R. L. Witter

Pathogenicity of variant Marek's disease virus isolants in vaccinated and unvaccinated chickens.

Mareks disease (MD) virus isolants Md/5 and Md/11, obtained from commercial broiler flocks vaccinated with turkey herpesvirus (HVT) but having excessive condemnation losses from MD, seemed similar to prototype MD viruses in oncogenicity for susceptible chickens, immunodepressive ability, and antigenicity. Compared with prototype MD viral strains JM/102W and GA/22, however, both field isolants were classed as biological variants on the basis of: 1) higher induction of acute cytolytic infection, characterized by atrophy of bursa and thymus and early death in the absence of lymphomas; 2) higher oncogenicity in genetically resistant chickens; and 3) higher oncogenicity in chickens immunized with HVT. A standard field dose (l103 plaque-forming units) of HVT vaccine protected 92-95% of chickens challenged with JM/ 102W but only 57-72% of chickens challenged with Md/5; the calculated dose of HVT required to protect 90% of chickens was at least 1000-fold as great for challenge with Md/5 as for challenge with JM/102W. The extent to which such variant MD viruses are responsible for MD vaccine failures in commercial flocks was not determined. These studies suggest that susceptibilities of chickens to acute cytolytic infection and MD lymphomas are mediated independently, and confirm that the severity of acute cytolytic infection in MD is influenced by virus strain.

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.

Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: Use of a pp38 mutant to validate the technology for the study of gene function

Mareks disease virus (MDV) genetics has lagged behind that of other herpesviruses because of the lack of tools for the introduction of site-specific mutations into the genome of highly cell-associated oncogenic strains. Overlapping cosmid clones have been successfully used for the introduction of mutations in other highly cell-associated herpesviruses. Here we describe the development of overlapping cosmid DNA clones from a very virulent oncogenic strain of MDV. Transfection of these cosmid clones into MDV-susceptible cells resulted in the generation of a recombinant MDV (rMd5) with biological properties similar to the parental strain. To demonstrate the applicability of this technology for elucidation of gene function of MDV, we have generated a mutant virus lacking an MDV unique phosphoprotein, pp38, which has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. Inoculation of Mareks disease-susceptible birds with the pp38 deletion mutant virus (rMd5Δpp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. This powerful technology will speed the characterization of MDV gene function, leading to a better understanding of the molecular mechanisms of MDV pathogenesis. In addition, because Mareks disease is a major oncogenic system, the knowledge obtained from these studies may shed light on the oncogenic mechanisms of other herpesviruses.

Polyvalent Marek's disease vaccines: Safety, efficacy and protective synergism in chickens with maternal antibodies 1

Three Mareks disease (MD) vaccines were evaluated for safety and protective efficacy in chickens with maternal antibodies against serotype 1, 2 and 3 MD viruses and in chickens with no maternal antibodies. The vaccines were: (1) Md11/75C, an attenuated serotype 1 MD virus, (2) a trivalent mixture of MD virus strains Md11/75C and SB-1, and turkey herpesvirus (HVT) strain FC 126, and (3) bivalent mixtures of these three viruses. These vaccines were compared with HVT or SB-1 vaccines in some trials. None of the vaccines was pathogenic or immunodepressive in susceptible chickens with or without maternal antibodies. No interference with in vivo HVT replication by additional viral components was noted, although some interference was demonstrated in vitro. Md11/75C replicated to limited titres in vivo and did not spread by contact, however, it acquired mild pathogenicity upon serial back-passage. Although Md11/75C provided good protection against highly virulent MD viruses in chickens without maternal antibodies, it was poorly protective in chickens with homologous maternal antibodies, and appeared more susceptible to in vivo neutralisation than did SB-1 or HVT. The trivalent vaccine, in contrast, was highly efficacious against very virulent MD virus challenge, even in chickens with maternal antibodies of all three serotypes, and was significantly more effective than monovalent vaccines. A bivalent vaccine composed of SB-1 and HVT was superior to the other two bivalent combinations of the three viruses. Protective synergism among all three vaccine viruses was confirmed. The efficacy of HVT was enhanced by as little as 80 PFU of SB-1, and even fractional doses of HVT and SB-1 together were superior to full doses of HVT alone.

Protection by attenuated and polyvalent vaccines against highly virulent strains of Marek's disease virus 1

Tests confirmed that turkey herpesvirus (HVT) vaccine protected chickens poorly against challenge with the highly virulent Md5 strain of Mareks disease (MD) virus, especially in chickens with homologous HVT antibodies. The naturally avirulent SB-1 vaccine virus was likewise poorly protective against challenge with the Md5 strain. Homologous antibodies reduced the protective efficacy of both vaccines, but SB-1 was not affected by HVT antibodies. In order to provide better protection against strains of MD virus poorly protected against by HVT, such as Md5, the Md11 strain of MD virus was attenuated by 75 cell culture passages and evaluated for protective efficacy. This vaccine virus, designated Mdl 1/75C, provided good protection against challenge with Md5 and most other highly virulent MD viruses tested, but was less efficacious against challenge with the JM/102W strain, a prototype MD virus protected against well by HVT and SB-1 vaccines. Furthermore, its efficacy was consistently lower in chicks with HVT antibody. Thus, although HVT, SB-1, and Md11/75C were all efficacious against certain MD viruses, none of these vaccines protected optimally against all MD challenge viruses in all chickens. A polyvalent vaccine composed of Md11/75C, HVT and SB-1 viruses protected chickens better against a battery of five highly virulent MD challenge viruses, including three strains poorly protected against by HVT, than any single vaccine and was not influenced by HVT antibody. These data suggest that vaccinal immunity may be partially viral strain specific.

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.

Differential effect of maternal antibodies on efficacy of cellular and cell-free Marek's disease vaccines.

The protective efficacy of cell-free and cell-associated turkey herpesvirus (HVT) vaccines against Mareks disease (MD) was determined by a quantitative protective dose 50% (PD(50)) assay in susceptible chicks from HVT-vaccinated, MD-exposed dams or from genetically comparable, isolation-reared, specific pathogen free dams. Comparison within trials showed that cell-associated and cell-free vaccines were equally efficacious in chicks lacking maternal antibodies (median PD(50)s were about 1-4 plaque-forming units (PFU) ). However, in chicks with maternal HVT/MD antibodies, PD(50)values were increased by 2- to 8-fold for cell-associated vaccine and by 15-to 80-fold for cell-free vaccine. The apparent requirement in antibody-positive chicks for greater doses of cell-free than of cell-associated vaccine to give 50% protection may be of practical importance in establishing the optimum number of PFU in a field dose.

Long-term studies of Marek's disease infection in individual chickens.

Parameters of infection with Mareks disease herpesvirus (MDV) were studied in a flock of naturally infected White Leghorn chickens during a 76-week period. Infection appeared to persist indefinitely in the host chicken although the virus titers were variable and frequently low. Transmission of infection by contact was demonstrated for some 76-week-old survivor birds and seemed related to the titer of viremia and presence of virus in the integument. Titers of Mareks disease (MD) precipitins peaked prior to the 28th week, and in a few cases eventually disappeared; however, antibody detected by the indirect fluorescent antibody test remained undiminished. The persistence of precipitins appeared related to persistence of viral antigen. Birds with high virus titers died more frequently of MD than other birds, but virus pathogenicity could not be implicated as a determinant of survival. Duration of survival was related directly, although not causally, to antibody titer. Response differences among sire families were noted, but progeny tests showed that absence of detectable viremia was unreliable as a marker for genetic resistance.

Retroviral insertional mutagenesis of a herpesvirus: A marek's disease virus mutant attenuated for oncogenicity but not for immunosuppression or in vivo replication

Our earlier studies have shown that retrovirus insertion into herpesvirus is an efficient process that engenders recombinant herpesviruses with altered biological properties. The RM1 clone is derived from the JM strain of Mareks disease virus (MDV) through retrovirus insertional mutagenesis and contains sequences of reticuloendotheliosis virus inserted at the junction of the internal repeat and unique short regions of the genome. In previous studies, the RM1 clone appeared attenuated for oncogenicity but caused marked atrophy of the thymic lobes. The present studies represent a detailed analysis of the biological characteristics of the RM1 clone in order to better understand mechanisms of oncogenicity and gene function of MDV. RM1 was almost fully attenuated for oncogenicity but retained other in vivo properties of virulent viruses such as thymic and bursal atrophy, early immunosuppression, early cytolytic infection followed by efficient replication, and contact spread--all normally absent in attenuated strains. This suggests that, for serotype 1 MDV, oncogenicity is not tightly linked with immunodepression or viral replication and that these properties may be controlled by different genes or mechanisms. The mutation was stable through serial passage of the virus in chickens as determined by molecular analysis. None of the mutant viruses demonstrated expansion of the 132-bp repeat region of the genome, indicating that such expansion is not required for attenuation. Chickens vaccinated with RM1 clones were protected against challenge with virulent MDV, and levels of protection exceeded those of other attenuated serotype 1 vaccine viruses. Thus, attenuation by selective mutation may be an advantageous strategy for development of serotype 1 Mareks disease vaccines.