Location: Avian Disease and Oncology Research2012 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.
3. Progress Report:
Substantial progress was made on all objectives of the project. To better understand how Marek’s disease (MD) virus (MDV) evolves, we have investigated the correlation between virus replication and virulence. We determined which time points and tissue samples showed the greatest difference in replication between a low and high virulent MDV strain and will use this to examine a broad collection of MDV strains with varying virulence. As some MD vaccines are produced by attenuating virulent MDV through blind passages in cultured chicken cells, it is important to identify the molecular changes that occur in MDV during such blind passages. In collaboration with researchers at Simon Fraser University, Canada, we identified all the sequence variants that occur during blind passages of some strains of MDV. This information should be useful to poultry vaccine manufacturers. We also investigated the specific role of macrophages in the pathogenesis of MD by depleting these phagocytic cells using a chemical named clodronate at 48 hours prior to exposure of chickens to MDV. Preliminary results indicate that treatment of chickens with clodronate significantly reduces the number of macrophages in the spleen and lungs, and consequently will influence the number of MDV particles being transmitted from the lungs to the lymphoid organs. This information is critical in understanding the immunological responses to MDV infection that will eventually lead to better strategies to control MD. To enable diagnostic laboratories in the USA and other countries to use a simple, specific and sensitive method for confirming the diagnosis of virus-induced tumorous diseases of poultry, we adapted a DNA-based test named polymerase chain reaction (PCR) for use in the diagnosis of MD and reticuloendotheliosis (RE) in formalin-fixed, paraffin-embedded (FFPE) tumorous tissues. Using PCR, MD and RE viruses were detected in FFPE tissues tested even in those stored for up to 20+ years; MDV was also detected in tissues that were just preserved in formalin for up to 7 weeks. The research proved that PCR is a sensitive and specific test that can be used in the diagnosis of MD and RE in affected tissues stored as FFPE tissues or in those only preserved in formalin. Preliminary data indicate that ADOL line alv6 chickens vaccinated in ovo or at hatch with the SB-1 strain of MDV developed more spontaneous tumors than chickens that did not receive the vaccine. Recently, we reported that inserting a genetic material known as long terminal repeat (LTR) from RE virus (REV), a virus that can cause tumors in chickens, into the genome of MDV lowered the pathogenicity of MDV. Results from a pilot study to determine protective ability of various passage levels of MDV with LTR insert showed that passage level 75 when used as a vaccine reduced MD lesions by 75% following challenge with a very virulent plus (vv+) strain of MDV. This information is critical for planning further protection studies to evaluate MDV with LTR insert as a vaccine against MD.
1. Elucidation of factors involved in evolution of Marek’s disease virus (MDV) to greater virulence. One of our major goals is to understand what traits are related to highly virulent MDV, to understand how and why the virus evolves and to better understand how to control MDV in the field. To understand the importance of virus replication with respect to virulence, we challenged chickens with a low and high virulent strain and measured virus replication from a variety of tissues at multiple time points. We found that replication was significantly higher in the virulent strain. Additional screening of replication rates with a comprehensive set of virus strains will confirm the importance of virus replication in relation to virulence and will allow us to use cheaper and faster methods for characterizing virulence of new field isolates.
2. Surveillance of field flocks for the presence of highly virulent Marek’s disease virus (MDV). In past years, we have been in close contact with our industry stakeholders and have isolated field MDV strains to monitor virus evolution in order to more quickly respond to shifts in virus virulence and to collect unique strains for a variety of research projects in our lab and around the world. During the last year ARS scientists in East Lansing, Michigan, pathotyped several MDV strains from Pennsylvania and Iowa and determined most of these isolates were highly virulent, although not more virulent than some of the past isolates in our collection. This information is valuable to the industry, as monitoring virulence of MDV field strains and identifying the source of problems during an outbreak should allow the owners of the flocks to reevaluate their vaccination programs to control the disease.
3. Development of a simple method for detection of Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) in formalin-fixed, paraffin embedded (FFPE) tissues. ARS scientists in East Lansing, Michigan, developed a simple DNA-based test termed polymerase chain reaction (PCR) for detection of MDV and REV in FFPE tissues. The test proved to be sensitive and specific in detection of MDV and REV in FFPE tissues. This simple protocol can be used as an alternative to currently available laborious and more expensive biological or molecular tests that require use of frozen samples; it will also alleviate the need for use of dry-ice for shipping frozen samples to diagnostic laboratories.
4. Influence of serotype 2 Marek’s disease virus (MDV) vaccine strain and route of vaccination on enhancement of spontaneous (non-viral) tumors noted in certain lines of chickens. ARS scientists in East Lansing, Michigan, found that serotype 2 MDV vaccine enhanced the development of spontaneous (non-viral) lymphomas that are seen in certain genetic lines, regardless whether chickens were vaccinated in ovo or at hatch. This information is important in understanding the pathogenesis of spontaneous tumors and will lead to better control of such tumors in commercial lines that exhibit such tumors.
Dunn, J.R., Silva, R.F., Lee, L.F., Witter, R.L. 2012. Competition between two virulent Marek's disease virus strains in vivo. Avian Pathology. 41(3):267-275. Available: http://dx.doi.org/10.1080/03079457.2012.677804.