Insertion of reticuloendotheliosis virus long terminal repeat into the genome of CVI988 strain of Marek's disease virus results in enhanced growth and protection.

Marek's disease (MD) is a lymphoproliferative disease of chickens caused by serotype 1 MD virus (MDV). Vaccination of commercial poultry has drastically reduced losses from MD, and the poultry industry cannot be sustained without the use of vaccines. Retrovirus insertion into herpesvirus genomes is an efficient process that alters the biological properties of herpesviruses. RM1, a virus derived from the virulent JM strain of MDV, by insertion of the reticuloendotheliosis (REV) long terminal repeat (LTR), was attenuated for oncogenicity but retains properties of the parental virus, such as lymphoid organ atrophy. Here we show that insertion of the REV LTR into the genome of vaccine strain CVI988 resulted in a virus (CVRM) that replicated to higher levels than parental CVI988 in cell culture and that remained apathogenic for chickens. In addition, CVRM showed protection indices similar or superior to those afforded by CVI988 virus in laboratory and field protection trials, indicating that it could be developed as a safe and efficacious vaccine to protect against very virulent plus MDV.

Optimization of the protocols for double vaccination against Marek's disease by using commercially available vaccines: evaluation of protection, vaccine replication, and activation of T cells.

Revaccination against Marek's disease is a widespread practice in some countries. The rationale of this practice is unknown, and there is no consensus in the protocols. Recently, we have demonstrated that administration of the first vaccine at 18 days of embryonation followed by a more protective second vaccine at hatch (18ED/1d) reproduced systematically the benefits of revaccination under laboratory conditions. Here, we have used the same model to optimize the revaccination protocols by using currently available vaccines and to determine whether two features associated with Marek's disease vaccine-induced protection (activation of T cells and replication of vaccine virus) are involved in the revaccination protocols. Protection conferred by three revaccination protocols (turkey herpesvirus [HVT] 18ED/HVT+SB-1 1d, HVT 18ED/CVI988 1d, and HVT+SB-1 18ED/ CVI988 1d) was evaluated. Revaccination protocols also were compared with single vaccination protocols (HVT 18ED, HVT+SB-1 18ED, HVT+SB-1 1d, CVI988 18ED, and CVI988 1d). Our results demonstrated that it is possible to improve efficacy of the currently available vaccines by using them in revaccination programs. Administration of HVT 18ED/CVI988 1d and HVT+SB-1 18ED/CVI988 1d were the two protocols that conferred the highest protection against a very early challenge (2 days of age) with very virulent plus Marek's disease virus strain 648A. In a separate experiment, we evaluated vaccine replication and activation of T cells in single and revaccination protocols. Our results demonstrated that replication of the second vaccine, although decreased compared with single vaccination, could be detected at 3 days (HVT, CVI988) or at 6 days (SB-1). Administration of the first vaccine (HVT) at 18ED resulted in a high percentage of activated T cells. Administration of a second vaccine (either HVT-SB-1 or CVI988) at 1d resulted in increased intensity of MHC-II stain in activated T cells.

Standardization of a model to study revaccination against Marek's disease under laboratory conditions.

Revaccination, the practice of administering Marek's disease (MD) vaccine a second time, has been used in commercial poultry flocks for many years. The rationale is largely anecdotal as the few published reports have failed to provide support for the value of the practice. In the present work, we have standardized a model to study MD revaccination under laboratory conditions. Nine bird experiments were conducted to evaluate homologous revaccination (same vaccine administered twice) and heterologous revaccination (administration of two different vaccines) with various challenge models. Our results demonstrated that heterologous revaccination (with a second vaccine more protective than the first vaccine) but not homologous revaccination provided a beneficial increase in protection. Administration of the first vaccine at 18 days of embryonation followed by a more protective second vaccine at hatch reproduced systematically the benefits of revaccination. In addition, our results show that revaccination protocols might aid in solving major drawbacks associated with various highly protective experimental MD vaccines; that is, lymphoid organ atrophy and residual virulence. Strain RM1 is one of the most protective vaccines against early challenge with highly virulent MD virus but it induces severe lymphoid atrophy in chickens lacking maternal antibodies against MD virus. In this study, strain RM1 did not induce lymphoid organ atrophy when administered as second vaccine in a revaccination protocol. Similarly, strain 648A100/BP5 maintains residual virulence in chickens lacking maternal antibodies against MD virus but did not induce any lesions when used as a second vaccine. Until now, arbitrary revaccination protocols have been occasionally proven useful to the poultry industry. The model developed in this study will allow for a better understanding of this phenomenon and its optimization. A more rational use of this practice will be of great help to control MD outbreaks until better vaccines are available.

Dynamic equilibrium of Marek's disease genomes during in vitro serial passage.

Attenuation of Gallid herpesvirus-2 (GaHV-2), the causative agent of Marek's disease, can occur through serial passage of a virulent field isolate in avian embryo fibroblasts. In order to gain a better understanding of the genes involved in attenuation and associate observed changes in phenotype with specific genetic variations, the genomic DNA sequence of a single GaHV-2 virulent strain (648A) was determined at defined passage intervals. Biological characterization of these "interval-isolates" in chickens previously indicated that the ability to induce transient paralysis was lost by passages 40 and the ability to induce persistent neurological disease was lost after passage 80, coincident with the loss of neoplastic lesion formation. Deep sequencing of the interval-isolates allowed for a detailed cataloguing of the mutations that exist within a single passage population and the frequency with which a given mutation occurs across passages. Gross genetic alterations were identified in both novel and well-characterized genes and cis-acting regions involved in replication and cleavage/packaging. Deletions in genes encoding the virulence factors vLipase, vIL8, and RLORF4, as well as a deletion in the promoter of ICP4, appeared between passages 61 and 101. Three mutations in the virus-encoded telomerase which predominated in late passages were also identified. Overall, the frequency of mutations fluctuated greatly during serial passage and few genetic changes were absolute. This indicates that serial passage of GaHV-2 results in the generation of a collection of genomes with limited sequence heterogeneity.

Competition between two virulent Marek's disease virus strains in vivo.

Previous studies have demonstrated the presence of multiple strains of Marek's disease virus simultaneously circulating within poultry flocks, leading to the assumption that individual birds are repeatedly exposed to a variety of virus strains in their lifetime. Virus competition within individual birds may be an important factor that influences the outcome of co-infection under field conditions, including the potential outcome of emergence or evolution of more virulent strains. A series of experiments was designed to evaluate virus competition within chickens following simultaneous challenge with two virulent serotype 1 Marek's disease virus strains, using either pathogenically similar (rMd5 and rMd5/pp38CVI) or dissimilar (JM/102W and rMd5/pp38CVI) virus pairs. Bursa of Fabricius, feather follicle epithelium, spleen, and tumour samples were collected at multiple time points to determine the frequency and distribution of each virus present using pyrosequencing, immunohistochemistry and virus isolation. In the similar pair, rMd5 appeared to have a competitive advantage over rMd5/pp38CVI, which in turn had a competitive advantage over the less virulent JM/102W in the dissimilar virus pair. Dominance of one strain over the other was not absolute for either virus pair, as the subordinate virus was rarely eliminated. Interestingly, competition between two viruses with either pair rarely ended in a draw. Further work is needed to identify factors that influence virus-specific dominance to better understand what characteristics favour emergence of one strain in chicken populations at the expense of other strains.

Replication ability of three highly protective Marek's disease vaccines: implications in lymphoid organ atrophy and protection.

The present work is a chronological study of the pathogenesis of three attenuated serotype 1 Marek's disease (MD) virus strains (RM1, CVI988 and 648A80) that provide high protection against MD but have been attenuated by different procedures and induce different degrees of lymphoid organ atrophy. All studied strains replicated in the lymphoid organs (bursa,x thymus and spleen) and a peak of replication was detected at 6 days post inoculation (d.p.i.). Differences, however, were observed among vaccine strains. RM1 strain replicates more in all lymphoid organs compared with CVI988 and 648A80 strains. In addition, replication of RM1 in the thymus did not decrease after 6 d.p.i. but continued at high levels at 14 d.p.i. and until the thymus was completely destroyed. Lung infection occurred very early after infection with all of the three vaccines and the level of replication was similar to that found in the lymphoid organs. Infected cells were very large and appeared scattered in the lung parenchyma and in the parabronchial lining. The study of the target cells for the early infection in cell suspensions of blood and spleen showed that both non-adherent cell populations (enriched in lymphoid cells) and adherent cells (enriched in monocytes/macrophages) supported MD virus infection. Infection in adherent cells was especially high at very early stages of the infection (3 to 6 d.p.i.). Atrophy of lymphoid organs is a major drawback in the production of highly protective vaccines against MD. A better understanding of the mechanisms associated with lymphoid organ atrophy will aid in overcoming this problem.

The effect of the time interval between exposures on the susceptibility of chickens to superinfection with Marek's disease virus.

Marek's disease virus (MDV) is ubiquitous within commercial poultry flocks because current vaccines do not prevent MDV infection or transmission. In order for newly-evolved MDV strains to become established within a flock, it seems inevitable that any new strain would need to infect and replicate in chickens previously infected with resident MDV strains. This phenomenon is difficult to detect and there is no clear evidence that it is even possible. Four experiments were performed to demonstrate superinfection and evaluate the effect of time between challenges on the effect of superinfection with the use of two pairs of fully virulent MDV strains that could be discriminated by novel technology: 1) JM/102W and rMd5//38CVI, and 2) rMd5 and rMd5//38CVI. Feather follicle epithelium (FFE), spleen, and tumor samples were collected at single or multiple time points from the same bird to determine the frequency and distribution of each virus present following superinfection, with the use of pyrosequencing and immunohistochemistry. Superinfection was observed in 82 of 149 (55%) FFE samples following short-interval challenge (24 hr) compared to only 6 of 121 (5%) samples following long-interval challenge (13 days), indicating a strong influence of challenge interval. In cases where the first inoculated virus was weak or delayed, the second inoculated virus was detected in 42 of 95 (44%) birds. In tumors from dually challenged birds, the second virus was again present much more often following short-interval challenge (68%) compared to long-interval challenge (11%). Virus mixtures in tumors were less common compared to those in FFE samples. Vaccination with turkey herpesvirus had no significant effect on the virus frequency for either virus pair or challenge time interval, suggesting these conclusions may be applicable to vaccinated chickens in the field. These studies demonstrated superinfection for the first time with two fully virulent MDV strains and suggest that short-interval challenge exposure and/or weak initial exposures may be important factors leading to superinfection--a prerequisite for the establishment of a second virus strain in the population. This model system should be useful to elucidate this important phenomenon further.

Factors influencing the attenuation of serotype 1 Marek's disease virus by serial cell culture passage and evaluation of attenuated strains for protection and replication.

This study was carried out to better understand factors that influenced the process of attenuation of Marek's disease (MD) virus by serial passage in cell cultures. Three virulent (v) pathotype and three very virulent plus (vv+) pathotype strains were passed by three techniques up to 131 times, and the passage level at attenuation was determined. The 18 attenuated or partially attenuated viruses were evaluated for protection against challenge with virulent MD virus, and the virus load (latent infection) in blood lymphocytes at 14-21 days postvaccination was determined. Viral pathotype strongly influenced the rate of attenuation. The mean passage level at attenuation for v and vv+ strains was 74 and >109, respectively. Full attenuation was achieved for nine of nine passage series with v pathotype strains but for only four of nine passage series with vv+ pathotype strains. Time to attenuation was not significantly influenced by multiplicity of infection at passage or by cell type, although a possible advantage of alternate high- and low-multiplicity passage was noted. Protection was not significantly influenced by pathotype or time to attenuation. Protection varied from 50% to 95% for the 18 passaged virus preparations; six attenuated viruses provided high protection that did not differ from that of the prototype Rispens strain. Virus load was not influenced by pathotype or by passage strategy and showed no positive correlation with protection. In several cases the most protective vaccines had the least virus load. This finding differs from previous reports and warrants further study. Variation among different strains within the same pathotype was documented for attenuation rate, protection, and virus load. Also, variation was evident when the same strain was passaged by different strategies, probably reflecting random changes during serial passage. Strain 596A (v pathotype) was the first to become attenuated, provided the best protection, and had one of the lowest virus loads. In contrast, strain 617A (v pathotype) provided the least protection and had one of the highest virus loads. Such strains provide fertile opportunities for further study.

Evaluation of Marek's disease field isolates by the "best fit" pathotyping assay.

Although determination of the pathotype is central to the study of Marek's disease (MD) field isolates, methods are not standardized and results from different laboratories may not compare well with the original Avian Disease and Oncology Laboratory assay. This study was designed to investigate the validity of the "best fit" pathotyping assay, a simplified method recently described for testing of field isolates of MD virus (MDV). Twenty serotype 1 MDV strains were isolated from 12 breeder and commercial flocks in eight regions of the Russian Federation and were pathotyped by the best fit assay using vaccinated and non-vaccinated chickens from Schelkovo specific pathogen free breeders. Lesion responses induced by field isolates were compared with those induced by reference strains JM/102W, Md5, and 648A representing pathotypes v, vv and vv+, respectively. Based on comparison with reference strains, we determined the pathotype of eight isolates as vv+, 11 isolates as vv and one isolate as v. Lesion responses induced by the three reference strains consistently differentiated the respective pathotypes in non-vaccinated chickens and in chickens vaccinated with FC126 (serotype 3) alone or with a bivalent FC126 + 301B/1 vaccine (serotypes 3 and 2, respectively). Variation between reference strain responses in replicate trials was minimal. In some cases, calculation of the proportional distance between pairs of reference strains aided in the classification of field isolates. These results indicate that the "best fit" pathotyping assay can be conducted with local chicken strains and, in the absence of statistical analysis, provides pathotype designations that are consistent with those obtained by the Avian Disease and Oncology Laboratory method. In addition, the pathogenicity of Russian isolates appeared comparable with that of United States isolates.

Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication.

Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in T cell lymphomas in visceral organs and peripheral nerves. Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV encode a basic leucine zipper protein, Meq, which structurally resembles the Jun/Fos family of transcriptional activators. Meq is consistently expressed in MDV-induced tumor cells and has been suggested as the MDV-associated oncogene. To study the function of Meq, we have generated an rMd5DeltaMeq virus by deleting both copies of the meq gene from the genome of a very virulent strain of MDV. Growth curves in cultured fibroblasts indicated that Meq is dispensable for in vitro virus replication. In vivo replication in lymphoid organs and feather follicular epithelium was also not impaired, suggesting that Meq is dispensable for lytic infection in chickens. Reactivation of the rMd5DeltaMeq virus from peripheral blood lymphocytes was reduced, suggesting that Meq is involved but not essential for latency. Pathogenesis experiments showed that the rMd5DeltaMeq virus was fully attenuated in chickens because none of the infected chickens developed Marek's disease-associated lymphomas, suggesting that Meq is involved in lymphocyte transformation. A revertant virus that restored the expression of the meq gene, showed properties similar to those of the parental virus, confirming that Meq is involved in transformation but not in lytic replication in chickens.

Comparison of serological and virological findings from subgroup J avian leukosis virus-infected neoplastic and non-neoplastic flocks in Israel.

Blood samples from nine broiler breeder flocks comprising five flocks clinically affected with myeloid leukosis tumours (ML+) and four tumour-free flocks from the same commercial background (ML-) were compared for avian leukosis virus subgroup J (ALV-J) serum antibodies by enzyme-linked immunosorbent assay (ELISA), for antigenemia (group-specific antigen) by antigen-trapping ELISA and for viremia. Group-specific antigen was detected in the sera of 58.1% of ML+ birds and 46.4% of the ML- birds (P=not significant), while 45.5% of ML+ birds and 24.1% of the ML- birds had ALV-J antibodies (P=0.065). In inoculated cell culture, 64.1% of the ML+ sera were viremic compared with 16.7% of the ML- sera (P=0.001). Similar significant differences were found between the two groups of flocks when ALV-J viremia was detected by immunofluorescence using a monoclonal env antibody (P=0.004), and for proviral DNA by polymerase chain reaction using two different sets of env-gene primers, H5-H7 (P=0.001) and R5-F5 (P=0.001). Using the primer pair R5-F5 the product size was approximately 1 kbp, while some heterogeneity in size among isolates was discernable. Our results indicate that a combination of diagnostic tests should be adopted in routine examination of tumour material in order to rule out false-negative findings.

Biocharacteristics shared by highly protective vaccines against Marek's disease.

Attenuated serotype 1 Marek's disease virus strains vary widely in their protection properties. This study was conducted to elucidate which biocharacteristics of serotype 1 MDV strains are related with protection. Three pairs of vaccines, each one including a higher protective (HP) vaccine and a lower protective (LP) vaccine originating from the same MDV strain, were studied. Two other highly protective vaccines (RM1 and CVI988/BP5) were also included in the study. Comparison within pairs of vaccines showed that marked differences existed between the HP and the LP vaccines. Compared with LP vaccines, HP vaccines replicated better in vivo. Also, they induced a significant expansion of total T cells and of the helper and cytotoxic T cell lineages (CD45(+)CD3(+), CD4(+)CD8(-), CD4(-)CD8(+)) as well as a marked increase in the expression of the antigens of MhcI and MhcII on T cells. Thus, our results show that in vivo replication and early stimulation of the T-cell lineage are two characteristics shared by HP vaccines. However, comparison among the four HP vaccines that provided protection equal to that of CVI988 (RM1, CVI988/BP5, CVI988 and 648A80) revealed variability, especially regarding in vivo replication. Strains RM1 and CVI988/BP5 showed much stronger replication in vivo than the other two vaccine strains (CVI988 and 648A80). Thus, no single set of characteristics could be used to identify the most protective Marek's disease vaccines, implying, perhaps, that multiple mechanisms may be involved.

Reticuloendotheliosis in Hungarian partridge.

A large commercial flock of Hungarian partridge (Perdix perdix) experienced elevated mortality associated with a wasting disease in May 1998. Postmortem examination of females consistently revealed a distended crop and abnormal gray-white tissue infiltrating the wall of the crop and thoracic esophagus. Neoplasia in male partridge was observed in the liver. Microscopic examination of the crop and thoracic esophagus revealed transmural masses of immature lymphocytes with frequent mitotic figures. Similar cells were observed in the liver of affected males. Virus particles consistent in size and morphology with reticuloendotheliosis virions were observed in neoplastic lymphoid cells via electron microscopy. Reticuloendotheliosis virus was isolated from each of four blood samples. This disease has not previously been reported in Hungarian partridges. Hungarian partridge may represent a potential reservoir of infection for other gamebirds, including prairie chickens.

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.

Marek's 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 Marek's disease-susceptible birds with the pp38 deletion mutant virus (rMd5 Delta pp38) 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 Marek's disease is a major oncogenic system, the knowledge obtained from these studies may shed light on the oncogenic mechanisms of other herpesviruses.