Protection against the JMV Marek's disease-derived transplantable tumour by Marek's disease virus-specific antigens

Chickens immunised with inactivated chicken kidney cells infected with Marek's disease (MD) virus were protected against the lethal effects of inoculation with the MD-derived tumour transplant JMV, which appears not to express viral antigens. It is concluded that the distinction between MD virus-specific and tumour-specific antigens is not as complete as has been thought.     

Failure of attenuated Marek's disease virus and herpesvirus of turkeys antigens to protect against the JMV Marek's disease-derived transplant able tumour

Chickens immunised with chick embryo fibroblasts infected with attenuated Marek's disease virus or with the herpesvirus of turkeys and inactivated with glutaraldehyde were not protected against the lethal effects of inoculation with the Marek's disease-derived tumour transplant, JMV, although such immunisation protected against experimentally induced Marek's disease. It is concluded that there may be virus-coded antigenic determinants that are peculiar to oncogenic Marek's disease viruses and lacking from the non-oncogenic variants.     

Stimulation of local solid tumour development of the nonproducer Marek's disease tumour transplant JMV by virus-induced immunosuppression

Chickens could be protected against lethal lymphoblastic leukaemia due to the nonproducer JMV Marek's disease (MD) tumour transplant by infection with the herpesvirus of turkeys (HVT) or various strains of MD virus. However, solid JMV tumours developed in MD virus-infected birds at the site of intramuscular or subcutaneous transplantation, but tumours never developed at the site of MD virus inoculation. The incidence and extent of local tumour growth, the development of metastases and the inhibition of tumour regression were related to the pathogenicity of the MD virus strains used for pre-treatment of the chickens. Infection of chickens with reticulo-endotheliosis virus (REV-C) or with chick syncytial virus (CSV), which are nonprotective against MD virus or JMV transplants, stimulated local tumour development of the attenuated JMV-A variant of the JMV transplant. Chickens which did not reject local tumours died of visceral JMV tumour metastases. A direct helper mechanism of viral infection on the oncogenicity of transplants was excluded. The results suggested that virus-induced immunosuppression stimulated the development of local JMV tumours which never occurred in normal chickens. Immunity to the JMV transplant, including resistance to lethal leukaemia and successful regression of local tumours, did not coincide with immunity to MD virus-induced visceral lymphomas or nerve lesions. Vaccinal induced tumour immunity evidently was defective. The significance of these results is discussed with reference to immunological functions of MD tumour-specific antigens.     

Local tumours induced in chickens by continuous cell lines of the JMV Marek's disease tumour transplant

Marek's disease (MD) tumour cell lines RPL-1 and JMV-1 were shown to express high oncogenic potential if inoculated intramuscularly. No more than 10(3) cells per dose were needed to cause primary tumours at the site of inoculation in 50% of 2-day-old chickens, but more than JO(6) to more than 10(7) cells were required to cause a 50% mortality due to lymphoblastic leukaemia as another parameter of oncogenicity. Herpesvirus of turkeys (HVT) was nonprotective against local tumour development of the transplants, and resistance to JMV tumours did not coincide with resistance to Marek's disease.     

Propagation and assay of virulent and attenuated JMV Marek's disease-associated tumour cells

JMV tumour cells were shown to cause a lethal lymphoblastic leukaemia in young chickens as well as in chicken embryos. The incubation period was very short but dose-dependent. Chickens died in 4 to 12 days, embryos in 7 to 14 days, after inoculation. Embryo-passaged attenuated JMV (JMV-A) caused the same lesions in embryos as virulent JMV. The dose-response relationship depended on the route of inoculation and on the quality of the tumour cell preparation. Intramuscular (i.m.) inoculation of leukaemic blood or embryo lymphoblasts provided the most satisfactory response. Intraperitoneal (i.p.) inoculation and lymphoblastic chicken spleens as a source of JMV were definitely less suitable. The dose-response curves obtained in yolk sac-inoculated embryos were similar to the curves obtained by i.m. inoculation of chickens. Only 4 to 10 lymphoblasts were needed per lethal dose (50%) in chickens and 50 to 80 in embryos. The pathogenicity and antigenicity of JMV and JMV-A were strictly cell-associated. No Marek's disease (MD) virus or any other avian virus could be detected, either by various virus isolation procedures, or by serological methods. Contact transmission of JMV to other chickens did not occur. Antibodies against surface antigens on JMV lymphoblasts were detected in JMV and JMV-A chicken hyperimmune sera. These sera reacted against MD lymphoblastoid cell lines (HPRS-1 & 2, MSB-1) as well as MSB-1 anti-serum, but all sera reacted also against thymus lymphocytes from normal chickens. The results of absorption tests suggested that the surface antigens of JMV lymphoblasts and of the tested cell lines were not identical. The majority of tumour cell surface antigens appeared to represent genetically specific histocompatibility or lymphocyte antigens. A common MD tumour-associated surface antigen (MATSA) could not be identified serologically (FA test) on the tumour cells studied.     

Potency tests with HVT-, MHV- and JMV vaccines against Marek's disease

Turkey herpesvirus (HVT)(FC 126) vaccine made in W. Germany, Marek's herpesvirus (MHV) (CVI 988) vaccine used in the Netherlands and experimental JMV vaccine were tested in laboratory and field trials for protection against Marek's disease. The tests were carried out with 780 SPF chicks and 3200 commercial white Leghorn chicks (with maternally derived antibodies to HVT and MHV). There were no differences in potency of HVT- and MHV vaccines. Both vaccines showed increased protection with an increased interval between vaccination and challenge. A significant protection (> 80%) resulted with both vaccine viruses after the 8th day following vaccination. JMV vaccine (embryo-adapted strain JMV-A-164) provided a 40 to 60% protection against Marek's disease depending on the time of challenge. However, there was a complete protection against the highly pathogenic JMV tumour cell inoculum. Hyperimmunisation of adult birds with JMV vaccine did not reduce the mortality in their progeny from Marek's disease.     

Effects of infectious bursal disease virus and reticuloendotheliosis virus infection of chickens on the incidence of Marek's disease and on local tumour development of the non-producer JMV transplant

Dual infection of chickens with the HPRS-16 or HPRS-B14 strains of Marek's disease (MD) virus and non-transforming reticuloendotheliosis virus (REV-C) was shown not to affect significantly the incidence of MD or the incidence of MD lymphomas. Simultaneous infection of chickens with a pathogenic strain of infectious bursal disease virus (IBDV) and the HPRS-B 14 strain of MD virus resulted in a consistently reduced incidence of MD and lengthening of the latent period, but the incidence of MD lymphomas was unaffected. Unmixed IBDV infection did not have any effect on the oncogenicity of the JMV non-producer MD tumour transplant or its attenuated variant JMV-A. In dually pre-infected chickens the JMV tumour development at the site of transplantation as stimulated by MD virus-induced immunosuppression was enhanced by IBDV infection. This phenomenon was attributed to an additive effect of MDV- and IBDV-induced immunosuppression.     

Differentiation between strains of Marek's disease virus and turkey herpesvirus by immunofluorescence assays

Two serological types of Marek's disease virus and a herpesvirus of turkeys have been differentiated by indirect immunofluorescence tests as (1) pathogenic strains of Marek's disease virus (MDV) and their attenuated variants: HPRS-16, HPRS-16/att, HPRS-B14, JM, JM/att, GA, VC and 'Oldenburg', a recent field isolate; (2) apathogenic strains HPRS-24 and HPRS-27 of MDV; (3) herpesvirus of turkeys strain FC126 and its HVT(A-) variant. Virus strains could not be distinguished on the basis of qualitative differences in immunofluorescent staining of intracellular virus-induced antigens. Results were similar whether chicken kidney, chicken embryo fibroblast or duck embryo fibroblast cell cultures were used. Fluorescence of virus-induced antigens was stronger with homologous than with heterologous antisera. Using the direct immunofluorescence technique Marek's disease virus and turkey herpesvirus infections could be distinguished. There were never any significant differences in the appearance and distribution of antigen in infected cells treated with homologous or heterologous antisera at dilutions of comparable activity using the indirect immunofluorescence technique. Antibody titres of antisera were 4 to 8-fold higher in the indirect immunofluorescence test against the homologous virus-induced antigens than against heterologous antigens. Cross-reactions between the 3 serological types could be prevented by absorption of antisera with the appropriate antigens. Cross-reactions could also be prevented by the appropriate dilution of antisera before use in the indirect immunofluorescence test.     

Antigenic characteristics of Marek's disease tumour cells

Surface antigens of Marek's disease lymphoblastoid cells were studied by indirect immunofluorescence (FA) tests and by cytotoxic antibody tests. The results of FA tests revealed genetic differences between cell lines of different sources with respect to their histocompatibility antigens. JMV and MSB-1 cells shared one of their alloantigens detectable by B blood group antisera. Differences between the cell lines were more pronounced when the respective hyperimmune sera were examined by cytotoxicity tests rather than by FA tests. The results of cross-absorptions of cell line antisera with cells of the different lines suggested that no identical common tumour-specific cell surface antigen was detectable serologically in addition to histocompatibility antigens or other normal cell surface antigens. The results of vaccination experiments with chicken embryo fibroblasts and with cells from various lymphoid tissues indicated that resistance against JMV lymphoblastic leukaemia could be induced by a number of different antigens which were not specific to Marek's disease.     

Relation between common antigen and membrane antigens associated with Marek's disease herpesvirus and turkey herpesvirus infections

Summary The appearance of two kinds of membrane antigen (MA) in Marek's disease herpesvirus (MDHV) or herpesvirus of turkey (HVT) infected cells was examined using antiserum to the common antigen (common-Ag). No early-appearing MA (EMA) was detected in these cultures until 16 hours post-inoculation, after which a number of cells in these cultures had late-appearing membrane antigen (LMA) as detected by immunofluorescence (IF). Fluids from cultures infected with these viruses were examined for the presence of the common-Ag by agar-gel precipitation (AGP) test. No common-Ag was detected until 16 hours, but after 24 hours, it was detected by AGP test.These results suggest that the common-Ag is related to LMA but not to EMA.With 2 Figures     

Comparative studies on Marek's disease virus and herpesvirus of turkey DNAs.

DNA of Marek's disease virus (MDV) was compared to that of herpes virus of turkey (HVT). Centrifugation of the two virus DNAs in neutral glycerol and CsCl density gradients showed that the MDV genome was slightly larger than that of HVT and that the buoyant density (1.705 g/ml) of MDV DNA in CsCl gradients was slightly lower than that (1.707 g/ml) of HVT DNA. MDV and HVT DNAs were digested with either EcoRI or HindIII restriction endonuclease and analysed by 0.5% agarose gel electrophoresis. The cleavage patterns of HindIII or EcoRI DNA digests of two strains of these two viruses showed general similarities between the strains, but not between MDV and HVT. However, a few fragments of EcoRI or HindIII digests of MDV DNA co-migrated with those of HVT DNA. DNA-DNA reassociation kinetics and DNA-RNA hybridization between the two viruses indicated that MDV and HVT DNAs share detectable homology, although it is less than 5%. The DNA of a HVT variant, which has lost the ability to protect chickens from Marek's disease, appeared similar to DNA of the vaccine strain in the size buoyant density and in its restriction endonuclease cleavage pattern.     

Efficacy of Marek's disease vaccines: protection and viraemia studies with turkey herpes virus vaccines

Two freeze dried turkey herpes virus vaccines A and C, of similar virus content were administered in graded doses to groups of day old chicks which were genetically susceptible to Marek's disease. Immediately after vaccination the chicks were challenged by contact with 4-weeks-old birds that had been infected at one day of age with virulent Marek's disease virus strain HPRS 16. Assessment of protection against Marek's disease was based on the absence of gross lesions after a 15 week observation period and a significant difference in the protective capacity of the 2 products was found. From the relationship between protection and vaccine dose that was obtained with vaccine C it was calculated that 0.48 of a field dose protected 50% of the birds. Vaccine A provided no significant protection at any dose level. Isolation of vaccinal virus 3 weeks after administration of graded doses of vaccines A and B revealed that a significantly higher dose of vaccine A was required to produce viraemia in vaccinated birds. Determination of viraemia at intervals following inoculation of one field dose of the vaccines demonstrated that Vaccine A also induced a less rapid response than the other 2 vaccines. These effects were not related to the virus content of the vaccines.     

Replication of Marek's disease virus in chicken feather tips containing vaccinal turkey herpesvirus DNA

The presence of herpesvirus of turkeys (HVT) DNA in the feather tips of chickens vaccinated with HVT was assessed by dot blot hybridisation with a probe specific for HVT and lacking homology to MDV DNA. Only small amounts of HVT DNA were detected in the feather tips of chickens that were vaccinated or left in contact with HVT vaccinated chickens. However when chickens were challenged with virulent MDV, HVT DNA was detected in the feather tips of vaccinated chickens and the largest amount was detected 35 days after vaccination. HVT DNA was recovered in significantly higher quantities from some of the MDV-infected chickens than from those infected by contact. This suggests that MDV infection may provide helper functions for HVT. MDV DNA was identified in the feather tips of MDV-challenged chickens from 25 to 45 days after challenge. Thus, immunisation of chickens with HVT did not prevent the replication of MDV in the feather tips but only diminished it.     

Multiplication of turkey herpes virus and Marek's disease virus in chick embryo skin cell cultures.

A simple method for the in vitro cultivation of chick embryo skin epithelial cells was developed and the replication of turkey herpes virus (HVT) and Marek's disease herpes virus (MDHV) in this system was studied. A high percentage of cells in monolayers inoculated with HVT (19·7 per cent) and MDHV (4·6 per cent) was infected on the fourth day post-incubation. Titres of infectious cells in the culture fluid or cell-free virus in the cell extract were low and cell-free virus was not detected in the culture medium. Enveloped particles of HVT, but not of MDHV, were detected by electron microscopy.     

Restriction enzyme map of herpesvirus of turkey DNA and its collinear relationship with Marek's disease virus DNA

The genome of herpesvirus of turkey (HVT) was shown to consist of long and short unique regions flanked by inverted repeats (J. Cebrian, Kaschka-Dietrich, C., Berthelot, N., and Sheldrick, P., 1982, Proc. Natl. Acad. Sci. USA 79, 555-558). In this paper we report the construction of the linkage map of HVT DNA for BamHI, HindIII, and PstI restriction endonucleases. The maps were constructed by hybridization of 19 cloned BamHI fragments of HVT DNA to electrophoretically separated digests of genomic DNA. Our results indicate that the terminal and internal inverted repeats (TRL and IRL) flanking the long unique sequences (UL) are spanned by BamHI-F fragment and a -F-related terminal fragment, respectively, whereas the terminal and internal inverted repeats (TRS and IRS) flanking the short unique sequences (US) are mostly contained in BamHI-A fragment. Both BamHI-A and -F showed a heterogeneity in size, suggesting the presence of amplification of certain sequences in the inverted repeats. We also report that the HVT genome is collinear with the genetically related Marek's disease virus (MDV) genome, as determined by hybridization of labeled cloned HVT DNA fragments with electrophoretically separated MDV DNA fragments.     

Further study on the three-dimensional structure of the core of Marek's disease virus and herpesvirus of turkey

Summary Three-dimensional structures of the core of Marek's disease virus and herpesvirus of turkey were examined by the tilting apparatus of an electron microscope. Various types of the core found in the infected cells were considered to represent developmental stages of the viruses. The basic structure of the core consisted of a toroid surrounding a cylindrical mass, which was clearly demonstrated by tilting the core in two directions. A cylindrical mass spooled by more than two toroids, which seemed to constitute a spiral band of 10 to 20 nm, was demonstrated. The maturation process of the cores of the viruses was also discussed.With 7 Figures     

Vaccination against Marek's disease: Immunizing effect of purified turkey herpes virus and cellular membranes from infected cells

One-day-old chickens susceptible to Marek's disease were vaccinated with experimental vaccines prepared from purified turkey herpes virus (HVT), inactivated HVT preparations or a membrane fraction isolated from HTV-infected chicken embryo fibroblasts, respectively. Purified HVT was found to be as effective in immunization against Marek's disease as cell-associated virus. The specific mortality of chickens twice vaccinated with cellular membranes from HVT-infected cells was reduced by 94%. These membranes also carried virus-specific antigens as shown by immunodiffusion tests. From the vaccination and serologic findings one may conclude that the immunologic prevention of Marek's disease by vaccination with the HVT is mediated by antibodies against viral envelope and virus-specific membrane antigens.     

Short- and long-term stability studies on four lyophilised and one cell-associated turkey herpesvirus vaccines against Marek's disease of chickens

One cell-associated and four lyophilised turkey herpesvirus (HVT) vaccines of different manufacturers were investigated comparatively in respect of the stability of their infectivity. All titrations were performed in primary chicken embryo fibroblast cell cultures and repeated seven times. The obtained data were computerised and statistically analysed. The long term stability study employed vaccine ampoules containing only lyophilised HVT. After appropriate storage in a refrigerator (4 degrees C) of all tested vaccine ampoules for 1 year no drop of titre was found. In contrast, storage at room temperature (24 degrees C) resulted in a loss of about 50% of the infectious units of HVT by 4 months. Marked differences between the products of different manufacturers were noted after 4 weeks of storage at 37 degrees or 45 degrees C. The infectivity dropped to values between 1/10 to 1/100 and 1/100 to 1/300, respectively. In the short term stability study cell-associated and lyophilised vaccine virus was resuspended in the manufacturer's diluent and maintained at temperatures of 4 degrees , 24 degrees , 37 degrees and 45 degrees C. The infectious units were titrated at 0, 2, 4, 6, 8 and 24 hours for estimation of the half life of the infectivity of HVT. The half life of infectivity of HVT at 4 degrees C was for three vaccines approximately 5 hours and for two vaccines between 1 and 3 hours. With increase of temperature the loss of infectivity increased resulting in half life times close to 1 hour at 45 degrees C.