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.     

Comparison of the sequence of the secretory glycoprotein A (gA) gene in Md5 and BC-1 strains of Marek's disease virus type 1

DNA fragments containing the secretory glycoprotein A (gA) gene of Marek's disease virus type 1 (MDV 1) were cloned from the DNA libraries of very virulent Md5 and virulent BC-1 strains and sequenced. Two open reading frames (ORF1 and ORF2) were identified for both strains. The ORF1 has the potential to code for a protein of 501 amino acids with a molecular weight of 56 kD that contains strong hydrophobic regions in both the amino and carboxyl termini, and nine potential N-linked glycosylation sites, while the ORF2 is capable of coding for a 24-kD protein. These results indicate that the ORF1 codes for the unprocessed form of gA. Between the Md5 and BC-1 strains, only two sequence mismatches exist in the DNA fragment. More differences appear to exist in the gA sequence of the MDV 1 GA strain (12), which lacks a strong hydrophobic anchor sequence. Similarities between the predicted amino acid sequences of the MDV 1 gA and the proteins of the other herpesviruses such as herpes simplex type I gC, pseudorabies virus gIII, and varicella zoster virus gpV were noted.     

Ultrastructure of the central nervous system in marek's disease and the effect of route of infection on lesion incidence in the central nervous system

The ultrastructure of the lesions of the central nervous system (CNS) of chickens was examined at intervals after intra-abdominal inoculation of Marek's disease virus (MDV). No transient paralysis occurred. Peri-vascular accumulations of lymphocytes and macrophages (cuffing) were accompanied by invasion of the CNS by these blood-borne leukocytes in the most severe lesions. Only minor damage to axons, myelin or glial cells was detected and no structural evidence for viral replication was observed. Intracerebral inoculation of cell-free MDV induced similar lesions in the CNS and there was no evidence for local exacerbation of their severity, although a few chickens developed transient paralysis. It is concluded that replication of MDV in the CNS is unlikely to be the direct cause of the CNS lesions.     

Comparative studies on pathogenical, virological and serological properties of Marek's disease virus isolated from Japanese quail and chicken

The QM-1 strain of Marek's disease virus (MDV) isolated from Japanese quail (JQ) induced higher mortality (96%) with MD lesions in quail than the JM strain of MDV isolated from chickens (C) (21%). In contrast both viruses induced MD in 90% of chickens. Pathological changes produced in quail inoculated with JQ-MDV or C-MDV were similar. However, the incidence of tumour lesions in the duodenum of quail inoculated with JQ-MDV was significantly higher. Neither virus induced tumour lesions in the duodenum of chickens. Appearance of viraemia in quail inoculated with MDV was delayed and its level was low as compared with chicken cases. MDV antigens in feather tips appeared in quail inoculated with JQ-MDV but not with C-MDV. No MDV was recovered from the MDV-inoculated quail by direct kidney cell cultures, although MDV was recovered by subpassage of the cultures into chicken embryo fibroblasts. No antibodies were detected in the affected quail by agar gel precipitation test.     

Sequential skin lesions in chickens experimentally infected with Marek's disease virus

Skin biopsies taken at weekly intervals from the same specific-pathogen free (SPF) chickens inoculated with Md/5 Marek's disease virus revealed two distinctive patterns of perifollicular cutaneous lesions, tumour-associated and non-tumour-associated. The tumour-associated pattern was subdivided into two types. The progressive type was manifested by a continuous increase of lymphoid cell aggregates (LCA) in the skin, resulting in the development of gross skin tumours with or without visceral tumours, and the regressive type showed initially increased and finally regressed LCA in the skin, associated with the development of visceral tumours. The non-tumour-associated pattern was characterized by initial transient small LCA in the skin without evidence of tumour formation. Birds with the tumour-associated pattern, regardless of type, had persistent nuclear inclusions (NI) and positive reactions against MDV1-specific phosphorylated polypeptides in the feather follicle epithelium (FFE) and initial R(1)-type (consisting mainly of small lymphocytes with a few lymphoblasts) to advanced T-type (consisting predominantly of lymphoblasts) feather pulp lesions (FPL). On the other hand, birds with the non-tumour-associated pattern formed transient NI and positive reactions against MDV1-specific phosphorylated polypeptides in the FFE and Ri-type to R(2)-type (consisting mainly of plasma cells with oedema) FPL. Antigen-positive lymphoid cells against MDV1-specific phosphorylated polypeptides were detected in both inflammatory and tumourous lesions, especially in the necrotic tumour lesions in the skin of birds showing the progressive type.     

Similarities and dissimilarities in the structure and expression of viral genomes of various virus strains immunologically related to Marek's disease virus

Various strains immunologically related to Marek's disease virus (MDV) have been subdivided into three serotypes: serotype 1, pathogenic strains of MDV and attenuated or apathogenic variants derived from them; serotype 2, naturally occurring apathogenic strains of MDV; serotype 3, herpesvirus of turkey (HVT). The viral genome structures of these three serotypes were compared by a simple, practical method using total DNA extracted from virus-infected cells instead of viral DNA purified from virions. The restriction endonuclease-cleavage patterns of serotype 2 viral DNA were found to differ from those of either serotype 1 MDV or serotype 3. Under stringent conditions, no significant DNA homology was detected among the three serotype viruses, except in a restricted portion of these viral genomes. Northern blot hybridization experiments suggested that virus-specific polyadenylated RNA of about 2.4 kilobases was transcribed from a restricted portion showing close homology in these viruses. Southern blot hybridization under less stringent conditions revealed that regions with weak homology were distributed over most of the viral genomes of the three serotypes. Two types of virus-specific glycoproteins, gA and gB, were identified in the immunoprecipitates of the culture medium and cell lysates, respectively, of serotype 2-infected cultures with monoclonal antibodies or hyperimmune antisera cross reactive with serotype 1 MDV and serotype 3 HVT, and detected on the surface of serotype 2-infected cells by the membrane immunofluorescence test. These results indicate a close evolutionary relationship among these three viral serotypes.     

Isolation of Marek's disease virus DNA from infected cells by electrophoresis on polyacrylamide gels

Summary Marek's disease virus DNA isolated from the nuclear fraction of infected chicken embryo fibroblasts and sucrose-purified particles was electrophoresed on 3 per cent polyacrylamide gels and was compared in its electrophoretical behaviour with isolated pseudorabies and herpes simplex DNA, strain HF. The DNA molecules eluted from the gel were identified by their sedimentation coefficient (53–55S) and buoyant density (1.707 g/ml) to be of viral origin. MDV DNA molecules were electrophoretically also detected and identified in DNA preparations of the lymphoblastoid Marek's disease tumour cell line MSB-1 which therefore has to be considered as a producer line. The electrophoresis of DNA preparations from Marek's disease virus-infected cells on polyacrylamide gels provides a semipreparative method for the isolation of MDV DNA.With 5 Figures     

Cross-protection between JMV Marek's disease-derived tumour transplant, Marek's disease and turkey herpesviruses

Cross-protection tests were conducted using attenuated JMV (JMV-A) Marek's disease-derived lymphoblasts, glutaraldehyde-treated JMV tumour cells, attenuated Marek's disease virus (MDV, strain HPRS-16/att) and turkey herpesvirus (HVT, strain FC126) as vaccines, and virulent JMV and MDV (HPRS-16) for challenge. The JMV and JMV-A preparations were free of MDV, leukosis and reticuloendotheliosis viruses. Vaccination of chickens with attenuated MDV or with HVT provided good protection against both JMV lymphoblastosis and Marek's disease (MD). In one experiment HVT (cell-free) caused a better resistance to JMV than to MD. Inoculation of JMV-A always resulted in a 100% resistance to virulent JMV. However, JMV-A did not induce any appreciable resistance to MD, even when the birds were challenged with MDV by contact exposure. Control experiments revealed that high doses of normal lymphocytes from uninfected chickens also had a protective effect against JMV. The 50% protective dose varied from 10(7) to 10(8) lymphocytes. JMV tumour cells inactivated by glutaraldehyde were used in different experiments but rarely caused a clear-cut protection against virulent JMV. The results of this study suggested that a one-way relationship exists in vivo between HVT or MDV and JMV lymphoblastic leukaemia. However, resistance induced against JMV tumour cells appeared to be related to histocompatibility antigens at least as much as to tumour-specific cell surface antigens. The results obtained failed to provide clear evidence for or against vaccinal resistance to MDV being dependent on the action of a common Marek's disease tumour-associated surface antigen (MATSA) additional to the immune response to viral antigens.     

Specific and nonspecific immune responses to Marek's disease virus

Marek's disease (MD) virus (MDV) has provided an important model to study immune responses against a lymphoma-inducing herpesvirus in its natural host. Infection in chickens starts with a lytic infection in B cells, followed by a latent infection in T cells and, in susceptible birds, T cell lymphomas develop. Non-specific and specific immune responses are important for the control of virus infection and subsequent tumor development. Interferon-gamma and nitric oxide are important for the control of virus replication during the lytic phase of infection and are also important to prevent reactivation of MDV replication in latently infected and transformed cells. Cytotoxic T cells (CTLs) are the most important of the specific immune responses in MDV. In addition to antigen-specific CTL against MDV proteins pp38, glycoprotein B (gB), Meq, and ICP4, ICP27-specific CTL can also be detected as early as 6 to 7 days post infection. The epitope for gB recognized by CTLs from P2a (MHC: B(19)B(19)) chickens has been localized to the Eco47III-BamHI (nucleotides 1515-1800) fragment. A proposed model for the interactions of cytokines and immune responses as part of the pathogenesis of MD is discussed.     

Novel criteria for the diagnosis of Marek's disease virus-induced lymphomas.

Several novel criteria have been tested to assist in the differential diagnosis of tumours induced by Marek's disease virus (MDV) from those induced by avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). A collection of tumours induced by inoculation of specific strains of MDV, ALV and REV, alone or in combination, were tested for quantification of MDV DNA by real-time polymerase chain reaction, expression of the MDV oncogene Meq, expression of several cell markers associated with transformation (CD30, Marek's disease-associated surface antigen, and p53), and level of DNA methylation in the tumour cells. In addition, tissues latently infected with MDV and non-infected tissues were tested as controls. Tumours induced by MDV had about 10(2)-fold more copies of MDV DNA than either tissues latently infected by MDV or tumours induced by retrovirus in MDV-vaccinated chickens. Moreover, the MDV antigen Meq was consistently expressed in all MDV tumours but it could not be detected in tissues latently infected with MDV or in tumours induced by retrovirus in MDV-vaccinated chickens. Other markers studied were not specific for MDV and therefore had limited value for diagnosis. Nonetheless, some of these markers might have potential value in research as they will help to identify transformed cells.     

Isolation of Marek's disease virus from Japanese quail with lymphoproliferative disease

Sixty-one Japanese quail from eight flocks with problems of recurring outbreaks of lymphoproliferative diseases resembling Marek's disease (MD), were examined aetiologically. Gross lymphomatous lesions were seen in 17 of the quail and 11 out of 56 quail had MD virus (MDV) feather tips antigens. MDV antibody was detectable in only one of 22 quail. None of 9 quail had antibodies against reticuloendotheliosis virus (REV). No MDV was isolated from the total 42 materials of quail using cell culture technique. No REV and avian leukosis virus (ALV) were isolated from some of them. However, specific-pathogen-free chicks inoculated with the blood materials revealed MD. and four MDVs were recovered from them. The isolates proved free from REV and ALV. The isolates were placed into serotype 1 by the indirect immunofluorescent antibody test. These results indicate that MDV is aetiologically involved in the present outbreaks of lymphoproliferative disease in Japanese quail.     

In ovo assay for Marek''s disease virus and turkey herpesvirus.

Marek's disease virus (MDV) and the turkey herpesvirus (HVT) may be assayed on the chorioallantoic membrane (CAM) of the chicken embryo after intravenous inoculation of chicken embryo fibroblasts (CEF) or chicken blood leukocytes infected with these viruses. Free HVT, MDV associated with Marek's tumor cells, and lymphoblastoid cell lines derived from Marek's tumors, may be assayed in the same way. The intravenous assay is quicker than the yolk sac assay and somewhat more sensitive than in vitro or conventional CAM assay after direct inoculation of the CAM. The optimal time for inoculation was day 10 of embryo incubation; therafter the log-10 CAM lesions decreased as a negative linear function of embryo age at the time of inoculation. The log-10 CAM lesions increased as a positive linear function of the time since inoculation. The optimal time for counts was day 5 after inoculation. The log-10 CAM lesions was a linear function of the log-10 cells in the inoculum; the slope was 1.0. Venous in ovo inoculation caused as increase in the weight of the spleen proportional to the number of CAM lesions. Repression of the splenomegaly, by prior X irradiation of the embryo, did not reduce the number of CAM lesions. Embryols from lines inbred for susceptibility to Marek's disease produced more CAM lesions than embryos from resistant lines. This difference did not depend on prior exposure of the mothers to MDV or HVT.     

Characterization of four very virulent Argentinian strains of Marek's disease virus and the influence of one of those isolates on synergism between Marek's disease vaccine viruses.

Isolates of Marek's disease virus (MDV) from vaccinated flocks in Argentina were characterized as very virulent (vv) and very virulent plus (vv+) strains. Experimental infection with these viruses caused a high incidence of Marek's disease in both resistant N-2a line and susceptible P-2a line birds. Vaccine viruses from each of the three Marek's disease viral serotypes were evaluated alone and in various combinations for protection against challenge with a vvMDV called NULP-1. Vaccination of P-2a birds with HVT did not protect satisfactorily against any of the vv and vv+MDV strains isolated. However, CVI988/Rispens vaccine alone or combined with serotype 2 and/or serotype 3 vaccine strains enhanced protection significantly against NULP-1. Serotype 2 plus serotype 3 vaccines also provided significant protection when challenged with this strain. This is one the first reports of the occurrence of vvMDV and vv+MDV in Argentina and Latin America. It is also a preliminary evaluation of the synergistic protective effect of different vaccine viruses with local MDV strains. However, further studies are needed to evaluate the real role of these and other Marek's disease isolates in 'vaccination failures' and the influence of serotype and virus strain on synergism between Marek's disease vaccine viruses.     

Replicating Marek's disease virus (MDV) serotype 2 DNA with inserted MDV serotype 1 DNA sequences in a Marek's disease lymphoblastoid cell line MSB1-41C

Summary We characterized the properties of herpes-type viruses which grew well in a Marek's disease lymphoblastoid cell line, MSB1-41C, inducing cytopathic effect characterized by the formation of syncytial giant cells. Examination of the infectious virus by field inversion gel electrophoresis revealed the presence of DNA of about 180 kbp in both the culture fluid and cell fractions of the infected MSB-41C cells. The DNA was found to consist of Marek's disease virus (MDV) serotype 2 (MDV2) and MDV serotype 1 (MDV1) DNA by Southern blot hybridization. The MDV1 DNA consisted of sequences mainly from the long inverted repeats including multiple copies of 132 bp direct tandem repeats. Molecular cloning of BamHI digests of the MDV2 DNA revealed a fragment of MDV1 DNA and MDV2 DNA fused together, indicating that the recombinant MDV2 DNA had been generated by genetic recombination with the latent MDV1 DNA.     

Further characterisation of the CVI 988 strain of Marek's disease virus

The CVI 988 strain of Marek's disease virus (MDV) has been studied for markers in vitro and in vivo. It was shown by indirect immunofluorescence assays that this strain belonged to the HPRS-16 serotype of MD viruses. The virus was positive for precipitating 'A' antigen. Medium plaques were produced in cell cultures. Therefore the CVI 988 strain could not be distinguished in vitro from other virus strains of the same serotype. The CVI 988 strain proved to be pathogenic for genetically susceptible Rhode Island Red chickens if inoculated at high doses equivalent to 10 times the field dose of the vaccine. The virus caused symptoms of classical Marek's disease in up to 28.5% of the inoculated chickens with gross lesions restricted to the peripheral nerves. Visceral tumours never occurred. The virus spread to uninoculated control chickens housed in the same pen. The CVI 988 strain can be classified as a mildly pathogenic classical Marek's disease virus of the HPRS-16 serotype.