresponding regions in SFV. Hybridization analysis and S1 nuclease mapping of RNA using viral DNA probes were used to define 5' and 3' ends of the various transcripts. The RNAs described here are transcribed in one direction in a densely arranged head to tail fashion similar to that described for some vaccinia virus early transcriptional units. At late times of infection the early SFV RNAs are not detected whereas the early MV RNAs are present in minor amounts. The early SFV and MV transcripts range in size from 3170 to 425 nucleotides (nt) long. All of the longer transcripts are produced as a result of read through transcription. Three MV transcripts contain fused SFV and rabbit myxoma virus sequences due to transcription through the recombination junction region in the MV genome. Two other MV transcripts are transcribed from a unique initiation site near another recombination junction region resulting in RNAs that are composed of SFV sequences having unique 5' ends.
Asunto(s)
Virus del Fibroma del Conejo/genética , Genes Virales , Myxoma virus/genética , Poxviridae/genética , ARN Viral/análisis , Transcripción Genética , Animales , Secuencia de Bases , Virus del Fibroma del Conejo/patogenicidad , Myxoma virus/patogenicidad , Hibridación de Ácido Nucleico , Conejos , Recombinación Genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Five strains of Shope fibroma virus (SFV), a strain of rabbit myxoma virus, and a strain of vaccinia virus were compared by restriction endonuclease digestion of their viral DNAs. Restriction digest patterns revealed that SFV and rabbit myxoma, both members of the Leporipoxvirus genus, were distinct from vaccinia, an Orthopoxvirus. All strains of SFV examined had a high degree of nucleotide sequence homology as shown by conservation of restriction sites within their genomes. However, restriction patterns of SFV and myxoma were quite different from one another suggesting that the genomes from these two viruses of the Leporipoxvirus genus do not share a large, highly conserved region of homology as do the viruses belonging to the Orthopoxvirus genus. Restriction mapping identified inverted terminal repeats of approximately 12 kb in length. Restriction fragments representing all but 400 bp of the termini were cloned in plasmid vectors.
Asunto(s)
Clonación Molecular , Virus del Fibroma del Conejo/genética , Genes Virales , Poxviridae/genética , Animales , Enzimas de Restricción del ADN , Plásmidos , Conejos , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Shope fibroma virus (SFV) causes a localized, self-limited, fibroblastic proliferation in adult rabbits. Extracts of Shope fibroma tumors were found to contain a second virus that induces a rapidly progressive disseminated tumor. Dissemination of this malignant fibroma is associated with activation of commensal mucosal infection with Pasteurella multocida, causing purulent conjunctivitis and rhinitis and resulting in death from nasal obstruction. We have isolated this new agent by two cycles of plaque purification. It is a poxvirus that is antigenically virtually identical to SFV as measured by a plaque reduction assay, but behaves differently both in vivo and in vitro. We have called this virus malignant rabbit fibroma virus (MV). Electrophoresis of restriction digests made with HIND III indicates that despite the antigenic similarity of SFV and MV, the locations of HIND III sites in the two viral genomes are quite different. These experiments have enabled us to determine that MV was present in small quantities in our initial uncloned stock of Patuxent strain SFV. Lymphocytes from rabbits bearing MV-induced tumors responded poorly to both B and T lymphocyte mitogens. This nonspecific immunologic dysfunction is evident at or before the time when metastases and Gram-negative infection develop, and it becomes more profound as the disease progresses. MV-induced tumors may provide a model for Gram-negative infection and decreased immunologic responsiveness associated with malignancies.