Genetic determinants of the demyelinating disease caused by Theiler's virus.
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Animals↗
Biomedical subjects
M Brahic
Publications and source records attributed to M Brahic.
Determinants of persistence and demyelination of the DA strain of Theiler's virus are found only in the VP1 gene.
The DA strain of Theiler's virus persists in the central nervous systems of mice and causes chronic inflammation and demyelination. The GDVII strain, on the other hand, causes an acute encephalitis that kills the host in a matter of days. We constructed a series of recombinants between two infectious cDNA clones of the genomes of DA and GDVII viruses. Analysis of the phenotypes of the recombinant viruses yielded the following results. (i) Determinants of persistence and demyelination are found only in the VP1 capsid protein of DA virus. (ii) Whereas the VP1 capsid protein of DA virus is able to fully attenuate the neurovirulence of GDVII virus and to allow the chimeric virus to persist and demyelinate, the VP1 capsid protein of GDVII virus is unable to render DA virus neurovirulent. (iii) The mere attenuation of the neurovirulence of GDVII virus does not allow it to persist and demyelinate.
Animals↗
In situ analysis of proteolipid protein gene transcripts during persistent Theiler's virus infection.
SJL/J mice inoculated intracranially with the DA strain of Theiler's virus exhibit a persistent demyelinating disease of the central nervous system. To investigate the effect of persistent infection of oligodendrocytes on the expression of myelin genes, we analyzed the level of PLP mRNA in infected as well as uninfected oligodendrocytes. This study was performed at the single-cell level using the simultaneous detection of viral antigens by immunocytochemistry and PLP mRNAs by in situ hybridization with 35S-labeled oligonucleotide probes. Our data indicate that viral infection of oligodendrocytes reduces the level of PLP mRNA by about 80%.
Animals↗
Isolation of a specific cellular mRNA by subtractive hybridization in Theiler's virus persistent infection.
Viruses change the mRNA repertoire of the tissues they infect. They add viral mRNAs and they specifically alter the expression of some host genes. These events can play important parts in pathogenesis. In principle, it should be possible to isolate viral mRNAs and to identify changes in host gene expression using subtractive hybridization. We tested this approach in the persistent infection of mouse central nervous system by Theiler's virus. A cDNA library was constructed with poly A+ RNA from infected mouse spinal cords. The library was screened with a subtracted probe. We identified one mitochondrial gene, coding for subunit 1 of cytochrome oxidase, which is overexpressed in infected tissues whereas another mitochondrial gene, URF 2, is not. Subtractive hybridization should prove to be invaluable in studying the pathogenesis of chronic human central nervous system diseases of unknown etiology.
Animals↗
Genetic mapping of the ability of Theiler's virus to persist and demyelinate.
Theiler's virus, a murine picornavirus, is responsible for two different types of disease: strains DA, BeAn, and WW persist for more than a year in the white matter of the central nervous system and cause primary demyelination; strains GDVII and FA, on the other hand, cause an acute encephalitis that kills the host in a matter of days. To map the regions of the viral genome responsible for persistence and demyelination, cDNA clones of the entire genomes of the DA and GDVII strains were constructed and cloned into Bluescript plasmid (A. McAllister, F. Tangy, C. Aubert, and M. Brahic, Microb. Pathogen. 7:381-388, 1989; F. Tangy, A. McAllister, and M. Brahic, J. Virol. 63:1101-1106, 1989). We constructed chimeric viruses obtained by exchanging regions between the cDNA clones. Analysis of the disease phenotypes produced by the chimeric viruses allowed us to map persistence and demyelination to a genome segment coding for the VP1 capsid protein and 27 amino acids of protein 2A.
Animals↗
Simultaneous in situ detection of two mRNAs in the same cell using riboprobes labeled with biotin and 35S.
We used 35S-labeled and biotinylated cRNAs (riboprobes) to detect simultaneously two different mRNAs by in situ hybridization. In a first step we established the conditions under which each type of probe achieved the same high level of sensitivity. We then used these conditions to hybridize BHK cells infected with Theiler's virus, a murine picornavirus, with a mixture of a virus-specific biotinylated riboprobe and a 35S-labeled riboprobe specific for beta-actin mRNA. Both mRNAs could be detected in the same cell, although the sensitivity achieved by the radiolabeled probe was reduced by about 40% by the simultaneous hybridization with the biotinylated probe.
Actins↗
Characterization of B lymphocytes present in the demyelinating lesions induced by Theiler's virus.
Theiler's virus, a murine picornavirus, persists in the central nervous system of SJL/J mice and causes inflammation and demyelination in the white matter of spinal cord. We isolated inflammatory cells from the central nervous system of infected animals and studied their functions in vitro. Flow microfluorimetry analysis showed the presence of all major lymphocyte subsets, namely CD4+ and CD8+ T cells as well as B lymphocytes. B lymphocytes were activated in vitro and the antigenic specificity of secreted Ig was determined by immunoblotting. Secreted Ig reacted strongly with viral capsid proteins VP1 and VP2 and had neutralizing activity. They reacted also with two nonviral white matter components which were present only in infected animals. Therefore, it is likely that Igs secreted at the site of infection play a role in limiting virus spread. It is also possible that virus induced autoreactive antibodies participate in demyelination.
Animals↗
Double-label techniques of in situ hybridization and immunocytochemistry.
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Animals↗
Molecular cloning of the complete genome of Theiler's virus, strain DA, and production of infectious transcripts.
We constructed a complete cDNA clone of the genome of Theiler's virus strain DA in a Bluescript plasmid. This recombinant plasmid, called pTMDA, was used to synthesize full length RNA transcripts of the viral insert. The RNA was infectious for BHK cells. Virus R1-DA, obtained from transfected BHK cells, caused the biphasic disease classically observed with this strain of Theiler's virus. SJL/J mice did not show clinical symptoms during the first week following intracranial inoculation, although viral antigens were found in a few neurons of brain and spinal cord. By 45 days post-inoculation, the mice had developed a chronic demyelinating disease and viral RNA and antigens could be found only in spinal cord white matter in areas surrounded by inflammatory infiltrates. At this stage no RNA or antigens were found in neurons. Therefore the phenotype of R1-DA was indistinguishable from that of genuine DA Theiler's virus.
Animals↗
Multiple sclerosis as a viral disease.
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Epidemiologic Methods↗
Molecular cloning of the complete genome of strain GDVII of Theiler's virus and production of infectious transcripts.
We constructed a complete cDNA clone of strain GDVII of Theiler's virus in Bluescript plasmid. This recombinant plasmid, called pTMGDVII, was used to synthesize full-length RNA transcripts of the viral insert. This RNA was infectious for BHK cells. Virus R1-GDVII, obtained from transfected BHK cells, caused rapidly fatal encephalomyelitis in BALB/c mice. High amounts of viral antigens were present in neurons. No antigens were found in white matter. Therefore, the phenotype of R1-GDVII was indistinguishable from that of genuine GDVII Theiler's virus.
Animals↗
Expression of Theiler's murine encephalomyelitis virus protease 3C and polymerase 3D in Escherichia coli and characterization of monospecific sera.
Defined DNA fragments of cloned Theiler's murine encephalomyelitis virus genome were used to construct procaryotic recombinant plasmids expressing viral genes 3C and 3D. In these plasmids (pEX-EMBL vectors), viral sequences are fused in-phase behind the Escherichia coli lac Z' gene which is under the control of the inducible lambda Pr promoter. Partially purified fusion proteins were used to immunize Balb/c mice. Sera monospecific for the viral protease 3C and polymerase 3D were obtained. These sera detected their corresponding antigens in situ in infected BHK cells using immunocytochemical reactions.
3C Viral Proteases↗
Minus-strand RNA synthesis in the spinal cords of mice persistently infected with Theiler's virus.
Theiler's virus, a murine picornavirus, causes a chronic neurological disease characterized by primary demyelination in SJL/J mice. The lesions are very reminiscent of those of multiple sclerosis. Theiler's virus persists in oligodendrocytes and to a lesser extent in astrocytes and macrophages throughout the disease. Viral RNA and capsid protein syntheses are minimal in these cells. This restriction could play a central role in the mechanism of virus persistence. By quantitating plus- and minus-strand RNAs in infected central nervous system cells, we showed that RNA replication was blocked at the level of minus-strand RNA synthesis.
Animals↗
Identification of Theiler's virus infected cells in the central nervous system of the mouse during demyelinating disease.
Theiler's virus is a picornavirus responsible for a persistent, demyelinating infection of mouse central nervous system. We examined the nature of infected cells during the course of this disease using a simultaneous immunoperoxidase-in situ hybridization assay. Cell types were identified with antigenic markers and infected cells were recognized by the presence of viral RNA. We found that, depending on the animal, approximately 10% of infected cells were migroglia-macrophages, 5 to 10% were astrocytes and 25 to 40% were oligodendrocytes. Approximately half of the infected cells could not be identified.
Animals↗
Quantitative in situ hybridization using initial velocity measurements.
In situ hybridization can be used to quantitate viral RNA at the single cell level by measuring levels of hybridization after saturation hybridization with an excess of cDNA probe has been achieved (1,2). In this paper we describe an alternative approach which consists in measuring the initial hybridization rate using a low concentration of cDNA probe and a short hybridization time. Under these conditions, we obtained a linear relationship between the number of autoradiographic grains and the number of viral genomes per cell in the range of 600 to 60,000 copies per cell of a 7-kb RNA genome. This approach allows an accurate measurement of copy number in a range for which saturation in situ hybridization is very difficult to achieve.
Autoradiography↗
Demyelinating lesions due to Theiler's virus are associated with ongoing central nervous system infection.
We used in situ hybridization and immunocytochemistry to look for a correlation between virus expression and white matter lesions during late demyelinating disease due to persistent Theiler's virus infection. We found the following. (i) Tissue lesions developed at the site of virus infection. This correlation was not explained by infection of lymphocytes and macrophages. (ii) Large differences in the extent of pathology existed between mice. The amount of inflammation paralleled the number of cells containing viral RNA or viral capsid antigens. (iii) C57BL/6 mice, which are resistant to demyelination, were able to eradicate the infection. Our results are strongly in favor of a mechanism of demyelination in which viral gene products play a central role.
Animals↗
Quantitation, with a new assay, of Theiler's virus capsid protein in the central nervous system of mice.
We developed a quantitative assay for antigens at the single-cell level. Tissue sections were reacted with a primary antibody, a biotinylated secondary antibody, or 35S-streptavidin. Binding of streptavidin to cells was quantitated by microscopic autoradiography. We showed that the number of autoradiographic grains was proportional to the amount of antigen per cell. With this assay, we studied the synthesis of Theiler's virus capsid proteins VP1, VP2, and VP3 in permissive BHK cells grown in vitro and in mouse central nervous system (CNS) cells during a persistent infection. We found that synthesis of the three capsid proteins was restricted in mouse CNS cells. Restricted virus replication could play a major role in the persistence of Theiler's virus in mouse CNS cells.
Animals↗