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Persistence and Expression of Marek's Disease Virus DNA in Tumour Cells and Peripheral Nerves Studied by in situ Hybridization

Norman L. J. Ross

Department of Microbiology and Immunology, University of California, San Francisco, California 94143
and¹ Infectious Disease Section, Veterans Administration Hospital, San Francisco, California 94121, U.S.A.

We have used cloned fragments of Marek's disease virus (MDV) DNA and in situ hybridization to search for virus DNA and study its expression in infected chick embryo fibroblasts (CEF), lymphoblastoid cell lines, tumours and neural lesions. DNA from the HPRS 16/att strain of MDV was cleaved with EcoRI endonuclease and several fragments were cloned in Escherichia coli using the vector PBR322. Seven fragments ranging in size from 2.6 to 11 kbp representing approx. 25% of the MDV genome were labelled in vitro and annealed to EcoRI digests of DNA from infected cells and tumours following separation and transfer according to the Southern blotting procedure. Most of the selected MDV DNA fragments hybridized to fragments of corresponding sizes in EcoRI digests of DNA from cell lines and tumours and failed to hybridize to digests of uninfected chick cell DNA. In situ hybridization using ³H-labelled DNA with specific activity of 10⁸ d/min/µg as probe showed intranuclear MDV DNA in infected CEF, in every cell of two lymphoblastoid cell lines and in the majority of infiltrating or proliferating lymphoid cells found in type ‘A’ lesions of grossly enlarged peripheral nerves. Both intranuclear and cytoplasmic RNA were detected in cells that contained virus DNA. However, comparatively little virus RNA appears to be transcribed in cell lines and in infected tissues from the regions of virus DNA (25% of genome) used as probe in this study. Our results favour the hypothesis that the accumulation of lymphoid cells in nerves is not the result of an inflammatory response to infected nerve cells but is rather the consequence of proliferating transformed cells.

Keywords: molecular cloning, Marek's disease, in situ hybridization

Present address: Houghton Poultry Research Station, Houghton, Huntingdon PE17 2DA, U.K.

Received 3 June 1981; accepted 10 August 1981.