Inhibitory effects of macrophages against Marek's disease virus plaque formation in chicken kidney cell cultures.

Inhibition of plaque formation by Marek's disease virus (MDV) in chicken kidney cell cultures was investigated with the use of peritoneal exudate cells (PEC) from chickens. PEC from MDV-infected White Leghorn chickens inhibited the formation of MDV plaques, whereas the inhibitory effect of PEC from chickens vaccinated with herpesvirus of turkey (HVT) or PEC from normal chickens was very weak. However, PEC from either normal chickens or HVT-vaccinated chickens inhibited the MDV plaque formation in the presence of serum from MDV-infected chickens but not from normal or HVT-vaccinated chickens. The capacity of PEC to inhibit plaque formation was significantly reduced when PEC was treated with carrageenan but not with antithymus or antibursa cell serum. These results indicate that macrophages may have a role in protection against Marek's disease by reducing the number of MDV-infected cells and thereby decreasing the spread of the virus in vivo.     

Properties of a chicken lymphoblastoid cell line from Marek's disease tumor.

Properties of a chicken lymphoblastoid cell line (MSB-1) from a Marek's disease tumor were studied. The cell line grew well at 41 degrees C in medium RPMI-1640 supplemented with 10% bovine fetal serum and had a doubling time of 8-12 hours. Cells grown in stationary suspension culture did not attach to the vessel and had the morphology of typical lymphoblasts. At 37 degrees C, the cell line grew initially but ceased to divide after several subcultures. In the subcultures maintained for 48-72 hours, 1-2% of the cells produced Marek's disease virus (MDV)-specific intracellular and mambrane antigens and contained herpesvirus particles when examined by the electron microscope. Cocultivation of these cells with duck or chicken embryo fibroblast cultures resulted in transfer of infection and production of microplaques typical of MDV. Peripheral nerve lesions and lymphoid tumors characteristic of Marek's disease were caused by inoculation of susceptible chicks with MSB-1 cells or duck cells infected with strain BC-1 of MDV recovered from the MSB-1 cell line. No specific tumors were produced at the site of inoculation, and infection was readily transmitted to cagemates. Tumors were also produced in the skeletal muscles and seemed to be largely virus induced. MSB-1 cell line was free of C-type virus particles.     

Characterization of an apparently nononcogenic Marek's disease virus.

A new isolate of Marek's disease virus (MDV) was described. This virus, SB, and a clone, SB-1, differed from pathogenic isolates in in vitro growth characteristics as described for other apathogenic isolates. Serologically, as with other apathogenic isolates, SB could be distinguished from pathogenic MDV and the avirulent turkey herpesvirus. SB failed to induce lesions characteristic of Marek's disease (MD) during a 6- to 11-week experimental period. Also, SB was nononcogenic in immunosuppressed chickens or in chickens inoculated with this virus in ovo. However, under those conditions, SB caused a cytolytic infection. The term "nononcogenic" rather than "apopathogenic" was therefore proposed to classify this and similar isolates. SB-1 protected chickens against challenge with either virulent MDV or the non-virus-producing MD tumor transplant, JMV. Possible mechanisms of protection are discussed.     

Effect of phosphonoacetate on Marek's disease virus replication.

Phosphonoacetate (PA), but not any of its analogues tested, effectively inhibited avian herpesvirus replication and viral DNA synthesis in cell cultures. At 100 mug/ml culture medium, PA completely inhibited the replication of Marek's disease virus (MDV), herpesvirus of turkeys, and owl herpesvirus, but had no measurable effect on normal cell growth. PA also inhibited DNA polymerases induced by these avian viruses. Enzyme inhibition was 50% at a PA concentration of 0.2 mug/ml. At a concentration of 3-6 mug/ml, the compound also effected a 50% inhibition of alpha (maxi) enzyme of the host DNA polymerase. It had no effect on the host beta (mini) enzyme. When administered to chickens, PA did not inhibit the replication of MDV, nor did it prevent the development of lymphoma.     

Development of cell-mediated immunity to Marek's disease tumor cells in chickens inoculated with Marek's disease vaccines.

Chickens inoculated with herpesvirus of turkeys or with apathogenic or attenuated vaccine strains of Marek's disease virus (MDV) developed a T-cell-mediated immune response to Marek's disease (MD) tumor cells. This immune response was detected in a 4-hour 51Cr-release assay in which effector cells obtained from spleens of vaccinated chickens were reacted with 51Cr-labeled target cells of an MD lymphoblastoid cell line (MSB-1). The cytotoxic effector cells generated by the vaccine viruses had characteristics similar to those noted previously for anti-MSB-1 effector cells generated by MDV. The immune response was specific to MSB-1 cells, because another target cell line (TLT) antigenically unrelated to MSB-1 cells was not lysed by the effector cells nor did the unrelated target cells inhibit the cytotoxicity of effector cells against MSB-1 target in a cold-target inhibition assay. Because MSB-1 cells contain MD tumor-associated surface antigen, we postulated that the immune response detected in the vaccinated chickens may be directed against this antigen and that the antitumor antigen immunity may play a role in the mechanism of vaccine protection against lymphoma development by pathogenic MDV.     

Marek's disease: a model for protection against herpesvirus-induced tumours

Marek's disease (MD) is a lymphoproliferative disease of chickens caused by a herpesvirus (MDV). Several effective vaccines have been developed and MD is therefore often considered as a model for studying antitumour vaccines. Key factors for the understanding of vaccine-induced immunity are discussed. Three serotypes have been characterized: serotype 1 or oncogenic MDV, serotype 2 or non-oncogenic MDV and serotype 3 or herpesvirus of turkeys. The three serotypes have clearly different genomes and proteins. The pathogenesis of infection with serotype 1 MDV can be divided into an early cytolytic phase, a latent phase and a second cytolytic infection combined with the development of tumours and permanent immunosuppression. The activation of T cells during the early cytolytic phase is important for establishing infection in the target cells for latency and transformation. Immune responses by B cells, and especially by T cells, are directed against viral infection. Evidence is presented that Marek's disease tumour-associated surface antigen is not involved in the antitumour responses. Moreover, the importance of antitumour immune responses in MD is questioned. Vaccinal immunity is dependent more on T cells than B cells and is directed against virus antigens. It is proposed that the evidence claimed for an antitumour response induced by vaccination actually relates to an antiviral response. The relevance of new, highly oncogenic strains causing MD in vaccinated chickens is discussed.     

Effects of levamisole on pathogenesis of Marek's disease

Levamisole was administered to chickens previously inoculated with Merek's disease virus (MDV) or infected by contact, and the influence of the drug on the mortality rate of Merek's disease (MD) was examined. The chickens inoculated with MDV and then administered levamisole (3 mg/bird) began to die earlier than chickens not treated with levamisole. However, the chickens infected with MDV by contact and then treated with the levamisole had delayed deaths. When the capacity of peritoneal exudate cells (PEC) to inhibit MDV plaque formation on chicken kidney cell (CKC) cultures was examined, the inhibitory activity of PEC from chickens inoculated with MDV and treated with levamisole at 4 days or 2 months of age was much weaker than that of PEC from chickens of the same ages that were not treated with levamisole. This effect was also noted when levamisole was added to CKC cultures to examine the inhibitory activity of PEC from MDV-infected chickens not treated with levamisole. These results indicate that the administration of levamisole to MDV-inoculated chickens in doses used in the present experiment suppressed the macrophage restriction on MDV replication and hastened the death of chickens by MD during the early course of infection.     

Establishment of B-lymphoblastoid cell lines from Marek's disease virus-induced tumors in turkeys

Four lymphoblastoid cell lines were established from tumors of turkeys inoculated with high doses of the GA strain of Marek's disease virus (MDV). Unlike other MD lymphoblastoid cell lines of chicken origin, these MDV transformed turkey lines appear to be B lymphocytes and produce immunoglobulin. Growth characteristics of these cell lines are slightly different; however, they all produce low levels of MDV-specific antigen and carry the complete genome of the virus as determined by virus rescue in the chicken or in duck embryo fibroblast cultures. These cell lines are pathogenic for chickens and produce virus-induced MD lesions. All four lines are free of the herpesvirus of turkeys, reticuloendotheliosis virus, and three lines are also free of avian leukosis virus. They all have typical normal turkey chromosomes and are positive for Marek's disease tumor-associated surface antigen.     

Transplantable Marek's disease lymphomas. III. Induction of MHC-restricted tumor immunity by lymphoblastoid cells in F1 hosts

F1 chickens, which were a cross between birds of the related inbred lines G-B1 and G-B2, were immunized with in vitro cultured virus-non-producer lymphoblastoid cells that were either syngeneic or allogeneic with the challenge tumor cells. The lymphoblastoid cells were derived from Marek's disease herpesvirus (MDV)-induced transplantable tumors. Previous findings showed that such immunization of G-B1 and G-B2 chickens prevented early mortality caused by the tumors. Lymphoblastoid cells syngeneic with the tumors were more effective than allogeneic cells, suggesting that an MHC-restricted immune response was induced, or, alternatively, that immune elimination of allogeneic cells prevented them from initiating strong immunity to MDV-associated tumor antigens. Immune elimination of the immunizing cells should not occur in F1 birds heterozygous for the 2 different parental MHC haplotypes (B6 and B13). Early mortality among F1 chickens immunized with lymphoblastoid cells that were syngeneic with the challenge tumor cells was significantly lower than for non-immunized control chickens or for birds immunized with lymphoblastoid cells that were allogeneic with the challenge tumor cells. Our results suggest that MDV-induced tumor antigens may be recognized by the host as altered-self MHC antigens.     

Identification of Marek's disease virus-specific antigens in Marek's disease lymphoblastoid cell lines using monoclonal antibody against virus-specific phosphorylated polypeptides

For identification of the antigens specific to Marek's disease virus (MDV) in virus-non-producer lymphoblastoid cell lines established from a tumor of Marek's disease (MD), hybridomas producing monoclonal antibodies (MAbs) against the antigens were isolated. Immunogens for preparation of the hybridomas were purified from the lysate of an MD-lymphoblastoid cell line, MSB1, by affinity chromatography coupled with chicken anti-MDV serum immunoglobulin G. Three of the MAbs obtained, MB1, MB2 and MB3, were specific to MDV by immunofluorescence test. An immunofluorescence test using MB2 antibody showed that immunofluorescence-positive cells in non-producer MD-lymphoblastoid line cells became detectable when the culture temperature was shifted from 41 degrees C to 33 degrees C or when treatment with 5-iodo-2-deoxyuridine (IUdR) was performed, indicating that the antigen reactive with MB2 antibody is an MDV-specific early antigen. This temperature shift or IUdR-treatment did not induce other MDV-specific antigens, such as late gene products of MDV, detected with MAbs. MB1 and MB2 antibodies immunoprecipitated 4 MDV-specific phosphorylated polypeptides with molecular weights (MWs) of 43,000 (43kd), 39kd, 36kd and 24kd from chick embryo fibroblasts productively infected with virulent MDV. In the place of 43kd, phosphorylated 44kd polypeptide was precipitated from avirulent MDV-infected fibroblasts. However, MB3 antibody did not precipitate any MDV-specific polypeptides from infected fibroblasts. These results suggest that the phosphorylated polypeptides are MDV-specific polypeptides predominantly expressed in non-producer MD-lymphoblastoid cell lines.     

Reduced incidence of Marek's disease gross lymphomas in T-cell-depleted chickens.

Chickens of line 7, highly susceptible to Marek's disease (MD), were depleted of T-cells by neonatal thymectomy, total-body gamma-irradiation, and multiple injections with antithymocyte serum. In two replicate experiments, significantly fewer gross lymphomas were present in T-cell-depleted chickens than in intact or in T-cell-depleted, reconstituted hatchmates; these findings provided evidence that T-cells may be the principal target for MD virus (MDV) transformation, T-cell depletion was not complete, and the presence of microscopic lesions in T-cell-depleted chickens was attributed to residual T-cells. Ten lymphomas from intact chickens and 2 lymphomas from a T-cell-depleted chicken were examined for cellular composition. All lymphomas consisted predominantly of T-cells. The results of this and other published studies indicated that T-cells may have a dual role in MD; They may serve as a target for lymphoma formation by MDV and also may participate in immune surveillance against the disease in resistant chickens.     

Immunization against Marek's disease using Marek's disease virus-specific antigens free from infectious virus.

Immunity against Marek's disease was conferred by the use of non-infectious materials extracted with non-ionic detergents from cells infected with the attenuated strain of Marek's disease virus (MDV). Antibody-free Rhode Island Red chicks were inoculated at 1 week of age with cell extracts emulsified in Freund's complete adjuvant and were given a second inoculation 1 week later without adjuvant. Protection against natural infection was obtained in groups inoculated with both soluble (not sedimented at 100,000 times g/2h) and insoluble antigens present in Nonidet P40 (NP40) extract, but only with the insoluble fraction of deoxycholate extract. The results suggest that the immunizing antigens can be partially solubilized with 0.5% NP40 and that the growth and spread of MDV are reduced in immunized chickens.     

Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus.

Three lymphoblastoid cell lines were established from splenic lymphocytes of a lymphomatous baboon (Papio hamadryas) by co-cultivation of the lymphocytes with X-irradiated cells of marmoset or baboon lymphoblastoid cell cultures; the baboon splenic lymphocytes failed to grow when cultured alone. A herpesvirus, associated with each cell line, was identified by immunofluorescence, molecular hybridization and electron microscopy. Antigenic comparison with Epstein-Barr virus (EBV) showed that the baboon herpesvirus and EBV shared cross-reacting viral capsid antigens (VCA): 20 of 20 (100%) anti-VCA (EBV)-positive human sera and 55 of 62 (89%) baboon sera reacted with the baboon lymphoblastoid cells and baboon sera stained EBV VCA in P3HR-1 and EB-3 cells. No nuclear antigen, as assayed by anti-complement immunofluorescence tests, was detected in baboon lymphoblastoid cells when human or baboon anti-VCA positive sera were used. Baboon anti-VCA-positive sera also failed to stain EBV nuclear antigens (EBNA) in Raji or P3HR-1 cells. Preliminary molecular hybridization studies showed only approximately 40% homology between viral DNA of baboon cell lines and DNA of EBV derived from P3HR-1 cells.     

Characteristics of JMV Marek's disease tumor: a nonproductively infected transplantable cell lacking in rescuable Virus.

Cells of the JMV Marek's disease (MD) tumor, originally produced by rapid serial passage of MD lymphoma cells in chickens, were characterized to determine whether they were of host or donor origin and to ascertain certain virus-host cell interrelationships. Differences noted in blood group B surface alloantigens between tumor cells and host lymphocytes indicated a probable nonhost origin (i.e., transplantability) of the tumor. JMV spleen tumors contained predominantly large lymphoblasts bearing MD tumor-associated surface antigen. DNA from JMV tumor cell suspensions hybridized significantly with MD virus cRNA, which indicated that JMV cells contained at least a portion of the MD virus genome. No MD virus was rescued from JMV tumors by techniques suitable for rescue of virus from MD lymphomas. The JMV tumor cells were also devoid of MD virus-specific antigens. These properties differed markedly from those of MD lymphoma cells and make the JMV tumor cell a unique, potentially valuable, tool for further study of oncogenic herpesvirus infection and tumor immunity in the chicken.     

Enhanced oncornavirus expression in Marek's disease tumors from specific-pathogen-free chickens.

An oncornavirus was recovered from cell cultures of kidney tumors from specific-pathogen-free chickens inoculated with Marek's disease herpesvirus (MDHV). The MDHV inoculum was free of infectious avian leukosis virus (ALV). Direct examination of a variety of tissues from MDHV-inoculated chickens demonstrated increased levels of ALV-specific RNA compared to tissues from diluent-inoculated (control) chickens. DNA from cultured kidney tumor cells annealed to an ALV complementary DNA probe at the same rate and exhibited the same extent of homology as DNA from cultured control kidney cells. This finding indicated the absence of exogenous ALV proviral sequences. As with vertically transmitted endogenous ALV of subgroup E, the oncornavirus recovered from kidney tumor cell cultures failed to replicate in chicken embryo fibroblast (CEF) cultures of the C/E phenotype, but did replicate in turkey embryo fibroblasts (TEF), which are permissive for replication of endogenous ALV of subgroup E. These oncornavirus particles served as a helper virus to form Rous sarcoma virus pseudotypes, which produced foci in TEF cultures but not in CEF cultures of the C/E phenotype. Whether enhanced expression of endogenous oncornavirus contributes to MDHV-induced tumorigenesis is not known.     

Further characterization of Marek's disease virus-infected lymphocytes. I. In vivo infection

Previous reports from this laboratory identified bursa-derived lymphocytes (B cells) and non-B cells as the predominant cell types respectively involved in the early cytolytic and subsequent latent infection of chickens with Marek's disease virus (MDV). It was not known whether these differences were qualitative or quantitative or if the method for detection of latent infection (viral antigen production after 48 h of in vitro cultivation) was sensitive enough. To further define the cells involved in the various phases of MDV infection, we used monoclonal antibodies which specifically react with B cells, or T cells, or la-antigen-bearing cells. Dual fluorescence tests to detect surface markers and viral internal antigen (VIA) were conducted with infected spleen cells freshly collected from MDV-infected chickens or after in vitro cultivation of those cells. The same antibodies were also used for a rosetting procedure to yield fractions enriched or depleted of T cells, B cells or la-bearing cells. These were examined directly for viral DNA by in situ hybridization or dot blot DNA hybridization and for VIA cultivation. We learned that infected T cells also comprise part of the early cytolytic phase of MDV infection but constitute a minority population (approximately 2-3%) compared to B cells (83-92%) at 3 or 4 days post infection. Latently infected cells were definitively identified as mostly la-bearing T cells, although a few (2-4%) were B cells. Prior to in vitro cultivation, latently infected cells apparently had insufficient viral DNA for detection by in situ hybridization, but the more sensitive dot blot procedure revealed viral DNA in fractions later found positive by VIA expression after in vitro cultivation. Viral DNA replication in latently infected cells apparently had occurred after 48 h cultivation because in situ hybridization detected infected cells at that time. Treatment of cell cultures with iodo-deoxyuridine, 12-O-tetradecanoyl phorbol-13-acetate or n-butyrate failed to increase the number of spleen cells which expressed VIA.     

Synthesis of immunoglobulin and Marek's disease virus antibody in susceptible and relatively resistant chickens.

The levels of IgM, IgY, and IgA and the development of specific antibody to Marek's disease virus (MDV) and sheep red blood cells (SRBC) in young chickens susceptible and resistant to Marek's disease were compared after exposure to MDV. No significant difference was noted in the immunoglobulin levels. However, the antibody response to MDV and SRBC occurred more rapidly in susceptible birds. The initial titer of antibody to these antigens was higher. These differences in response, however, were transient. At 3 weeks post exposure, the levels of IgM antibodies to MDV and antibodies to SRBC were similar in the two lines of chickens. At 6 weeks, the levels of IgY antibodies to MDV and antibodies detected by the agar gel precipitation test were similar.     

Demonstration of Marek's disease tumor-associated surface antigen in chickens infected with nononcogenic Marek's disease virus and herpesvirus of turkeys.

The presence of Marek's disease tumor-associated surface antigen (MATSA) was demonstrated on spleen cells from P-line chickens inoculated 5--6 days earlier with herpesvirus of turkeys and SB-1 (a nononcogenic Marek's disease virus). Antisera against MATSA expressed on five Marek's disease lymphoblastoid cell lines were able to recognize the MATSA present on SB-1-infected spleen cells. No viral membrane antigens and only a low incidence of viral internal antigens could be demonstrated.     

Ultrastructural characterization of a new human B lymphotropic DNA virus (human herpesvirus 6) isolated from patients with lymphoproliferative disease

A new DNA virus, designated "human B lymphotropic virus" or "human herpesvirus 6" (HBLV), has been isolated from the peripheral blood leukocytes of patients with various lymphoproliferative disorders, in some instances also associated with human immunodeficiency virus (HIV-1) infection. HBLV, propagated in vitro in human cord blood lymphocytes, was found to be ultrastructurally similar to members of the herpesvirus family. It is an enveloped virus with an icosahedral nucleocapsid made up of 162 capsomeres. Unenveloped nucleocapsids in the cytoplasm wee always coated with a tegument, a feature also described for human cytomegalovirus (HCMV). However, the nucleoprotein core of HBLV does not have the beaded appearance as that of HCMV, nor do HBLV-infected cells contain the skein-like structure. Immune electron microscopy demonstrated the presence of specific antibodies to viral envelope and internal antigens in sera of infected patients, indicating that this virus is a possible human pathogen. These and previously reported characteristics are consistent with the HBLV being a new and unique DNA virus morphologically belonging to the herpesvirus family.