2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Identify host and/or viral genetic determinants that control pathogenicity, transmission, and drive the evolution of new strains of avian tumor viruses.
Subobjective 1.2: Identify the genetic determinants in the MDV genome that account for vitro attenuation.
Subobjective 1.3: Factors that influence the development of spontaneous ALV-like tumors.
Subobjective 1.4: Confirming an association between MDV replication rate and pathotype.
Objective 2: Develop diagnostics for detecting new strains of avian tumor viruses.
Subobjective 2.1: Evaluation of MDV BAC clones as standardized reagents for MDV research.
Subobjective 2.2: Surveillance for virulent strains of avian tumor viruses in field flocks and develop improved diagnostics for new strains.
Subobjective 2.3: Development of reliable techniques for immunohistochemistry (IHC) using paraffin-fixed sections.
Objective 3: Elucidate the genetic determinants that modulate MDV interactions with the avian immune system.
Subobjective 3.1: Identification and characterization of host/viral genes that mediate production of cell-free enveloped infectious virus particles in the FFE
Subobjective 3.2: Role of NK cells in vaccine-induced immunity against MD.
Subobjective 3.3: Role of macrophages and T cells in viral transport to lymphoid organs and FFE.
Objective 4: Discover safe and highly effective vaccine platforms that convey protection against emerging MDV strains.
Subobjective 4.3: Determine protective ability of high passage levels of a BAC clone of strain Md5 of MDV containing LTR from REV.
Subobjective 4.4: Evaluation of vaccine competition using HVT 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;.
2)a long-term strategy designed to reduce the ongoing emergence of new virulent MDV through multiple barriers or reduction in viral load and shedding; and.
3)better procedures to detect and control new ALV recombinants. 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 avian retroviruses, primarily 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 and role of MDV vaccines in enhancement of spontaneous non ALV-induced tumors. The four objectives are highly interrelated and interface in a manner that should not only identify new basic knowledge but also translate this knowledge to practical use in control programs. The end product will be a better understanding of viral gene function, virus-host interactions and the development of materials and improved methodology for diagnosis and control of avian tumor viruses.
Substantial progress was made on all objectives of the project. Sequence variants that are associated with in vitro attenuation of Marek’s disease viruses (MDVs) were tested in recombinant MDVs. Of the 5 viruses tested, a single base change in a single gene resulted in almost complete loss of virulence, suggesting that such approach could be used in preparation of next generation MD vaccines. Our studies to determine the correlation between MDV replication and virulence indicated that the lowest virulent strains had significantly lower virus replication compared to viruses in the two higher virulence pathotype groups, but viruses in the higher two groups were unable to be distinguished. Results from evaluating interference between HVT-vectored vaccines suggested that insertion of another viruses’ gene into the HVT vaccine reduces the ability of the virus to replicate in birds. We also characterized several highly virulent MDV strains from current outbreaks; we plan to sequence these isolates in hopes of identifying correlations between DNA sequence and virulence as an alternative to slower and more costly evaluations in birds. Two experiments were conducted to test the protective efficacy of an experimental recombinant MDV vaccine that contains LTR from reticuloendotheliosis virus (REV) termed rMd5/REV LTR BAC. Results from both experiments indicate that this recombinant MDV was as effective as Rispens vaccine, the most effective currently available commercial MDV vaccine. Data obtained from our studies to understand the role of subgroup E avian leukosis virus (ALV-E) and serotype 2 MDV vaccine in enhancement of spontaneous ALV-like tumors, suggest that ALV-E and serotype 2 MDV plays an important role in the enhancement of spontaneous ALV-like tumors in certain lines of chickens. This year, our studies to determine the role of macrophages (MQ) in MD revealed that depletion or partial reduction of MQ population was inversely correlated with number of virus particles and infected cells; also reduction in MQ population had an adverse effect on the expression pattern of cytokines/chemokines in the MDV-infected tissues. Also, our investigations to study role of natural killer (NK) cells in MD vaccinal immunity suggested that vaccination arms NK cells by increasing the production of cytotoxic granule proteins (granzyme and perforin) and interferon gamma, and that a functional marker for NK cell degranulation (called CD107a) was significantly increased in expression at seven days post vaccination.
Characterization of new highly virulent field isolates of Marek’s disease virus (MDV). To understand virus evolution and be able to more quickly respond to newly evolved and more virulent MDV strains, it is critical to characterize virus isolates from current outbreaks occurring throughout the country. ARS researchers in East Lansing, MI have isolated and pathotyped several MDV strains of high virulence. These isolates will undergo further sequencing analysis in hopes of identifying relationships between DNA sequence and virulence. This information is valuable to the industry for both demonstrating the virulence of current circulating field strains and sequencing results may lead to cheaper and faster tests for characterizing virulence of field strains, and consequently improved disease detection and control.
Characterization of commercially available herpes virus of turkeys (HVT)-vectored vaccines. HVT, a serotype 3 Marek’s disease (MD) vaccine virus has been used by commercial poultry vaccine manufacturers as a vector to insert genes from other viruses to immunize chickens against diseases induced by these viruses. Such HVT-vectored vaccines, also known as multivalent vaccines can provide protection against MD virus as well as other diseases of interest. The interaction between multiple virus or vaccine strains is important for understanding both the benefits of multivalent vaccine products as well as understanding the interference and reduced protection as reported with mixtures of certain HVT-vectored vaccines. To study interference between HVT vaccines, ARS researchers in East Lansing, MI have evaluated individual HVT strains and found that insertion of foreign genes into the vectored vaccines reduces the replication of the vaccine in chickens compared to standard HVT vaccines. This finding offers a potential explanation for the reduced protection of certain HVT-vectored vaccine strains when mixed, and will be evaluated in upcoming experiments using vaccine mixtures. Also, this information is important to poultry vaccine manufacturers and should lead to development of better HVT-vectored vaccines.
Efficacy of an experimental recombinant Marek’s disease virus (MDV) vaccine. Recently, ARS researchers in East Lansing, MI developed a recombinant MDV that contains parts of the genome of another avian tumor virus, reticuloendotheliosis virus. This year, experiments were conducted to test efficacy of this recombinant MDV termed rMd5/REV LTR BAC. Results from these experiments proved that this recombinant MDV vaccine was as effective as the most effective currently available commercial MDV vaccine named Rispens. This experimental vaccine not only protected chickens from MD tumors, but also from atrophy or damage of two important immune organs known as bursa and thymus. Also, results from both experiments indicate that this experimental MD vaccine had a higher protective efficacy in chickens lacking maternal antibodies than in chickens with maternal antibodies. This information is important to poultry vaccine manufacturers who are searching all the time for more effective vaccines against MD.
Spontaneous avian leukosis virus (ALV)-like tumors in chickens. Certain lines of chickens such as the Avian Disease and Oncology Laboratory (ADOL) line named RFS are susceptible to development of spontaneous ALV-like tumors. ARS researchers in East Lansing, MI investigated role of endogenous ALV and serotype 2 Marek's disease virus (MDV) vaccines in enhancement of these spontaneous tumors. Results demonstrated that chickens that harbor endogenous ALV (ALV-E) are likely to develop a relatively high incidence of spontaneous ALV-like tumors than chickens that lack ALV-E following vaccination with serotype 2 MDV at 1 day of age. This information is important to primary breeders who are interested in reducing or eliminating spontaneous ALV-like tumors in certain lines of their breeders.
Role of macrophages (MQ), an important cell of the chicken immune system, in Marek’s disease (MD). Vaccinal immunity against MD, an economically important herpesvirus-induced cancer like disease of chickens is not clearly understood. ARS researchers in East Lansing, MI demonstrated that depletion or partial reduction of MQ population was inversely correlated with number of MD virus particles and infected cells. Also, depletion of MQ led to reduction in blood level of nitric oxide, an important soluble factor in the control of virus particles in infected chickens. Reduction in MQ population also had adverse effect on the expression pattern of cytokines/chemokines (soluble components of the immune system that orchestrate immunity-related activities) in the MDV-infected tissues. This information is critical to the understanding of role of important host immune cells, namely MQ in MD and should lead to the establishment of new immunomodulatory approaches to enhance MQ function , and consequently provide better vaccinal protection against this important disease.
Role of Natural Killer (NK) cells, an important component of the chicken immune system in Marek’s disease (MD). Vaccinal immunity against MD, an economically important herpesvirus-induced cancer like disease of chickens is not clearly understood. Recent studies by ARS researchers in East Lansing, MI provided evidence that vaccination against MD arms NK cells by increasing the production of cytotoxic granular proteins (granzyme and perforin) as well as interferon gamma, another important soluble immune component. The study also revealed that a functional marker for NK cell degranulation, named CD107a was significantly increased in expression seven days post vaccination; also, that a pathogenic strain of MD virus interferes with different aspect of the biological function of NK cells, rendering them ineffective against both infected and tumor cells. This research provided insight into the critical role of NK cells in vaccinal immunity against MD, and should lead to establishment of immunomodulatory approaches for enhancing functional activities of NK cells, and consequently provide better vaccinal protection against this important disease.
Correlation between virulence of Marek’s disease virus (MDV) and replication rate. To understand how and why viruses evolve and to better understand how to control MDV in the field, we must understand what traits are related to highly virulent MDV strains. To understand the importance of virus replication with respect to virulence, ARS researchers in East Lansing, MI challenged groups of chickens with one of fifteen MDV strains ranging from low to high virulence and measured virus replication. MDV strains are classified into three pathotypes: virulent (vMDV), very virulent (vvMDV) and very virulent plus (vv+MDV). Replication rates were significantly higher for vvMDV and vv+MDV strains compared to vMDV, although there was no difference between vvMDV and vv+MDV strains. This information is important to poultry diseases diagnosticians and researchers and may lead to cheaper and faster screening tests for characterizing virulence of new field isolates into two groups, vMDV in one group and vv and vv+ MDV in another group. However, existing pathotyping methods should still be used for differentiation between vv and vv+ MDV.
Pandiri, A.R., Gimeno, I.M., Mays, J.K., Reed, W.M., Fadly, A.M. 2012. Reversion to subgroup J avian leukosis virus viremia in seroconverted adult meat-type chickens exposed to chronic stress by adrenocorticotrophin treatment. Avian Diseases. 56:578-582.
Xu, M., Fitzgerald, S.D., Zhang, H., Karcher, D.M., Heidari, M. 2012. Very virulent plus strains of MDV induce acute form of transient paralysis in both susceptible and resistant chicken lines. Viral Immunology. 25(4):306-323.
Dunn, J.R., Silva, R.F. 2012. Ability of MEQ-deleted MDV vaccine candidates to adversely affect lymphoid organs and chicken weight gain. Avian Diseases. 56:494-500.
Plotnikov, V.A., Grebennikova, T.V., Yuzhakov, A.G., Dudnikova, E.K., Norkina, S.N., Zaberezhny, A.D., Aliper, T.I., Fadly, A.M. 2012. Molecular-genetic analysis of field isolates of Avian Leucosis Viruses in the Russian Federation. Problems of Virology. 57(5):39-43.
Lee, L.F., Heidari, M., Sun, A., Zhang, H., Lupiani, B., Reddy, S.M. 2013. Identification and in vitro characterization of a Marek’s disease virus encoded ribonucleotide reductase. Avian Diseases. 57:178-187.
Lee, L.F., Kreager, K., Heidari, M., Zhang, H., Lupiani, B., Reddy, S.M., Fadly, A.M. 2013. Properties of a meq-deleted rMd5 Marek’s disease vaccine: protection against virulent MDV challenge and induction of lymphoid organ atrophy are simultaneously attenuated by serial passage in vitro. Avian Diseases. 57(2):491-497.
Dunn, J.R., Gimeno, I.M. 2013. Current status of Marek’s disease in the United States & worldwide based on a questionnaire survey. Avian Diseases. 57(2):483-490.
Sun, A., Lee, L.F., Khan, O., Heidari, M., Zhang, H., Lupiani, B., Reddy, S. 2013. Deletion of Marek’s disease virus large subunit of ribonucleotide reductase (RR) impairs virus growth in vitro and in vivo. Avian Diseases. 57(2):464-468.
Lupiani, B., Lee, L.F., Kreager, K.S., Witter, R.L., Reddy, S.M. 2013. Insertion of reticuloendotheliosis virus long terminal repeat into the genome of CVI988 strain of Marek’s disease virus results in enhanced growth and protection. Avian Diseases. 57(2):427-431.
Reddy, S.M., Sun, A., Khan, O.K., Lee, L.F., Lupiani, B. 2013. Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome. Avian Diseases. 57(2):469-473.