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Detection of rabies RNA by in situ hybridization
82
CYTOKERATIN INFECTION
BY
ALTERATIONS
RESPIRATORY
AFTER
"IN VITRO"
SYNCYTIAL. VIRUS
EPITHELIAL
CELL
(PSV)
B. GARCIA-BARRENO, C. LOPEZ-GALINDEZ and J.A. MELERO Virologia e Immunologia Sanitarias, Centro National de Microbiologia, Majadahonda, Madrid, Spain
Extracts of Hep-2 cells infected by RSV have been analyzed by SDS-PAGE. A protein band (Mr 46,000) was consistently obtained in low yield when compared to uninfected cell extracts. This polypeptide has been identified as a cytokeratin of intermediate filaments : 1) The protein band was present in extracts of epithelial but not fibroblast-like cell lines. 2) It showed solubility properties characteristic of cytokeratins. 3) Monospecific antisera raised against the isolated polypeptide gave an immunofluorescence staining pattern characteristic of intermediate filaments. In addition, "pulse-chase" experiments indicate that the 46K polypeptide has a shorter half-life in RSV infected that uninfected cells. Also, viral nucleoprotein and matrix protein were present in cytokeratin-enriched extracts from RSV-infected Hep-2 cells. We suggest that RSV components associate with the cell cytoskeleton leading to inestabilization of this structure.
83
DETECTION
OF RABIES
RNA BY IN SITU HYBRIDIZATION
A. ERMINE, Laboratoire
E. CECCALDI,
de la RAGE, Institut
0. POCH, N. TORDO Pasteur
75015 Paris, FRANCE
We cloned the rabies genome by priming with an octadecanucleotide complementary to the 3' end of the RNA genome. We used these clones to adapt in situ hybridization techniques to rabies infected BHK cells and to frozen brain sections of inoculated mice. The cells and frozen section were fixed with ethanol-acetic acid and treated with HCl 2N and proteinase K to increase diffusion of probes through the cells. Background, dueto non specific hybridization of the probe with cellular RNA, was reduced by adding cellular RNA extract and PolyC, PolyG. Hybridization was run at laboratory temperature (20"-22°C) during 24h to 72h in the presence of 50% of formamid. In BHK cells we detected an important specific hybridization of 35 S probes 6h after infection. At this time, rabies antigenes could not be detected with rabies fluorescent antibody. Viral antigenes were detected betwen 14h and 18h after infection when the same conditions were used. In situ hybridization with 35 S labeled probes allowed to detect infected cells earlier than with rabies fluorescent antibody. Localisation of specific hybridization in cells and in brain was discussed. 42
CYTOKERATIN INFECTION
BY
ALTERATIONS
RESPIRATORY
AFTER
"IN VITRO"
SYNCYTIAL. VIRUS
EPITHELIAL
CELL
(PSV)
B. GARCIA-BARRENO, C. LOPEZ-GALINDEZ and J.A. MELERO Virologia e Immunologia Sanitarias, Centro National de Microbiologia, Majadahonda, Madrid, Spain
Extracts of Hep-2 cells infected by RSV have been analyzed by SDS-PAGE. A protein band (Mr 46,000) was consistently obtained in low yield when compared to uninfected cell extracts. This polypeptide has been identified as a cytokeratin of intermediate filaments : 1) The protein band was present in extracts of epithelial but not fibroblast-like cell lines. 2) It showed solubility properties characteristic of cytokeratins. 3) Monospecific antisera raised against the isolated polypeptide gave an immunofluorescence staining pattern characteristic of intermediate filaments. In addition, "pulse-chase" experiments indicate that the 46K polypeptide has a shorter half-life in RSV infected that uninfected cells. Also, viral nucleoprotein and matrix protein were present in cytokeratin-enriched extracts from RSV-infected Hep-2 cells. We suggest that RSV components associate with the cell cytoskeleton leading to inestabilization of this structure.
83
DETECTION
OF RABIES
RNA BY IN SITU HYBRIDIZATION
A. ERMINE, Laboratoire
E. CECCALDI,
de la RAGE, Institut
0. POCH, N. TORDO Pasteur
75015 Paris, FRANCE
We cloned the rabies genome by priming with an octadecanucleotide complementary to the 3' end of the RNA genome. We used these clones to adapt in situ hybridization techniques to rabies infected BHK cells and to frozen brain sections of inoculated mice. The cells and frozen section were fixed with ethanol-acetic acid and treated with HCl 2N and proteinase K to increase diffusion of probes through the cells. Background, dueto non specific hybridization of the probe with cellular RNA, was reduced by adding cellular RNA extract and PolyC, PolyG. Hybridization was run at laboratory temperature (20"-22°C) during 24h to 72h in the presence of 50% of formamid. In BHK cells we detected an important specific hybridization of 35 S probes 6h after infection. At this time, rabies antigenes could not be detected with rabies fluorescent antibody. Viral antigenes were detected betwen 14h and 18h after infection when the same conditions were used. In situ hybridization with 35 S labeled probes allowed to detect infected cells earlier than with rabies fluorescent antibody. Localisation of specific hybridization in cells and in brain was discussed. 42