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

Abstract

SUMMARYWe 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//dg as probe showed intranuclear MDV DNA in infected CEF, in every celt 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.

Patterns of Single- and Double-stranded Hepatitis B Virus DNA and Viral Antigen Accumulation in Infected Liver Cells

“…However, our findings contrast sharply with similar in situ hybridization studies with papovaviruses (D6rries et al, 1979;Langelier et al, 1975;Neer et al, 1977), adenoviruses (Moar & Jones, 1975) and herpesviruses (Moar & Klein, 1978;Ross et al, 1981 ;Volsky et al, 1981). In each of these studies with viruses known to replicate their DNA in the nucleus, autoradiographic grains representing viral DNA were confined to the nuclei of infected cells.…”

Methylation of Marek's Disease Virus DNA in Chicken T-lymphoblastoid Cell Lines

“…T-lymphoblastoid cell lines established from malignant lymphomas of chickens with Marek's disease (MD) have been shown to contain multiple copies of the Marek's disease virus serotype 1 (MDV1) genome (Nazerian & Lee, 1974;Tanaka et al, 1978;Hirai et al, 1981;Ross et al, 1981). The latent virus genomes in non-producer MD lymphoblastoid cell lines were shown to exist as closed circular DNA of full length (Tanaka et al, 1978;Hirai et al, 1981;Rziha & Bauer, 1982) or in an integrated form (Kaschka-Dierich et al, 1979).…”

Nucleotide Sequence and Characterization of the Marek's Disease Virus Homologue of Glycoprotein B of Herpes Simplex Virus

“…Digests of genomic DNA were separated by electrophoresis in 0.7~ agarose gels, transferred to nitrocellulose and hybridized with 32p-labelled probes in the presence of 50~ formamide as described previously (Ross et al, 1981). Probes used to identify restriction enzyme fragments that contained the gB gene of MDV were 'prime cut probes' prepared by primer extension of appropriate M 13 single-stranded clones (Farrell et al, 1983).…”

“…An RNA ladder (0-24 to 9.5 kb, Bethesda Research Laboratorie~Gibco) served as size markers. Hybridization with 32p-labelled probes was carried out in the presence of 50% formamide at 42 °C as described previously (Ross et al, 1981).…”