Christine Aubert

Identification of Theiler's virus infected cells in the central nervous system of the mouse during demyelinating disease

Theilers 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.

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 Theilers 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 Theilers 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 Theilers virus.

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 Theilers 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.

In situ analysis of proteolipid protein gene transcripts during persistent Theiler's virus infection.

SJL/J mice inoculated intracranially with the DA strain of Theilers 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%.

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 Theilers 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.