Identification of Marek''s disease virus nuclear antigen in latently infected lymphoblastoid cells.

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.     

Pathogenicity for chicks of line cells from lymphoma of Marek's disease.

Pathogenicity for chicks of the MSB-1 line, a cell line derived from the tumorous tissue of a chick with Marek's disease (MD) and established by Akiyama & Kato, was studied. Five groups, including a control one, of 20 chicks each were inoculated with 1 X 10(3), 1 X 10(4), 1 X 10(5), 1 X 10(6) and no cells of a 180-day culture of the cell line at one day of age. They were housed all together in an isolation unit. An attempt was first made successfully to isolate MD virus (MDV) directly in culture of kidney cells 3 weeks after inoculation. Horizontal infection was first detected 4 weeks after inoculation. From 3 weeks after inoculation on, the disease with almost the same clinical and pathological pictures as the infection with a virulent strain of MDV showed a high incidence. Morbidity was closely related to the number of MSB-1 line cells inoculated. Parenchymal destruction was conspicuous in the central lymphoid organs of four chicks given the largest number of MSB-1 line cells and sacrificed in extremis about 4 weeks after inoculation. Establishment of MD in chicks inoculated with MSB-1 line cells carrying MDV genome seemed to be initiated under the circumstances where the line cells which had come into contact with susceptible cells in the peritoneal cavity released virulent MDV per se. Then host chicks might be infected with MDV and suffer from MD at a high rate. There was no great difference in oncogenic potential between MSB-1 line cells cultivated in vitro for 180 days and virulent MDV serially passaged through one-day-old chicks.     

Suppression of mitogen-induced proliferation of normal spleen cells by macrophages from chickens inoculated with Marek's disease virus

Spleen cells from chickens 7 days after inoculation with Marek's disease virus (MDV) responded poorly to stimulation by phytohemagglutinin (PHA). Addition of these cells to syngeneic normal spleen cells caused of marked suppression of the PHA response of the normal cells. The MDV spleen cells also inhibited the DNA synthesis of MSB-1 lymphoblastoid cells in vitro. The suppressive activity is attributed to the presence in MDV spleen cells of a population of suppressor cells with characteristics typical of macrophages. The suppressor cell activity was not removable by treatment with anti-T or anti-B serum with C, but it was reversible by treatment with carrageenan or carbonyl iron/magnet, by passage through glass wool column, and by adherence to plastic Petri dishes. The adherent MDV spleen cells also showed strong suppressor cell activity against syngeneic normal spleen cells.     

Identification of a unique Marek's disease virus gene which encodes a 38-kilodalton phosphoprotein and is expressed in both lytically infected cells and latently infected lymphoblastoid tumor cells.

The identification of unique Marek's disease (MD) virus (MDV) antigens expressed not only in lytically infected cells but also in latently infected MD lymphoblastoid tumor cell lines is important in understanding the molecular mechanisms of latency and transformation by MDV, an oncogenic lymphotropic herpesvirus of chickens. Through cDNA and nucleotide sequence analysis, an open reading frame (designated the pp38 ORF) which encodes a predicted polypeptide of 290 amino acids was identified in BamHI-H. Demonstration that the pp38 ORF spans the junction of the MDV long unique and long internal repeat regions (MDV has an alphaherpesvirus genome structure) precludes the presence of the gene encoding the B-antigen complex (gp100, gp60, and gp49) in the same region of BamHI-H, where it was originally thought to exist. Duplication of the complete pp38 ORF was not observed in BamHI-D, but part of it (encoding 45 amino acids) was found in the long terminal repeat region of the fragment. By use of trpE-pp38 fusion proteins, antisera against pp38 were prepared. By immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a predominant virus-specific 38,000-dalton polypeptide (designated pp38) and a minor 24,000-dalton polypeptide (designated p24) were found. No precursor-product relationship was found between pp38 and p24 by pulse-chase analysis, and only pp38 was detected by Western blot (immunoblot) analysis with antiserum to pp38. pp38 was found to be phosphorylated and present in oncogenic serotype 1-but not nononcogenic serotype 3-infected cells. Expression of the gene encoding pp38 was relatively insensitive to phosphonoacetic acid inhibition, suggesting that pp38 may belong to one of the early classes of herpesvirus proteins. pp38 was also detected in the latently infected MSB-1 lymphoblastoid tumor cell line. The detection of antibody against pp38 in immune chicken sera indicates that pp38 is an immunogen in birds with MD. Most of the properties described here for a protein detected by methods based on finding the ORF first are identical to those of a 38-kDa phosphoprotein reported by others, suggesting that they are the same. Collectively, the data reported here provide (i) more definitive information on the complete ORF of another MDV gene and the protein that it encodes, (ii) clarification of the gene content within a specific region of the MDV genome, and (iii) the molecular means to conduct further studies to determine whether pp38 plays a role in MDV latency and transformation.     

Copy number of tandem direct repeats within the inverted repeats of Marek's disease virus DNA

We previously reported that DNA of the oncogenic strain BC-1 of Marek's disease virus serotype 1 (MDV1) contains three units of tandem direct repeats with 132 base pair (bp) repeats within the inverted repeats of the long regions of the MDV1 genome, whereas the attenuated, nononcogenic viral DNA contains multiple units of tandem direct repeats (Maotani et al., 1986). In the present study, the difference in the copy numbers of 132 bp repeats of oncogenic and nononcogenic MDV1 DNAs in other strains of MDV1 was investigated by Southern blot hybridization. The main copy numbers in different oncogenic MDV1 strains differed: those of BC-1, JM and highly oncogenic Md5 were 3, 5 to 12 and 2, respectively. The viral DNA population with two units of repeats was small, but detectable, in cells infected with either the oncogenic BC-1 or JM strain. The MDV1 DNA in various MD cell lines contained either two units or both two and three units of repeats. The significance of the copy number of repeats in oncogenicity of MDV1 is discussed.