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

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Abstract
Summary 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.