Effect of native chicken interferon on MDV replication

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus. Its specific phosphorylated protein, pp38 has been implicated in MDV oncogenesis. In order to check whether the known anti-viral or anti-proliferative actions of interferon (IFN) are of importance in Marek's disease (MD), chicken embryo fibroblasts (CEFs) were infected with attenuated serotype-1 MDV strain CVI988, or with herpesvirus of turkeys (HVT). Different concentrations of native chicken IFN were added to the cell cultures, prior to their infection. After incubation, MDV plaques were counted. Analysis by flow cytometry for pp38 expression was performed by using three monoclonal antibodies (MAbs) and for HVT by using an anti-glycoprotein B (gB) MAb. Increasing IFN quantities caused a reduction in a stepwise manner of plaque numbers as well as a suppression of pp38 and gB expression in the CVI988- and HVT-infected cells, respectively.     

Molecular-biological characterization of Marek's disease virus. II. Differentiation of various MDV and HVT strains

Immunological cross-reactions were found between each of the four major virus-specific polypeptides of Marek's disease virus (MDV) strains CVI 988, K and HPRS-16/att, and herpesvirus of turkey (HVT) Fc126 by one-dimensional (1D) gel analysis of immunoprecipitates from the lysate or culture medium of infected cells. The results, however, did not allow a serotype classification of MDV and HVT strains. Comparison of the two-dimensional (2D) gel patterns of virus-specific polypeptides of nine MDV and HVT strains with different biological properties revealed many similarities. Strain CVI 988 provided the reference pattern, consisting of 35 polypeptides, 18 of which were glycosylated. Based on their similarities in migration characteristics (size, charge, and heterogeneity of spots) during 2D gel electrophoresis, 11 virus-specific polypeptides or polypeptide complexes were identified in the patterns of nearly all virus strains analyzed. One of these polypeptides was glycoprotein gp5, the putative A antigen of MDV and HVT, which was also detected in the medium of cells infected with HPRS-16/att, a strain which was reported to have lost its capacity for production of A antigen. In addition to the similarities mentioned above, differences were found in migration behavior of virus-specific polypeptides (complexes) p4/p5/p6, gp3, gp5, and gp8/gp9, which were confirmed by coelectrophoresis experiments. The most conspicuous difference was found between three patterns of gp5 which seem to be characteristic for three groups of viruses: (I) high- and low-virulent MDV strains and their attenuated variants; (II) avirulent and apparently nononcogenic MDV strains; (III) herpesviruses of turkey. The minor differences found for the other polypeptides mentioned above further substantiated this molecular-biological classification of MDV and HVT strains and, in addition, enabled the differentiation of two HVT strains within molecular-biological group III.     

Pathogenicity and Antigenicity of Clones from Strains of Marek's Disease Virus and the Herpesvirus of Turkeys

Virus was extracted by filtration from chicken embryo fibroblast cultures infected with the JM, high passage JM(JMHP), GA, and RPL39 strains of Marek's disease virus (MDV) and from the herpesvirus of turkeys (HVT) and purified by cloning. The plaques produced by clones of HVT, JMHP, and other MDV strains differed in morphology from one another. Clones of MDV varied greatly in pathogenicity for chickens, but JMHP and HVT were nonpathogenic. Two pathogenic clones of JM virus and a clone of JMHP virus lacked the A precipitin antigen present in all other clones tested. All clones had at least one B antigen in common. HVT and MDV clones with and without the A precipitin antigen could be distinguished from each other by the indirect fluorescent antibody test. Changes in virus-host cell relationships, loss of pathogenicity, and loss of the A antigens were independent events.     

Serological relationship between a pathogenic strain of Marek's disease virus, its attenuated derivative and herpes virus of turkeys.

Precipitating antigens present in extracts of chick embryo cells infected with the HPRS-16 attenuated strain of Marek's disease virus (att-MDV) were separated by gel filtration on Sephadex G200 and some of their properties determined. The two main antigens detected with convalescent MD serum, referred to as 'B' and 'C' antigens, had mobilities of 0-55 and 0-25 respectively relative to phenol red on electrophoresis in 7-5% acrylamide gel. The B antigen was relatively stable and of low mol. wt. in comparison with the C antigen. B and C antigens were in some instances also detected in culture medium of infected cells, but were distinguishable from the A antigen, a major glycoprotein antigen released into the culture medium of cells infected with HPRS-16. The results of immunodiffusion studies suggest that B antigen is common to MDV and strains of herpes virus of turkeys(HVT) and that at least 2 antigens (including C) are MDV specific. The A antigen was also common to MDV and HVT strains. It was noted however that the capacity of HPRS-16/att to synthesize A antigen was considerably reduced in comparison with HPRS-16 and HVT strains, and in some preparations the A antigen could not be detected. Evidence was also obtained for the presence of HVT-specific antigens associated mainly with the cell fraction.     

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.     

No homology detectable between Marek's disease virus (MDV) DNA and herpesvirus of the Turkey (HVT) DNA

The relation of four different strains of MDV and two strains of HVT was analyzed by gel electrophoresis of viral DNA digested by various restriction endonucleases and by filter hybridization of viral DNA with complementary RNA.The four MDV strains showed fragment patterns completely different from those of HVT upon digestion of the viral DNA with Bam H I, Eco R I, Hind III, Hpa I, and Xho and separation of fragments on agarose gels.The cleavage patterns of the four MDV strains showed great similarities among each other as well as some differences between the individual strains. In the cleavage patterns of HVT a similar close relationship was observed between the two HVT strains with slight divergence between both.Filter hybridizations of viral DNA with labelled complementary RNA prepared from the DNA of the GA strain of MDV or from the DNA of the PH-THV1 strain of HVT revealed no cross-hybridization between the MDV and the HVT strains.cRNA prepared from the DNA of an MDV strain hybridized only to restriction enzyme fragments of the MDV strains transferred to nitrocellulose filters, but not to fragments of HVT DNA, and vice versa.     

A quantitative sequential study of viraemia and neutralising antibody to HVT and MDV in a commercial flock vaccinated with HVT

Levels of viraemia due to the herpesvirus of turkeys (HVT) and neutralising antibodies to HVT and Marek's disease virus (MDV) were followed in a flock of commercial broiler breeders held in commercial premises and vaccinated at 1-day-old with HVT. The flock was sampled at 3, 5 and 8 weeks of age and then at 4-weekly intervals until 32 weeks of age. The mean viraemia titre was highest at 3 and 5 weeks and then fell to a low level by 16 weeks where it remained until 32 weeks of age. At all sampling periods there was great variability in individual viraemia titres and a significant proportion with no detectable viraemia. Neutralising antibody titres to both HVT and MDV were highest at 1 day old. The geometric median titres fell rapidly and only started to rise slowly between 8 and 12 weeks of age. The titre of individuals within a sample varied greatly and from 16 weeks of age between 3% and 43% had no neutralising antibody to MDV. An examination of the factors affecting the accuracy of the assay for HVT viraemia indicated that the bird effect was greatest suggesting the variability in viraemia was not due to errors in the assay. Results similar to the field study were obtained when the same strain of chicken was vaccinated under laboratory conditions indicating that faulty vaccination was not responsible for the variable response to vaccination. There was a positive correlation between HVT viraemia and neutralising antibody to HVT and MDV. Groups of 1-day-old progeny of HVT vaccinated parents and grandparents of three laying strains and one broiler strain derived from a number of farms were examined for levels of neutralising antibody to HVT. Both the geometric mean titres of each group and the titres of individuals within a group varied greatly. The strain of chicken had no effect on the neutralising antibody response to vaccination with HVT and probable field exposure to MDV but the farm of origin affected both the geometric mean titre and the variability in titre of neutralising antibody in individuals. It is suggested that the exceptionally high levels of neutralising antibody to HVT noted in a proportion of the 1-day-old chicks of the field study could be responsible for a poor response of some individuals to vaccination with HVT.     

Efficacy of a combination vaccine containing MDV CVI 988 strain and HVT against challenge with very virulent MDV

With the emergence of very virulent Marek's disease virus (MDV) strains, vaccines based on herpesvirus of turkeys (HVT) appear to be not powerful enough to confer full protection, whereas in chicken flocks vaccinated with MDV CVI 988 strain protective immunity sometimes is generated not early enough for full protection. For this reason combination vaccines containing HVT as well as CVI 988 have been developed. In this paper the beneficial effect of combining both types of virus strains in one vaccine for early protection is shown in a vaccination challenge experiment, in which one-day-old chickens were vaccinated with suboptimal dosages of the monovalent vaccines and the same dosages in a combination vaccine. After 5 days the chickens were challenged with a very virulent MDV strain and subsequently observed for a period of approx. 50 days. It appeared that the combination vaccine provided better early protection than the monovalent vaccines. In addition, the combination vaccine was tested as vaccine administered in ovo. It appeared that after in ovo vaccination the vaccine conferred adequate protection against challenge with a very virulent MDV strain, 5 days after hatch, and that protection after in ovo vaccination was similar to that obtained after subcutaneous vaccination with the same combination vaccine.     

Kinetics of Marek's disease virus (MDV) infection in broiler chickens 1: effect of varying vaccination to challenge interval on vaccinal protection and load of MDV and herpesvirus of turkey in the spleen and feather dander over time

Two experiments in commercial broiler chickens vaccinated with herpesvirus of turkeys (HVT) and challenged with Marek's disease virus (MDV) investigated the effects of the vaccination-to-challenge interval (VCI) on vaccinal protection against Marek's disease, and the kinetics of MDV and HVT load in the spleen and feather dander determined using real-time quantitative polymerase chain reaction. Experiment 1 in isolators tested VCI of 2, 4 and 7 days, while Experiment 2 in floor pens tested VCI of 0, 2, 4, 7 and 10 days. MDV challenge induced gross Marek's disease lesions in 14% to 74% of chickens by 56 days post-challenge. Vaccinal protection increased from ～40% to ～80% with increasing VCI between days 2 and 7 in both experiments, but not thereafter. MDV was detected in both the spleen and dander at 7 days post-challenge and increased rapidly to approximately 21 days post-challenge, after which levels plateaued, rose or fell gradually depending on treatment. HVT was also shed in significant amounts, 1 to 2 logs lower than for MDV, with a clear peak around 14 to 21 days post-vaccination. Vaccination significantly reduced the log₁₀MDV load in the spleen (vaccinated, 2.99±0.20/10⁶ spleen cells; unvaccinated, 4.60±0.23/10⁶ spleen cells) and dander (vaccinated, 5.28±0.13/mg; unvaccinated, 6.00±0.18/mg) from infected chickens. The MDV load had a significant negative association with the VCI and the level of vaccinal protection. Measurement of dander production in Experiment 1 and the dust content of air in Experiment 2, combined with determination of the MDV load in these, enabled estimation of total daily shedding rates of MDV per chicken and of the MDV load in air for the first time.     

A comparison of the biological properties of type 2 plaque-producing agent derived from the Cal-1 strain of Marek's disease virus with other related viruses

Summary The serological and biological properties of the type 2 plaque-producing agent (PPA) derived from the Cal-1 strain of Marek's disease virus (MDV) were compared with those of reference strains of the three serotypes of MDV and herpesvirus of turkeys (HVT) groups; namely JM, HPRS-24 strains of MDV and the FC-126 strain of HVT for serotype 1, 2, and 3, respectively. By agar gel precipitation (AGP), indirect fluorescent antibody and virus neutralization tests, type 2 PPA was related but not identical to the FC-126 strain. By the AGP test, type 2 PPA showed a poor ability to synthesize B antigen and the A antigen was different from that of strain FC-126. To compare the virological characteristics of type 2 PPA with the reference strains, the release of cell-free virus into supernatants of infected cell cultures and titers of cell-free virus extracted sonically from infected cell cultures were examined. Cell-free type 2 PPA virus was easily detected in the supernatants and extracted from infected cell cultures. These properties were similar to reference strains of serotype 2 and 3. Next, the structural similarities of viral DNAs of type 2 PPA and strain FC-126 were examined by Southern blot hybridization. The restriction endonuclease-cleavage patterns of DNA of type 2 PPA were very similar but not identical to those of the FC-126 strain. In chickens inoculated with type 2 PPA, splenic lymphocytes supported a non-productive latent infection as did also those from chickens inoculated with the FC-126 or HPRS-24 strains. From these results, type 2 PPA appears to belong to serotype 3 of MDV and HVT groups. The origin of type 2 PPA is discussed.With 3 Figures     

Immunosuppressive effects of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) in broiler chickens and the protective effect of HVT vaccination against MDV challenge

Much of the impact of Marek's disease in broiler chickens is considered to be due to immunosuppression induced by Marek's disease virus (MDV). The present study evaluates the effects of an Australian isolate of pathogenic MDV (strain MPF 57) and a non-pathogenic vaccinal strain of herpesvirus of turkeys (HVT) (strain FC 126) on the immune system of commercial broiler chickens for 35 days following challenge at days 0 or 3 of age. It also investigates the extent of protection provided by HVT vaccine against MDV-induced immunosuppression. Immune system variables, including relative lymphoid organ weight, blood lymphocyte phenotype (CD45+/CD3+, putatively T, and CD45+/LC+, putatively B) and antibody production following vaccination against infectious bronchitis (IB) at hatch, were used to assess the immune status of chickens. Immunosuppression was also assessed by susceptibility to secondary challenge with pathogenic Escherichia coli on day 29 post-MDV challenge. MDV infection reduced the weight of the thymus and bursa of Fabricius, the numbers of circulating T lymphocytes and B lymphocytes, and IB antibody titre. The timing of these effects varied. MDV infection greatly increased susceptibility to E. coli infection. HVT alone caused mild depletion of T and B lymphocytes but no effect on immune organ weight or IB titre. Vaccination with HVT provided good protection against most of the immunosuppressive effects of MDV but not against MDV-induced growth impairment and reduced responsiveness to IB vaccination, suggesting that recent Australian strains of MDV may be evolving in virulence to overcome the protective effects of HVT.     

The expression of Marek's disease tumour-associated surface antigen in various avian species

Marek's disease tumour-associated surface antigen (MATSA) was detected on lymphoid cells from chickens infected with Marek's disease virus (MDV) or the herpesvirus of turkeys (HVT), from a chicken-quail hybrid infected with MDV, and from turkeys infected with the GA strain of MDV, but not on cells from turkeys infected with HVT or the HPRS-16 strain of MDV or from MDV-infected Japanese quails or bobwhite quails, despite the presence of lymphomas in some Japanese quails. Two complementary explanations are offered for these observations/Firstly, certain combinations of virus strain and host species may not result in malignant transformation although infection has occurred; secondly, if, as seems likely, MATSA is a modified host antigen, antigens on malignant cells arising in a foreign host may not be recognised by an antiserum prepared against MATSA of chicken origin.     

Development of a cell line system susceptible to infection with vaccine strains of MDV

Despite reliance on the need to continually prepare fresh cultures of chick embryo fibroblasts (CEFs) to make Marek's disease (MD) vaccines, MD vaccines are the most widely used vaccines in the poultry industry. Preparation of CEF's accounts for approximately 40% of the costs associated with producing MD vaccines. A significant reduction in MD vaccine production costs could be realized if a continuous cell lines were available for MD vaccine production. Recently, we reported development and characterization of a cell line system (OCL) that supports growth and replication of oncogenic serotype 1 Marek's disease virus (MDV). Here we report development of three cell line systems for production of MD vaccine. These cell lines support the growth and replication of attenuated serotype 1 MDV (CVI-OCL), serotype 2 MDV (SB1-OCL) and serotype 3 MDV (HVT-OCL). MDV is maintained in a stable state in the OCL cells and the infected cells can be continuously grown. The vaccines made from these cell lines are safe and protect White Leghorn chickens against challenge with very virulent serotype 1 MDV, similar to traditional vaccines made from CEF cells. These cell line systems can significantly reduce the costs associated with MD vaccine production. Furthermore, the increased stability of MDV and the potential for positive selection of recombinant MDV suggest that OCL will be ideal for production of more effective MDV vaccines using recombinant DNA technology.     

Quantitative pathogenicity of Marek's disease virus and vaccine virus strains in chickens: macroscopical aspects

Two virulent strains (JM and K) and one vaccine strain (CVI 988) of Marek's disease virus (MDV), together with two vaccine strains of the herpesvirus of turkeys (HVT) (FC 126 and PB-THV 1), all in the cell-associated state, were administered intramuscularly at 3.7 log TCID50 per dose to day-old SPF White Leghorn chickens. A control group of chicks received uninfected cells. The pathological parameters studied were onset and duration of clinical symptoms, mortality, bird weight and macroscopical lesions of peripheral nerves and visceral organs. Data were obtained from females autopsied at the age of 3, 8 and 20 weeks, and from chickens which died. Virological and serological data were procured mainly from males taken at various ages. The results indicate a clear distinction between virulent and vaccine strains. MD vaccines had no significant influence on bird weight and caused no mortality or macroscopical lesions, whereas the virulent MDV strains produced all these effects. Macroscopical lesions caused by the virulent MDV strains were seen predominantly in nerves (in about 50% of birds succumbing to MD) and gonads (in 0% to 80% of such birds depending on sex and on strain of MDV). Differences between the two virulent strains could be demonstrated. Strain JM induced earlier incidence and shorter duration of clinical disease. With strain JM death occurred earlier in females than in males. Strain K caused significantly more macroscopical lesions in gonads, heart and liver. Under the conditions of the experiment, detection of macroscopical lesions after inoculation with a virulent MDV strain was possible 3 weeks after inoculation.     

Complete nucleotide sequence of the Marek's disease virus ICP4 gene.

The Marek's disease virus (MDV) gene encoding a homologue to the ICP4 protein of herpes simplex virus has been mapped to BamHl fragment A based on the physical map of the MDV genome (Fukuchi et al., 1984). The gene lies completely within the inverted repeat flanking the unique short region of the genome. The complete nucleotide sequence of the MDV ICP4 gene has been determined. The coding region is 4245 nucleotides long and has an overall G+C content of 52%. The MDV ICP4 protein is predicted to have a structure similar to that of ICP4-like proteins of other herpesviruses in that it has five distinct regions, the second and fourth of which are highly conserved. In addition, the protein contains the characteristic run of serine residues located toward its amino terminus. The MDV ICP4 gene is expressed in MDV-infected chicken embryo fibroblasts.     

Molecular-biological characterization of Marek's disease virus. I. Identification of virus-specific polypeptides in infected cells

Cells infected with Marek's disease virus (MDV) were labeled with [⁴⁵S]methionine or [³H]glucosamine and virus-specific polypeptides were analyzed after immunoprecipitation from the cell lysate or culture medium by one- or two-dimensional (1D and 2D, respectively) gel electrophoresis followed by autoradiography. Four major and several minor virus-specific polypeptides were present in each of the 1D gel patterns of MDV strains CVI 988, K, and HPRS-16/att and of herpesvirus of turkey (HVT) Fc126. The molecular weights of the major virus-specific polypeptides of these strains were identical. High-resolution 2D gel electrophoresis of immunoprecipitates from lysates of cells infected with strain CVI 988 revealed 35 virus-specific polypeptides, 18 of which were glycosylated. A tentative nomenclature for these virus-specific polypeptides is suggested. One of the glycoproteins, gp5, was secreted rapidly and in large amount in the culture medium and is most probably identical to the A antigen of MDV. Although a pulse-chase experiment showed that several virus-specific polypeptides could not be detected in their mature form after 15 min pulse labeling, distinct precursor-product relations among the virusspecific polypeptides were not detected. It was shown that the migration behavior of the virus-specific polypeptides during 2D gel electrophoresis did not depend on the stage of infection or the host cell used. The latter observation is essential, as it allows comparing 2D-polypeptide patterns of various MDV and HVT strains with different multiplication properties in cell culture.     

Molecular characteristics of very virulent European MDV isolates

Marek's disease virus (MDV) strains with increasing virulence have been reported from many parts of the world. Many of these recent MDV isolates produce an acute early cytolytic disease with high mortality and severe atrophy of the lymphoid organs, thymus and the bursa of Fabricius. Although the degree of the atrophic changes and the virulence of the virus are correlated, the molecular basis of the increased virulence is not known. We examined the characteristics of the disease induced by 3 such MDV isolates, C12/130, MR36 and MR48, isolated from Europe. All the three viruses produce high early mortality and atrophy of the lymphoid organs. As a first step in understanding the determinants of the increased virulence of these isolates, we have compared the sequences of MEQ and the ICP4 genes of these three viruses with that of the published sequences. Some of the amino acid changes seen within the Meq and ICP4 proteins were conserved in all the three isolates and could account for the increased virulence characteristics.