Deoxyribonucleic Acid of Marek''s Disease Virus in Virus-Induced Tumors

DNA was extracted from [(3)H]thymidine-labeled Marek's disease virus (MDV) and purified by two cycles of CsCl gradient centrifugation in a fixed-angle rotor. The DNA was transcribed in vitro into (32)P-labeled complementary RNA (cRNA). MDV cRNA did not hybridize with DNA from chicken embryo fibroblast cultures or from chicken spleen, but hybridized efficiently with DNA from MDV particles or MDV-infected cell cultures. Five Marek's disease tumors from different chickens and different organs (ovary, liver, testis) were all found to contain MDV DNA sequences. The relative amount of MDV DNA varied from tumor to tumor and was between 3 and 15 virus genome equivalents per cell. The content of virus DNA per cell in spleens from tumor-bearing chickens was much lower than in tumors from the same animals. MDV-infected cell cultures contained a large proportion (28-59%) of virus antigen-positive cells, as measured by immunofluorescence, but tumor cells were negative in this respect (<0.02% positive cells). These data indicate that MDV is present in a provirus form in tumor cells.     

Augmentation of retrovirus-induced lymphoid leukosis by Marek''s disease herpesviruses in White Leghorn chickens.

Our objective was to determine whether the cell-associated herpesvirus vaccines used in chickens to control Marek's disease tumors can augment development of lymphoid leukosis (LL) induced by exogenous avian leukosis virus (ALV). Various single or mixed Marek's disease vaccines were inoculated at day 1, and ALV was injected at 1 to 10 days, with chickens of several experimental or commercial strains. Development of LL was monitored at 16 to 48 weeks in various experiments. In several strains of chickens we repeatedly found that the widely used serotype 3 turkey herpesvirus vaccine did not augment LL in comparison with unvaccinated controls. However, LL development and incidence were prominently augmented in several chicken strains vaccinated with serotype 2 vaccines, used alone or as mixtures with other serotypes. In one chicken strain, augmentation was demonstrated after natural exposure to ALV or serotype 2 Marek's disease virus viremic shedder chickens. Augmentation of LL by virulent or attenuated Marek's disease viruses of serotype 1 was intermediate in effect. Serotype 2 Marek's disease virus augmentation of LL was prominent in three laboratory lines and one commercial strain of White Leghorns, but it was not observed in an LL-resistant laboratory line or four commercial strains susceptible to ALV infection. Chickens developed similar levels of viremia and neutralizing antibodies to ALV regardless of the presence of augmentation of LL, suggesting that the mechanism of enhanced LL did not result from differences in susceptibility or immune response to ALV. We postulate that the serotype 2 herpesviruses may augment LL through one of several possible influences on bursal cells that are subsequently transformed by exogenous ALV.     

Map location of homologous regions between Marek''s disease virus and herpesvirus of turkey and the absence of detectable homology in the putative tumor-inducing gene.

The DNA region having homology between Marek's disease virus and herpesvirus of turkey was assigned to the restriction map of Marek's disease virus by Southern blot hybridization. Under moderate conditions at the level of 15% mismatching, homology was found to be distributed throughout the Marek's disease virus genome. The long inverted-repeat regions (TRL and IRL), which are considered to play a significant role in tumorigenicity, did not show any homology to herpesvirus of turkey DNA.     

Replicatin of the resident marek''s disease virus genome in synchronized nonproducer MKT-1 cells

MKT-1, a virus nonproducer lymphoblastoid cell line established from a Marek's disease tumor, was synchronized by double thymidine block to determine the sequence of events in the synthesis of cellular and latent marek's disease virus DNA. Cellular DNA synthesis was measured by incorporation of [3H]thymidine, whereas viral DNA synthesis was determined by DNA-DNA reassociation kinetics. The results of these studies indicate that the resident Marek's disease viral DNA in MKT-1 cells replicates during the early S phase of the cell cycle, before the onset of active cellular DNA synthesis. This observation is similar to that seen in the replication of resident Epstein-Barr virus DNA in synchronized Raji cells.     

Demonstration of a tumor-associated surface antigen in Marek's disease.

Surface antigenic markers were detected on three classes of Marek's disease (MD) tumor cells, i.e., MD lymphoma cells, cultured cells of the MSB-1 lymphoblastoid cell line, and JMV lymphoblastic leukemia cells, by indirect membrane immunofluorescent staining with serum from chickens immunized with JMV cells or from rabbits immunized with MSB-1 cells. This surface antigen was not detected on normal chicken lymphocytes, RPL-16 tumor cells (tranedormed by an avian RNA virus, or MD virus-infected fibroblasts that were positive for viral membrane antigen (MA). Furthermore, the surface antigen appeared unrelated to embryonic or histocompatibility antigens. This antigen is provisionally designated as a Marek's disease tumor-associated surface antigen (MATSA). The MATSA's on JMV, MSB-1 and MD lymphoma cells were related but not identical as demonstrated by antiserum titration, absorption and blocking tests with homologous and heterologous systems.     

In vitro cytotoxicity against Marek's disease lymphoblastoid cell lines after enzymatic removal of Marek's disease tumor-associated surface antigen.

Marek's disease tumor-associated surface antigen (MATSA) has been claimed to be the target of cytotoxic lymphocytes in in vitro tests for Marek's disease immunity. Treatment with papain, but not with trypsin or mixed glycosidases, removed MATSA from certain Marek's disease lymphoblastoid cell lines. Tumor cells with and without MATSA were used as target cells for in vitro studies on cell-mediated immune responses with sensitized spleen cells in a chromium release assay. The removal of MATSA did not influence the results of the chromium release assay. Attempts to block the cell-mediated cytotoxicity in vitro by coating tumor cells with an anti-MATSA serum failed. It was concluded that cell-mediated immune responses against Marek's disease tumor cells are directed against an as yet undefined antigen(s).     

The pp38 gene of Marek's disease virus (MDV) is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV

Marek's disease virus has a unique phosphoprotein, pp38, which is suspected to play an important role in Marek's disease pathogenesis. The objective of the present study was to utilize a mutant virus lacking the pp38 gene (rMd5Deltapp38) to better characterize the biological function of pp38. This work shows that the pp38 gene is necessary to establish cytolytic infection in B cells but not in feather follicle epithelium, to produce an adequate level of latently infected T cells, and to maintain the transformed status in vivo.     

Pathological changes in chickens inoculated with reticuloendotheliosis-virus-contaminated Marek's disease vaccine.

A disease characterized by delayed growth, anemia, abnormal feathers, and leg paralysis occurred among chickens inoculated with Marek's disease vaccine over a period from spring to fall in 1974. These chickens were recognized among flocks inoculated with the vaccine produced by two vaccine makers. The affected ones were examined pathologically. Gross examination revealed a slight enlargement of peripheral nerves and atrophy of the spleen, thymus, and bursa of Fabricius. Histopathologically, the peripheral nerves had a mild cell infiltration of lymphoid and plasma cells, edema, degeneration of nerve fibers with Schwann's cell proliferation. Perivascular cuffings consisting mainly of lymphoid cells were seen in the brain and spinal cord. Atrophic changes displayed by prominent reduction of lymphocytes were recognized in the spleen, thymus, and bursa of Fabricius. Etiological examination suggested that most of the chickens examined might have been infected with reticuloendotheliosis virus and not with Marek's disease virus. The pathological changes observed in the peripheral nerves and central nervous system, however, were not distinguishable from those of Marek's disease.     

Identification with monoclonal antibodies of glycoproteins of Marek''s disease virus and herpesvirus of turkeys related to virus neutralization.

By use of monoclonal antibodies cross-reactive with Marek's disease virus and herpesvirus of turkeys, three glycoproteins (for Marek's disease virus, gp115/110, gp63, and gp50; for herpesvirus of turkeys, gp115, gp62 and gp52) related to virus neutralization were identified. Immunization of chickens or rabbits with these glycoproteins purified by affinity chromatography resulted in production of neutralizing antibodies.     

Isolation and identification of Marek's disease virus by fluorescent antibody technique.

Cell-associated herpesvirus related to Marek's disease (MD) was isolated from the direct culture of kidney cells of naturally infected chickens at Taoyuan or by inoculation of clinical specimens to chick kidney (CK) and chick embryo fibroblast cells. The virus isolates replicated in CK or chick embryo kidney cell cultures were identified to be MD by the fluorescent-antibody technique.     

Altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirus-infected arterial smooth muscle cells

We describe herein the effects of Marek's disease herpesvirus (MDV) on cholesterol and cholesteryl ester metabolism in cultured chicken arterial smooth muscle cells. Infection of arterial smooth muscle cells from specific pathogen-free chickens with MDV, but not a virus control, herpesvirus of turkeys led to a 7-10-fold increase in the accumulation of free and esterified cholesterol and a 2-fold increase in phospholipids. The cellular lipid changes observed in the MDV-infected arterial smooth muscle cells resulted, in part, from the following: decreased low-density lipoprotein-cholesteryl ester hydrolysis due to decreased lysosomal (acid) cholesteryl ester hydrolytic activity; increased de novo synthesis of cholesterol; decreased excretion of free cholesterol; and, both increased cholesteryl ester synthetic activity and decreased cytoplasmic (neutral) cholesteryl ester hydrolytic activity which resulted in increased incorporation of oleic acid into cholesteryl ester. Other changes noted in the MDV-infected cells as compared to uninfected cells included a 2-fold increase in both total protein synthesis and lysosomal and microsomal marker enzyme activities. These alterations in lipid and protein metabolism in MDV-infected arterial smooth muscle cells may explain in part our in vivo findings that herpesvirus (MDV) infection of specific pathogen-free chickens fed a normocholesterolemic diet will induce arterial thickening and lipid accumulation resembling human atherosclerosis.     

Genomic expansion of Marek''s disease virus DNA is associated with serial in vitro passage.

An EcoRI restriction endonuclease pattern of Md11 virus DNA, a very virulent strain of Marek's disease virus (MDV), was obtained by using total cellular DNA from infected cells. With the EcoRI restriction endonuclease pattern and a published BamHI map of MDV (Fukuchi et al., J. Virol. 51:102-109), we constructed a partial EcoRI map of a series of MDV clones (gift from H. J. Kung). The clones were used to identify a region of the Md11 genome which is altered as the oncogenic virus is passaged in vitro. This region was mapped into a 1.8-kilobase segment in the inverted-repeat sequences flanking the long unique region of the virus genome. The alteration appeared to result from multiple DNA insertions that produced an increase of 0.6 to 5.4 kilobases. Although the expansion of this region did not diminish the ability of MDV to replicate in vitro, it may be associated with the loss of Marek's disease oncogenicity.     

Isolation of a reticuloendotheliosis virus from chickens inoculated with Marek's disease vaccine.

Over a period from spring to fall in 1974, a disease with delayed growth, anemia, abnormal feathers, and leg paralysis as main symptoms broke out in flocks of chickens inoculated with Marek's disease vaccine. A virus was isolated from affected birds in the field and the same lot of Marek's disease vaccine as inoculated into these birds. It had a common antigenicity to the T strain of reticuloendotheliosis virus (REV) and could not be discriminated from this strain on the basis of morphology or property. When chicks were inoculated with it, they presented essentially the same symptoms as the birds affected in the field. Since the disease was reproduced in this manner, it was presumed to have been caused by REV contained in the vaccine as contaminant. The virus persisted in the body for long time and also induced horizontal infection.     

Marek's disease virus (MDV) ICP4, pp38, and meq genes are involved in the maintenance of transformation of MDCC-MSB1 MDV-transformed lymphoblastoid cells.

An antisense strategy has been used to identify genes important for the maintenance of transformation of MDCC-MSB1 (MSB1) Marek's disease virus-transformed lymphoblastoid cells. Oligodeoxynucleotides antisense to the predicted translation initiation regions of ICP4 and pp38 mRNAs inhibited proliferation of MSB1 cells but not MDCC-CU91 (CU91) reticuloendotheliosis virus-transformed cells. Control oligodeoxynucleotides having the same base composition but a different sequence did not inhibit MSB1 cell proliferation. In addition, ICP4 and pp38 antisense oligodeoxynucleotides resulted in 77- and 100-fold reductions in colony formation by MSB1 cells in soft agar, respectively. To extend and corroborate these results, a novel system based on efficiently regulated expression of eukaryotic genes by a chimeric mammalian transactivator, LAP267 (S. B. Baim, M. A. Labow, A. J. Levine, and T. Shenk, Proc. Natl. Acad. Sci. USA 88:5072-5076, 1991), was used. MSB1-derived stably transfected cell lines in which RNA antisense to Marek's disease virus ICP4, pp38, or meq could be induced by treatment of the cells with isopropyl-beta-D-thiogalactopyranoside (IPTG) were constructed. Control cell lines in which expression of ICP4 sense or pUC19 sequences could be induced by IPTG were also constructed. Induction of the cell lines indicated that ICP4 antisense RNA, but not ICP4 sense RNA or pUC19 RNA, inhibited proliferation of MSB1 cells. Induction of ICP4, meq, or pp38 antisense RNAs, but not ICP4 sense or pUC19 RNAs, had a dramatic effect on relative colony formation by MSB1 cells in soft agar. These results indicate that ICP4, pp38, and Meq are all involved in the maintenance of transformation of MSB1 cells.     

A repeat sequence, GGGTTA, is shared by DNA of human herpesvirus 6 and Marek''s disease virus.

Some regions of the genomes of human B-lymphotrophic virus (HBLV), also designated as human herpesvirus 6, and Marek's disease virus were found to hybridize to each other under moderate to stringent conditions, scoring from 10 to 30% base-pair mismatch. Nucleotide sequence analysis showed that a 6-base-pair repetitive sequence, GGGTTA (DR2), present in the IRS-IRL junction region of the Marek's disease virus genome, was also reiterated in the HBLV genome. The function(s) of such a sequence is unknown, but this is the first report of homology between HBLV and a nonhuman herpesvirus.     

Persistence of genomes of both herpesvirus of turkeys and Marek's disease virus in a chicken T-lymphoblastoid cell line

A cell line tentatively designated as MDCC-BO1(T), was established from spleen cells of an apparently healthy chicken inoculated with herpesvirus of turkey (HVT). BO1(T) cells were T lymphoblastoid cells and the more than 95% of them had Marek's disease (MD) tumor-associated surface antigen (MATSA). However, no viral internal antigens or membrane antigens could be demonstrated in them by immunofluorescence tests using chicken anti-HVT and -MD virus (MDV) sera. The virus could be rescued from BO1(T) cells by co-cultivation with chick embryo fibroblasts (CEF). The DNA of the rescued virus was characterized as HVT DNA by its sedimentation profile in a neutral glycerol gradient and its endonuclease Hind III cleavage-pattern. Ultrastructural studies on CEF infected with the rescued virus revealed the presence of HVT-like virions. However, DNA-DNA reassociation kinetics showed that the BO1(T) cells contained a few copies of NVT and also MDV genomes.     

Lyophilization of Cell-Free Marek''s Disease Herpesvirus and a Herpesvirus from Turkeys

Cell extracts of the JM and GA strains of Marek's disease herpesvirus and the FC 126 strain of turkey herpesvirus were lyophilized with various stabilizers. Much higher virus titers were obtained with stabilizer than without stabilizer. Titers increased even further in the case of the Marek's disease virus strains by the addition of a chelating agent, disodium ethylenediaminetetraacetate.