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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
Identify genetic predictors of Marek's disease virus (MDV) virulence. Identify host-viral genetic determinants that control avian tumor virus pathgenicity and shedding. Elucidate the genetic determinants that modulate MDV interactions within the avian immune system. Elucidate host-viral interactions that drive the evolution of new virulent strains of avian tumor viruses. Discover safe and highly effective vaccine platforms that convey protection against emerging MDV strains.

1b.Approach (from AD-416)
Avian tumor viruses of economic importance include:.
1)Marek’s disease virus (MDV), a herpesvirus that induces a lymphoproliferative disease of chickens that, in the absence of effective control measures, is capable of causing devastating losses in commercial layer and broiler flocks; and 2) avian retroviruses, namely avian leukosis virus (ALV) and reticuloendotheliosis virus (REV), both are associated with neoplastic diseases and other production problems in poultry. Also, both ALV and REV are potential contaminants of live-virus vaccines of poultry. Critical needs are:.
1)better MDV vaccines to protect against the current and next generation of virulent field strains of MDV; and.
2)a long-term strategy designed to reduce the ongoing emergence of new virulent MDV, and creation of recombinant ALVs through multiple barriers or reduction in viral load and shedding. The primary emphasis will be on molecular approaches to better understand which viral genes are important for immunopathogenesis and shedding of MDV. Parallel studies will monitor the virulence of field strains of MDV and ALV. Studies are also aimed at characterization of new virus isolates and on improving assays for their detection; additional efforts will be devoted to better understand MDV immunity. The project also emphasizes studies on:.
1)elucidating factors involved in creation of recombinant ALVs; and.
2)determining whether REV genome insertion into MDV and fowlpox virus influences transmission and epidemiology of REV. The end product will be a better understanding of viral gene function, virus-host interactions and the development of materials and improved methodology for control of avian tumor viruses.

3.Progress Report
Substantial progress was made on all objectives of the project. A brief description of selected accomplishments is listed below. This year, we investigated characteristics of individual Marek’s disease virus (MDV) strains that may enhance virus evolution by challenging birds simultaneously with two MDV strains. Dominance, as defined by a majority frequency of a particular MDV strain, was consistently demonstrated for the more virulent strain when challenged simultaneously with the less virulent strain; also, dominance was not observed between two similar strains. Our results suggest that viral dominance and temporal relationships may be important factors that influence the outcome of coinfection under field conditions, including the potential outcome of emergence or evolution of more virulent strains. Recently, we have reported on the development and pathogenicity of a bacterial artificial chromosome (BAC) clone of MDV with an insert of long terminal repeat (LTR) of reticuloendotheliosis virus (REV). We have also shown the presence of REV LTR in all viruses tested in vitro even in those used at levels higher than the 40th passage, but not in buffy coat (BC) cells obtained from infected chickens at 3 and 8 weeks post-infection when tested directly by PCR. This year, we were able to demonstrate that REV LTR was retained by the rMd5 BAC virus as early as 1 week following infection of one-day old chickens. Further, because this rMd5 BAC with REV LTR is less pathogenic than the parent strain or strain lacking REV-LTR we plan to explore the possibility of using high passage level of this virus as a vaccine. In our attempts to survey field flocks for MDV of unusual pathogenicity, we have received samples in 2011 from a broiler breeder flock in Pennsylvania as well as from a layer flock in Iowa experiencing high mortality. Pathotyping experiments are currently ongoing for selected 2011 isolates. Also, this year, in our surveillance of field flocks affected with avian leukosis virus (ALV)-like tumors, using routine molecular, virological, serological assays, we were not able to demonstrate the presence of exogenous ALV in blood or tumors from affected chickens. Only an endogenous ALV named AF-227 was isolated. We plan to biologically and molecularly characterize this isolate and study its role in induction of such spontaneous ALV-like tumors in chickens vaccinated with various serotypes of Marek's disease (MD) vaccine. Studies of immunological basis for resistance or susceptibility to MD demonstrated an increase in the transcriptional activity of CTLA-4 gene in line 7-2 is suggestive of another possible immunological basis for the susceptibility of this line. In general, a vigorous T helper 1-associated type of immune response is more frequently observed in the resistant line than in the susceptible line. Most recently, we have determined that codon and di-codon bias in MDV genes are probably important in virus replication and/or pathogenesis. We plan to explore altering the codon or di-codon bias of critical MDV genes as a novel approach to vaccine development.

1. Factors leading to the evolution of Marek’s disease virus (MDV). Previous studies by ARS scientists in East Lansing, MI investigating superinfection with two fully virulent MDV strains suggest that short interval between virus exposures and/or weak initial exposures may be important factors leading to superinfection; a prerequisite for the establishment of a more virulent second virus strain in the population. To further investigate characteristics of individual virus strains that may enhance virus evolution, we challenged birds simultaneously with two MDV strains. Dominance, as defined by a majority frequency of a particular MDV strain, was consistently demonstrated for the more virulent strain when challenged simultaneously with the less virulent strain and even occurred with two similar strains. Viral dominance and time between multiple infections may be important factors that influence the outcome of mixed infections in chickens, including the potential outcome of emergence or evolution of more virulent strains. The information is important in understanding what conditions may help slow the evolution and establishment of new strains within poultry flocks.

2. Artificial insertion of genetic materials from reticuloendotheliosis virus (REV) into Marek’s disease virus (MDV) reduces its pathogenicity. MDV and REV are both avian viruses that belong to two different groups of oncogenic viruses, MDV is a DNA virus whereas, REV is an RNA virus; both viruses can cause cancer-like disease in chickens. It has been reported that under certain circumstances part of the genetic material from REV known as long terminal repeat (LTR) can be inserted in and be part of the genome of MDV. The effect of REV-LTR insertion into the genome of MDV on the pathogenicity (disease-inducing potential) of MDV is poorly understood. Recently, using a DNA-based technology termed bacterial artificial chromosome (BAC) we were able to artificially insert REV-LTR into a clone of very virulent MDV. The pathogenicity of this BAC clone of MDV with and without REV-LTR was compared in susceptible chickens. The results confirmed those obtained from our previous experiments indicating that BAC clone of MDV containing REV-LTR was less pathogenic than that without LTR or the wild type MDV. The data also demonstrated the REV-LTR was retained in MDV recovered from inoculated chickens, but only for one week after infection. The information is important, as it adds significantly to the knowledge in the area of retroviral gene insertion into large DNA viruses, an important new area of research in the molecular biology of avian tumor viruses; the information will also help in understanding the molecular basis for pathogenicity and shedding of these viruses.

3. Immunological basis for resistance or susceptibility to Marek's disease (MD). To shed some light on the molecular mechanism of resistance to MD, we compared immunological responses to MD virus (MDV) infection between a highly susceptible (7-2) and a relatively resistant (6-3) chicken lines. Our studies revealed that a population of a sub-set of immune cells named T-lymphocyte (target cells for MDV) is much higher in line 7-2 than in line 6-3. Also, a significant down regulation of a specific protein termed CD8 antigen on the surface of killer T cells of the susceptible line, 7-2, was observed. Analysis of a panel of soluble components of immune system known as cytokine and chemokine and other immune-related genes between the two lines, revealed major differences in response to MD. In general, the data indicate that a vigorous T cell-mediated immune response is observed more frequently in the resistant line when compared with the susceptible line, suggesting an immunological role in the genetic resistance to MD. This information is important in understanding the pathogenesis and immunity of MD; and should help vaccine manufacturers as well as poultry breeders and growers to design more effective vaccine platforms.

4. Molecular mechanism of Marek's disease vaccine protection. Although Marek’s disease (MD) vaccines have been in use for many decades, the molecular mechanism of their protection is poorly understood. There is growing evidence that a subset of immune cells known as natural killer (NK) cells play a critical role in vaccine-induced protection, genetic resistance, and anti-tumor immunity. To elucidate the functional activities of NK cells in vaccine-induced protection, we compared the biological role of NK cells in vaccinated birds with that in vaccinated/challenged, challenged, and control uninfected chickens. Expression analysis of several NK-specific genes indicates that vaccination activates NK cells that provide protection against virulent MD challenge viruses. This information is essential in understanding the mechanism of vaccine-induced protection, and leading to the development of more effective vaccines against MD by immune-modulation of NK cells.

5. Surveillance of field flocks for Marek's disease (MD) virus (MDV) of unusual pathogenicity. In past years we have been in close contact with our industry stakeholders and have received blood samples from layer and broiler breeder flocks experiencing high MD mortality in Pennsylvania and Iowa, although vaccinated with the Rispens strain of MDV, the most effective currently available commercial vaccine. Two Pennsylvania virus isolates from 2010 pathotyped as virulent (v) and very virulent+ (vv+) MDV and interestingly shared a specific mutation in the MDV gene termed pp38 similar to Pennsylvania isolates from 2007 and 2009. Although the pathotyped strains were not unusually virulent, this unique mutation in combination with increased MD incidence in several surrounding flocks indicate that there may be a mutated MDV strain circulating in Pennsylvania. These results suggest that MDV field isolates from 2010 did not cause high incidence of disease as the result of an evolutionary shift in virulence.

6. Improving the safety of an efficacious recombinant (new generation) Marek's disease vaccine. Deletion of the gene responsible for induction of tumors (Meq gene) of Marek's disease virus (MDV) rendered the virus non-oncogenic; in both laboratory and field trials, the new Meq-deleted virus has been shown to be an efficacious vaccine. However, the vaccine caused atrophy (loss of size and weight) of lymphoid organs that are responsible for maintaining a proper immune system in the host. Scientists at ADOL developed a method to rid the most effective vaccine against Marek's disease from this serious side effect, namely immunosuppression resulting from its negative effects on lymphoid organs. Serial passage of this Meq-deleted Marek’s disease vaccine for up to 80 cell culture passages resulted in elimination of its negative effects on lymphoid organs beginning at the 40th cell culture passage. This development is important, as it will assist vaccine manufacturers to proceed with their plans for commercializing the vaccine.

7. Bursal and thymic atrophy induced by Marek's disease (MD) vaccines. While it has been discovered that Meq-deleted Marek's disease viruses (MDVs) were better vaccines than Rispens, the best commercially available MD vaccine, the Meq-deleted viruses induced significant reduction in size or atrophy in two organs vital for maintaining an adequate immune system, bursa of Fabricious and thymus. Commercial Rispens vaccines did not induce atrophy. The ability of a virus to induce thymic atrophy directly correlated with the virus’s capacity to replicate to high titers in the thymus. This work demonstrated that the ability of MDV to induce tumors and disease is separate from its ability to induce atrophy. Consequently, we should be able to target specific MDV genes for either deletion or mutation and eliminate the atrophy.

8. Demonstration of di-codon bias in chicken genes. ARS scientist at the Avian Disease and Oncology Lab (ADOL) in East Lansing, MI, have demonstrated that chicken genes utilize a pronounced preference for some but not all nucleic acids, referred to as di-codon bias. They found that di-codon bias in chickens is similar to but slightly different from the bias in human genes. Scientist at the ADOL also calculated the di-codon bias for every gene in Marek's disease virus (MDV) and demonstrated that MDV genes utilize a distinct di-codon bias that is uniquely different from the bias in chicken or human genes. This work indicated that di-codon bias in MDV genes may be important in virus replication and/or pathogenesis. To determine whether di-codon bias is important for efficient virus replication ADOL scientists designed a computer program that would replace frequently used di-codons with synonymous infrequently used di-codons. Using this program, they altered the di-codon frequencies in UL48, an MDV gene essential for viral gene control, thus demonstrating that the MDV with the altered UL48 was still able to replicate efficiently in cell culture. Preliminary results indicate that the mutant MDV may not be as pathogenic as the unmodified virus. Thus, di-codon de-optimization may be an unexplored means to generate novel vaccines that still retain all the antigenic epitopes present in the parental pathogenic virus.

Review Publications
Fadly, A.M. 2010. Neoplasms. In: Kahn, C. M., Line, S., editors. The Merck Veterinary Manual. 10th edition. Whitehouse Station, NJ: Merck & Co., Inc. p. 2449-2457.

Mays, J.K., Silva, R.F., Lee, L.F., Fadly, A.M. 2010. Characterization of reticuloendotheliosis virus isolates obtained from broiler breeders, turkeys, and prairie chickens located in various geographical regions in the United States. Avian Pathology. 39(5):383-389.

Haq, K., Brisbin, J.T., Thanthrige-Don, N., Heidari, M., Sharif, S. 2010. Transcriptome and proteome profiling of host responses to Marek's disease virus in chickens. Veterinary Immunology and Immunopathology. 138(4):292-302. available:

Dunn, J.R., Witter, R.L., Silva, R.F., Lee, L.F., Finlay, J., Marker, B.A., Kaneene, J.B., Fulton, R.M., Fitzgerald, S.D. 2010. The Effect of the Time Interval Between Exposures on the Susceptibility of Chickens to Superinfection with Marek Disease Virus. Avian Diseases. 54(3):1038-1049.

Lee, L.F., Zhang, H., Heidari, M., Lupiani, B., Reddy, S. 2011. Evaluation of factors affecting vaccine efficacy of recombinant Marek's disease virus lacking the Meq oncogene in chickens. Avian Diseases. 55(2):172-179.

Kim, T., Mays, J.K., Fadly, A.M., Silva, R.F. 2011. Artifically inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes. Virus Genes. 42(3):369-376.

Last Modified: 7/23/2016
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