K. Nazerian

Studies on the etiology of Marek's disease. II. Finding of a herpesvirus in cell culture.

Summary Duck embryo fibroblast (DEF) monolayer cultures seeded with whole blood from birds inoculated with the JM strain of Mareks disease (MD) and which showed a cytopathic effect (CPE), also contained intranuclear herpes-like virus particles. Complete virus particles with envelope essential for infectivity of herpesviruses were not found in either the infected cells or the extracellular materials of cultures containing such cells. All cultures showing CPE contained virus particles, and when inoculated into susceptible chicks caused MD. Cytopathic effect and viral synthesis could not be induced in fresh DEF cultures with spent media from infected cultures passed through 450 mμ Millipore filters. The perfect correlation obtained between CPE, presence of virus particles, and reproduction of MD by these cells suggests a cause and effect relationship and circumstantially implicates this virus in the etiology of MD.

Studies on the Etiology of Marek's Disease. I. Propagation of the Agent in Cell Culture

Summary A focal cytopathic effect (CPE) was observed in duck embryo fibroblast (DEF) cultures 11-25 days postinoculation with blood from the JM strain of Mareks disease (MD). All cell suspensions from such cultures reproduced MD when inoculated into chicks, while all of the morphologically normal DEF cultures were noninfectious. The CPE and infectivity were maintained in DEF cultures for 182 days. Both the induction of CPE in cell cultures and MD in chickens required intact cells in the inoculum. These data indicate that the JM strain of MD was successfully propagated in DEF cultures and produced a characteristic CPE.

A Nonproducer T Lymphoblastoid Cell Line from Marek's Disease Transplantable Tumor (JMV)

A continuous lymphoblastoid cell line was established from a JMV tumor transplant related to Mareks disease (MD). It is designated RPL1 (JMV) lymphoblastoid cell line. This cell line contains DNA sequences complementary to MD virus DNA and has an antigen similar to MD-tumor-associated surface antigen (MATSA). However, it lacks any MD virus (MDV) rescuable in vivo or in vitro. The cell line has surface antigens typical of chicken thymus cells (T cells) and histocompatability antigens different from those of the host chicken.

Deoxyribonucleic Acid of Marek's Disease Virus in a Lymphoblastoid Cell Line from Marek's Disease Tumours

Summary DNA extracted from a chicken lymphoblastoid cell line (MSB-1) originally derived from a Mareks disease tumour was examined for the presence of the Mareks disease virus genome. DNA cRNA membrane filter hybridization experiments established the presence of 60 to 90 genome equivalents in these cells.

Ultrastructural studies of a herpesvirus of turkeys antigenically related to Marek's disease virus

Abstract Morphological changes induced by a herpesvirus of turkeys (HVT) in duck embryo fibroblast, chicken embryo fibroblast, and chicken kidney cell cultures were studied by light and electron microscopy and compared with those of high cell culture passaged Mareks disease virus (MDV) in cell culture. Both viruses produced somewhat similar microplaques with different degrees of polykaryocytosis depending on the type of cell culture used. A high concentration of 35 nm nuclear particles, occasionally in crystalline form, were seen in cells infected with HVT. These cells also demonstrated crystalline arrays composed of particles about the same size and electron density of ribosomes. Morphologically, virions of HVT were similar to those of MDV and went through the same stages of maturation. However, a high percentage of naked virions of HVT were morphologically distinct; i.e., they lacked a single dense core and had an inner structure resembling an electron lucent cross.

Studies on the etiology of lymphomas in turkeys: isolation of reticuloendotheliosis in virus.

Studies of tissues from 25 normal turkey flocks and of 15 selected turkey lymphoid tumors revealed no Mareks disease or lymphoid leukosis viruses, although reticuloendotheliosis viruses (REV) were isolated from 3 flocks (2 with a history of neoplastic disease). The REV isolates were culturally and antigenically similar to the prototype REV strain T but were of low pathogenicity. Attempts failed to transmit 6 lymphoid tumors of diverse origin by inoculation into 2 strains of recipient turkeys. These data support an etiological role for REV in some though not all forms of turkey neoplastic disease.

Selective inhibition by phosphonoacetic acid of MDV DNA replication in a lymphoblastoid cell line.

Abstract The effect of phosphonoacetic acid (PA), an inhibitor of herpesvirus DNA polymerase, was studied on replication of Mareks disease virus (MDV) DNA in a lymphoid cell line (MSB-1) originally established from an MD tumor. PA had no significant effect on the growth of MSB-1 cells at concentrations of 100 μg/ml and lower. Also, PA had no effect on synthesis of early virus antigens at concentrations up to 400 μg/ml. PA did, however, suppress the synthesis of virus specific DNA, as indicated by a decrease in the number of virus genomes per cell, and inhibited the synthesis of late virus products, as indicated by the lack of virus assembly. Minimal virus DNA was produced in cells treated with up to 400 μ/ml of PA. Removal of PA from MSB-1 cultures and further propagation of the cells in the absence of PA resulted in an increase in the number of virus genomes per cell and synthesis of late virus products.

Comparative studies of six avian herpesviruses.

Six avian herpesviruses were compared as to DNA base composition, electron-microscope properties, and in vitro cultural characteristics. Mareks disease virus (MDV), herpesvirus of turkeys (HVT), and infectious laryngotracheitis virus (ILT) were found to have lower DNA buoyant densities than owl herpesvirus (OHV), pigeon herpesvirus (PHV), or Lake Victoria Cormorant virus (LVC). Also, the cell-associated MDV and HVT produced less enveloped viruses and grew less rapidly in cell culture than OHV, PHV, LVC, or ILT.

Structural proteins of Marek's disease virus

Abstract Mareks disease virus (MDV) was propagated in roller-bottle cultures of duck embryo fibroblasts and partially purified by sucrose gradient centrifugation. Analysis of viral protein by polyacrylamide gel electrophoresis revealed that at least eight proteins (designated VPI-VPVIII) were present in MDV. The VPI is the major viral protein. At least two viral proteins, VPII and VPIV, with glucosamine label could be detected. These two peaks may represent viral glycoprotein and may be associated with the viral envelope. No host cell proteins coelectrophoresed with any viral proteins. Similar electropherograms were obtained by coelectrophoresis of MDV and herpes simplex virus proteins and of MDV and pseudorabies virus proteins.

Development and characterization of a Marek's disease transplantable tumor in inbred line 72 chickens homozygous at the major (B) histocompatibility locus.

A serially transplantable Mareks disease (MD) tumor, designated MDCT-RP-3, was developed from an MD-virus-induced lymphoma (GA strain) in a pedigreed female chicken of the inbred B-histocompatible (B2/B2), line 72, and is the first MD tumor transplant to be developed in chickens both syngeneic and selected for susceptibility to MD. The MDCT-RP-3 tumor maintained its female karyotype through at least 80 passages in male 72 chickens. High doses of tumor cells caused progressively growing tumors at 5 days postinoculation and death of young 72 chicks in 7-10 days, whereas allogeneic chicks of other lines were less susceptible. Tumors frequently regressed when doses of tumor cells were low or older chickens were used. MD virus was rescued from MDCT-RP-3 cells in cell culture, and chickens surviving the early transplant response sometimes developed MD lymphomas. The tumor cells expressed MD-tumor-associated surface antigen (MATSA) and T-cell surface antigens. A serum raised in rabbits against MDCT-RP-3 cells and absorbed with normal 72 cells appeared to be reactive against MATSA on all MD tumor cells tested and is probably monospecific. Sera raised against MDCT-RP-3 in chickens also contained MATSA antibodies reactive against heterologous but not homologous MD tumor cells. Protection against transplantation of MDCT-RP-3 cells was not afforded by immunization with turkey herpesvirus vaccine. Some unvaccinated chickens that regressed MDCT-RP-3 transplant appeared to be partially immune to later development of MD lymphomas.