RESUMEN
The hemagglutinating-inhibition (HI) test was used to detect antibodies for Theiler's murine encephalomyelitis virus (TMEV), and the virus was isolated from sero-positive mice derived from colonies in Japan. HI antibody was detected in conventional mice (38.7%; 137/354) at titers ranging from 1:8 to 1:512, but it not in SPF mice (0/90). To isolate the virus, weanling mice inoculated intracerebrally with samples obtained from sero-positive mice were sacrificed and 10% brain homogenates were subcultured. New isolates designated as YOC and AB strains were obtained, and their physicochemical and biological properties were characterized. The results indicated that the new isolates were similar to Theiler's original (TO) strain according to the following observations of persistent paralysis of the hind limbs, resistance to ether treatment, a particles size of 10 approximately 50 nm in diameter, stability at pH 3, a density of 1.35 g/cm3 and three major and one minor viral proteins, (VPO; 38 Kd, VP 1; 33 Kd, VP2; 32Kd, VP3; 25 Kd). Immunoblotting analysis also showed that VP 2 of YOC and encephalomyocarditis virus of the Cardiovirus group, reacted strongly with the antisera against the viruses as well as with the GDVII strain. These results suggest that TMEV infection does exist in conventional mouse colonies in Japan, and that these viruses resemble the TO strain of TMEV.
Asunto(s)
Infecciones por Enterovirus/microbiología , Virus Maus Elberfeld/aislamiento & purificación , Animales , Anticuerpos Antivirales/análisis , Western Blotting , Infecciones por Enterovirus/inmunología , Femenino , Pruebas de Inhibición de Hemaglutinación , Japón , Masculino , Virus Maus Elberfeld/análisis , Virus Maus Elberfeld/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Endogámicos , Proteínas Virales/análisisRESUMEN
Chronic Theiler's murine encephalomyelitis virus infection of susceptible mice is an animal model for human demyelinating diseases. Previously we described an altered and diminished pattern of central nervous system disease in immunocompetent SJL/J mice infected with a variant virus. This variant virus H7A6-2 was selected with a neutralizing mAb recognizing the capsid protein VP-1 of Theiler's virus. Here we characterize the variant virus by ELISA and neutralization assays and by sequencing selected regions of the viral RNA genome and relate the alteration to disease. The variant virus contains one single point mutation within a neutralizing epitope of VP-1. This nucleotide change lead to an amino acid replacement at amino acid 101 of VP-1, a threonine (wild type) to an isoleucine (variant). Model building based on sequence alignments and the known structure of the related Mengo virus indicates that the altered amino acid is located in an exposed loop on the surface of the virus at the periphery of a site that has been proposed to be the receptor binding site. The results of ELISA, neutralization assay, and direct RNA sequencing provide for the first time an opportunity to precisely map an important structural determinant of neurovirulence.
Asunto(s)
Cápside/análisis , Enterovirus/patogenicidad , Virus Maus Elberfeld/patogenicidad , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Cápside/genética , Proteínas de la Cápside , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Virus Maus Elberfeld/análisis , Pruebas de Neutralización , ARN Viral/análisis , Relación Estructura-ActividadRESUMEN
The Theiler's murine encephalomyelitis viruses (TMEV) are important neurotropic picornaviruses because they persist in the central nervous system (CNS) and produce an inflammatory demyelinating disease in the mouse, their natural host. Insight into the pathogenesis of this disease may come from studying the genetic and biochemical compositions of these viruses; therefore, in this report, the structural and nonstructural proteins specified by both highly and less virulent TMEV were examined. Using two-dimensional gel electrophoresis, structural and nonstructural proteins, originating from each of the three regions of the picornavirus genome (Kitamura et al., 1981; Rueckert and Wimmer, 1984), from nine TMEV isolates were compared on the basis of isoelectric points (pI). Proteins of two virulent TMEV (GDVII and FA viruses) had almost indistinguishable pI values, whereas two of the three major capsid proteins of the less virulent TMEV varied considerably. For example, the structural proteins VP1 and VP3 from seven less virulent viruses ranged from pI 6.3 to 6.9 and 6.5 to 8.3, respectively. On the other hand, the pI values of VP2 and nonstructural proteins from the less virulent TMEV varied relatively little. In general, structural proteins of each TMEV group had pI ranges unique to their respective biological group, while most nonstructural proteins were similar for all TMEV. The virus-specified proteins of Vilyuisk virus, which is serologically related to the TMEV and a possible cause of encephalomyelitis in man, had pI values similar to the less virulent TMEV. Finally, VP3 not only showed the greatest variation in pI among the less virulent TMEV, but it also was preferentially radioiodinated in intact virus from each of the two biological groups using the lactoperoxidase technique.
Asunto(s)
Enterovirus/análisis , Virus Maus Elberfeld/análisis , Proteínas Virales/análisis , Animales , Cricetinae , Electroforesis en Gel de Poliacrilamida , Radioisótopos de Yodo , Focalización Isoeléctrica , Virus Maus Elberfeld/patogenicidad , Proteínas Estructurales Virales , VirulenciaRESUMEN
We have combined in situ hybridization and immunocytochemistry to detect RNA and proteins in the same cell. We envision a wide range of applications for this method, and, as one example, we show that viral genomes and capsid antigens can be detected simultaneously in paraffin sections of the central nervous system of mice infected with Theiler's virus, the causative agent of a slow demyelinating disease.
Asunto(s)
Antígenos Virales/análisis , Enterovirus/análisis , Virus Maus Elberfeld/análisis , ARN Viral/análisis , Proteínas Virales/análisis , Animales , Antígenos Virales/genética , Cápside/análisis , Línea Celular , Cricetinae , Genes Virales , Riñón , Virus Maus Elberfeld/genética , Ratones , Hibridación de Ácido Nucleico , ARN Viral/genética , Coloración y EtiquetadoRESUMEN
Growth and neurovirulence of a number of neurotropic viruses show pronounced differences after passage in cell culture compared with continued in vivo passage in the central nervous system. The DA strain of Theiler's murine encephalomyelitis virus provides a model for studying these issues since DA virus grown in mouse brain produces acute neuronal disease in weanling mice, but tissue culture-passed DA virus does not. In addition, DA virus grown in mouse brain has a greater 50% mouse lethal dose/50% tissue culture infective dose ratio than tissue culture-passed DA virus. Comparison of these viruses required the analysis of virus purified directly from infected mouse brain, without tissue culture passage. Capsid proteins from DA virus grown in mouse brain were resolved on sodium dodecyl sulfate-polyacrylamide gels and shown to have the same profile as tissue culture-passed DA virus. Viral RNAs were the same size, with no evidence of defective interfering particle production. Two-dimensional gels of in vitro-labeled RNase T1-digested RNA showed that virus variants were more apparent during acute in vivo passage. These genomic differences may be critical in determining the biological behavior of the virus.
Asunto(s)
Encéfalo/microbiología , Infecciones por Enterovirus/microbiología , Enterovirus/análisis , Virus Maus Elberfeld/análisis , Animales , Cápside/análisis , Cricetinae , Ratones , Oligonucleótidos/análisis , ARN Viral/análisis , Ribonucleasa T1/metabolismoRESUMEN
The genetic changes occurring in the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) during persistent infection in the mouse central nervous system (CNS) were studied. RNase T1-oligonucleotide fingerprinting of the RNAs of 28 BeAn viruses isolated at various times postinfection (p.i.) demonstrated that mutation occurred throughout the infection. Although plaque-purified BeAn virus was used to inoculate mice intracerebrally, genetically different viruses were recovered from the CNS. One to three oligonucleotide changes were found up to Day 152 p.i., but all three viruses isolated at Day 180 had four to nine oligonucleotide changes. No pattern of oligonucleotide changes occurring in different virus isolates was found, yet three viruses isolated from different animals at Day 180 had the same four new oligonucleotides. Overall, the number of oligonucleotide changes represented a 0.1 to 1.2% change in the virus genome. In addition, the analytical two-dimensional gel technique of P.Z. O'Farrell, H.M. Goodman, and P.H. O'Farrell (Cell 12, 1133-1142, 1977) suggested that mutation occurred in all virus isolates. In nine isolates, one to three proteins were found to have charge changes, and in general, as many nonstructural proteins had charge changes as structural proteins. P20, a nonstructural protein probably equivalent to the protease described for encephalomyocarditis virus, was found to have shifted cathodally in six different viruses. Several virus isolates had doublet patterns, suggesting the possibility that within the CNS, subpopulations existed which had proteins of slightly different charge or that virus-specified proteins had been modified after translation. Finally, antigenic variation of neutralizing site(s) on BeAn virus isolates as a way for virus to evade immune surveillance and thereby maintain the persistent state was studied. The ability of mouse serum to neutralize persisting virus isolates was not significantly different from the ability to neutralize the infecting virus. Therefore, antigenic variation does not appear to be a factor in TMEV persistence.
Asunto(s)
Enfermedades del Sistema Nervioso Central/microbiología , Infecciones por Enterovirus/microbiología , Enterovirus/genética , Genes Virales , Virus Maus Elberfeld/genética , Animales , Antígenos Virales/inmunología , Epítopos , Masculino , Virus Maus Elberfeld/análisis , Virus Maus Elberfeld/crecimiento & desarrollo , Ratones , Mutación , Pruebas de Neutralización , Oligorribonucleótidos/análisis , ARN Viral/análisis , Temperatura , Ensayo de Placa Viral , Proteínas Virales/análisisRESUMEN
GDVII and DA picornaviruses, which represent two biologically distinct subgroups of Theiler's murine encephalomyelitis viruses, were analyzed to detect the presence of a RNA-linked protein, VPg. It was found that the single-stranded RNA genomes isolated from the virions of both viruses contain a protein of approximately 7,000 daltons, which is covalently linked to the 5' end of the RNA. Like in other picornaviruses (e.g., poliovirus and encephalomyocarditis virus), the terminal nucleotide adjacent to the protein was found to be pUp. The possibility that differently charged VPg species exist is also mentioned.
Asunto(s)
Enterovirus/análisis , Virus Maus Elberfeld/análisis , ARN Viral/análisis , Proteínas del Núcleo Viral , Proteínas Virales/análisis , Secuencia de Bases , Virus Maus Elberfeld/genética , Ribonucleótidos/análisisRESUMEN
The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.
Asunto(s)
Enterovirus/análisis , Virus Maus Elberfeld/análisis , Proteínas Virales/aislamiento & purificación , Animales , Cápside/metabolismo , Línea Celular , Cricetinae , Dactinomicina/farmacología , Electroforesis en Gel de Poliacrilamida , Riñón , Mesocricetus , Biosíntesis de Proteínas , Tripsina/metabolismo , Virión/análisisRESUMEN
The appearance of histological lesions and the localization of viral RNA in the central nervous system of mice infected with tissue culture-adapted Theiler's murine encephalomyelitis virus (WW strain) (TMEV-WW) was studied. Viral RNA was detected by autoradiography after in situ hybridization, using a (3)H-labeled DNA probe complementary to virion RNA, which was applied to deparaffinized sections of central nervous system tissues from infected mice. Subjacent histological sections of tissues were used to assess the location and extent of lesions. Lesions were first observed at 20 days post-inoculation and appeared to enlarge throughout infection. They consisted of infiltrates of mononuclear cells and lymphocytes in spinal cord white matter and leptomeninges; at 78 days post-inoculation severe necrotizing and demyelinative myelitis and gliosis were observed. In contrast to the pathogenesis of brain-derived TMEV-WW-infected mice, no lesions were found in the central nervous system gray matter of mice infected with tissue culture-adapted TMEV-WW at any time post-infection. Tissue culture-adapted viral RNA was found in the cells of spinal cord white matter throughout infection; only one neuron in close proximity to the injection site was found to contain viral RNA shortly after infection. At early times after infection, spinal cord white matter cells containing viral RNA were found before development of inflammatory lesions; at later days post-inoculation, positive cells were found within, at the periphery of, or at a distance from lesions. The number of infected cells and the amount of viral RNA per cell appeared to remain constant from 20 to 78 days post-inoculation despite the increasing intensity of the inflammatory response. The nearly exclusive spinal cord white matter tropism of tissue culture-adapted TMEV-WW appeared to directly correlate with the disease-inducing potential of this virus.
Asunto(s)
Infecciones por Enterovirus/microbiología , ARN Viral/análisis , Médula Espinal/análisis , Animales , Aracnoides/análisis , Aracnoides/microbiología , Aracnoides/patología , Técnicas de Cultivo , Infecciones por Enterovirus/patología , Virus Maus Elberfeld/análisis , Ratones , Ratones Endogámicos ICR , Neuroglía/análisis , Neuroglía/microbiología , Neuroglía/patología , Neuronas/análisis , Neuronas/microbiología , Neuronas/patología , Hibridación de Ácido Nucleico , Piamadre/análisis , Piamadre/microbiología , Piamadre/patología , Médula Espinal/microbiología , Médula Espinal/patologíaRESUMEN
Theiler's murine encephalomyelitis viruses are usually included in the enterovirus genus of the family Picornaviridae, although there is little physicochemical evidence to support this classification. In this report, the size of the RNA of highly virulent and less virulent representatives of the Theiler's group of viruses has been determined by sucrose gradient centrifugation and electrophoresis in agarose to be the same as that of other enteroviruses. The absence of a poly(C) residue provides evidence that these viruses are not cardioviruses or aphthoviruses. The base composition of the two members are similar to each other but differ from those of other enteroviruses. However the one- and two-dimensional maps of the ribonuclease T1 hydrolysates of the two virus RNAs show considerable differences despite their close serological similarity. Virus-specified RNA synthesis in cells infected with the more virulent strain of the virus was almost 10 times greater than that induced by the less virulent strain, in accord with the yields of virus particles.
Asunto(s)
Enterovirus/análisis , Virus Maus Elberfeld/análisis , ARN Viral/análisis , Composición de Base , Centrifugación por Gradiente de Densidad , Cinética , Virus Maus Elberfeld/metabolismo , Virus Maus Elberfeld/patogenicidad , Oligorribonucleótidos/análisis , Poli C/análisis , ARN Viral/biosíntesis , Ribonucleasa T1RESUMEN
The WW strain of Theiler's murine encephalomyelitis virus (WW-TMEV) was purified from homogenates of acutely infected mouse brain. Infectious WW-TMEV was found to have an estimated sedimentation coefficient of 156 (s20,w) and a density of 1.35 g/cm3 in CsCl. Electron microscopy revealed a homogeneous population of 26-nm nonenveloped particles. Iodination of sodium dodecyl sulfate (SDS)-disrupted virions revealed four major capsid proteins with molecular weights of 58,000, 37,000, 34,000, and 27,000. A 6,000-dalton polypeptide was observed after long exposures of autoradiograms. The 37,000-, 24,000-, 27,000-, and 6,000-dalton polypeptides corresponded to picornaviral VP1, VP2, VP3, and VP4 capsid polypeptides, respectively. Comparison of autoradiograms of virions radiolabeled before and after SDS disruption indicated that the 58,000-dalton protein, VP2, and VP3 preferentially bound 125I under the labeling conditions used. Direct evidence was obtained that VP2 and VP3 were derived from the 58,000-dalton polypeptide by isolation of the 58,000-dalton polypeptide from polyacrylamide gels run under nonreducing conditions and subjecting it to reelectrophoresis under reducing conditions. The effect of trypsin on purified virions and their polypeptides was also investigated. Trypsin-sensitive sites were found in the 58,000-dalton protein, VP1, and VP2. Our results indicate that, in addition to the four typical picornaviral capsid polypeptides, there is a 58,000-dalton polypeptide present in WW-TMEV, which is sensitive to trypsin and can be reduced into two of the capsid proteins, VP2 and VP3.
Asunto(s)
Encéfalo/microbiología , Cápside/análisis , Infecciones por Enterovirus/microbiología , Enterovirus/análisis , Virus Maus Elberfeld/análisis , Proteínas Virales/análisis , Animales , Electroforesis en Gel de Poliacrilamida , Virus Maus Elberfeld/aislamiento & purificación , Ratones , Peso Molecular , Péptidos/análisis , Tripsina/farmacologíaRESUMEN
Theiler's murine encephalomyelitis viruses (TMEV) are separable into two groups based on their biological behavior: those highly virulent isolates which are unable to cause persistent infection and the less virulent isolates which regularly produce persistent central nervous system infection in mice. Two highly virulent and five less virulent TMEV were found to have the same buoyant density (1.34 g/ml) on isopycnic centrifugation and virion structure by electron microscopy. Negatively stained virus particles purified in Cs(2)SO(4) gradients appeared to have icosahedral symmetry and measured 28 nm in diameter. Mature virions were found to possess three major structural polypeptides, VP1, VP2 and VP3, in the range of 25,000 to 35,000 daltons, and a smaller fourth major polypeptide, VP4, of 6,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The precursor of VP2 and VP4, VP0, which is a minor polypeptide of mature picornavirus particles, was also identified. However, a slight but consistent difference in several of the capsid polypeptides between the highly virulent and less virulent TMEV was found. VP1 was slightly larger (34,000 versus 33,500 daltons) and VP2 was slightly smaller (31,000 versus 32,000 daltons) for the highly virulent strains compared to the same polypeptide species in the less virulent viruses. VP0 was also slightly smaller (35,500 versus 36,000 daltons) for the highly virulent isolates compared to their less virulent counterparts. Finally, trypsin which was used initially in our purification procedure resulted in preferential cleavage of a 2,000-molecular-weight fragment or fragments from VP1 of only the less virulent isolates.
Asunto(s)
Enterovirus/análisis , Virus Maus Elberfeld/análisis , Proteínas Virales/análisis , Centrifugación por Gradiente de Densidad , Virus Maus Elberfeld/aislamiento & purificación , Virus Maus Elberfeld/patogenicidad , Peso Molecular , Péptidos/análisis , Tripsina/farmacología , Virión/análisisRESUMEN
The polypeptides of encephalomyocarditis, Mouse-Elberfeld and type 5 rhinoviruses behave similarly when chromatographed on calcium phosphate (brushite), each being eluted by a linear phosphate buffer gradient containing sodium dodecyl sulphate in three major peaks, CI, C2 and C3. Analysis of the peaks by polyacrylamide gel electrophoresis suggests that the major capsid polypeptides of these three picornaviruses elute in the order: delta (peak CI), gamma with (peak C2) and alpha (peak C3).