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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF AVIAN TUMOR VIRUS PATHOGENICITY, TRANSMISSION, AND EVOLUTION

Location: Avian Disease and Oncology Laboratory

Title: Research update: Avian Disease and Oncology Laboratory avian tumor viruses

Authors
item Fadly, Aly
item Cheng, Hans
item Dunn, John
item Heidari, Mohammad
item Hunt, Henry
item Lee, Lucy
item Silva, Robert
item Zhang, Huanmin

Submitted to: United States Animal Health Association Proceedings
Publication Type: Proceedings
Publication Acceptance Date: October 18, 2012
Publication Date: October 18, 2012
Citation: Fadly, A.M., Cheng, H.H., Dunn, J.R., Heidari, M., Hunt, H.D., Lee, L.F., Silva, R.F., Zhang, H. 2012. Research update: Avian Disease and Oncology Laboratory avian tumor viruses. United States Animal Health Association Proceedings. 513-516.

Technical Abstract: Genomics and Immunogenetics Use of genomics to identify QTL, genes, and proteins associated with resistance to Marek’s disease. Marek’s disease (MD), a lymphoproliferative disease caused by the highly oncogenic herpesvirus Marek's disease virus (MDV), continues to be a major disease concern to the poultry industry. The fear of MD is further enhanced by unpredictable vaccine breaks that result in devastating losses. The field of genomics offers one of the more exciting avenues for enhancing control of MD. By identifying genes that confer genetic resistance, it should become possible to select for birds with superior disease resistance. Genetic resistance to MD is a complex trait controlled by many genes. Identification of these genes is a major challenge despite the existence of the chicken genome sequence and ever increasing number of tools, especially next generation sequencing. Thus, we have been implementing and integrating genomic approaches that identify QTL, genes, and proteins that are associated with resistance to MD. The rationale for using more than one approach is that the strengths of each system can be combined to yield results of higher confidence. Another justification is that given the large volume of data produced by genomics, each method provides an additional screen to limit the number of targets to verify and characterize in future experiments. Some highlights of this year’s findings include: (1) analysis of RNA seq datasets indicates both the Toll-like receptor and JAK/STAT pathways are conserved responses to MDV infection in commercial broilers and experimental layers, and genes at the start of each pathway can be selected to modulate the response, (2) Meq binds AP-1 sites to regulate expression of genes that influence immunological responses including MAPK signaling, which is also needed to maintain growth in low serum, and (3) a complete list of polymorphisms and genes in the MDV genome associated with in vitro attenuation has been compiled, and testing of recombinant MDVs indicates that a SNP in UL5 (helicase/primase) has significant impact on viral virulence. Host Genetics and Vaccinal Protective Efficacy against MD. Vaccinal protective efficacy against vv+MDV challenge was studied in MD resistant and susceptible chickens. Chickens from a MD resistant line (63) and a susceptible line (72) were either vaccinated or vaccinated followed by vv+MDV challenge. Chickens from both lines that were only vaccinated with either HVT or CVI988/Rispens did not develop any tumor and survived throughout the experiment. Chickens that vaccinated followed by vv+MDV challenge resulted in differential MD incidence and protective index (PI). Both HVT and Rispens conveyed comparable protection against the vv+MDV challenge with PI 91.2 and 86.7 percent in line 63, respectively. In comparison, CVI988/Rispens conveyed 80 percent protection while HVT achieved significantly lower protection (25%) in line 72. This result confirms our previous report that host genetics plays a vital role in modulating vaccinal protective efficacy. Furthermore, next generation RNA sequencing data suggest vaccine, MDV, and vaccine plus MDV differentially up- or down-regulated global gene expression in both MD resistant and susceptible chickens. RNA samples were collected from both MD resistant and susceptible lines either vaccinated, MDV challenged, and both. RNA libraries were constructed following standard procedures for next generation RNA sequencing with Illumina’s HiSeq platform. The RNA reads data suggested that the global gene expression differed between the MD resistant and susceptible chickens and differentially up- or down-regulated by each vaccine, MDV, or the combination of both vaccine and MDV. This finding suggests host genetics effect on vaccinal protective efficacy may be partially explained by differential globe gene expression upon vaccination and MDV challenge of chickens with different genetic backgrounds. Marek’s Disease Virus Evolves to Higher Virulence in Birds with Limited Genetic Variation. MD is still a major concern as MDV continues to evolve to higher virulence. Most studies addressing the evolution of MDV virulence have concentrated on the virus while largely ignoring the hosts’ influence. The host system called the major histocompatibility complex (MHC) represents a highly polymorphic system designed to defend the species from extinction by the fast paced evolution of a parasite. In natural chicken populations, there are hundreds of different MHC haplotypes that oscillate in response to pathogen evolution, but commercial poultry breeding has limited the number of MHC haplotypes to six or less. Our current work has shown that MDV can evolve to higher virulence in birds with a single MHC haplotype. We are evaluating the effects of resistant and susceptible MHC haplotypes on MDV evolution. Our results suggest that MDV evolves to higher virulence in the susceptible MHC haploytpe. The virus passed in the resistant MHC haplotype does not overcome the resistance but is more virulent in the susceptible haplotype than the parental virus. Thus, the virus can evolve to more virulence in resistant MHC haplotypes but this increased virulence is only observed in the more susceptible MHC haplotypes. This may help explain sporadic outbreaks of MDV in flocks segregating for resistant and susceptible MHC haplotypes. Immunopathogenesis, Diagnosis and Control of Marek’s Disease Pathotyping of new field strains of MDV. Pathotyping of new field strains of MDV requires both a long period of time and a large number of birds. Confirming a positive correlation of virus replication and pathotype may lead to faster and cheaper alternative pathotyping methods or as a screening assay for choosing isolates to be pathotyped. Past studies have found differences in replication rates between selected vMDV and vv+MDV, but this correlation has not been evaluated using a broad selection of virus strains. Our first trial evaluated replication rates of five virus strains from each virulent pathotype (v, vv & vv+) using maternal antibody positive chickens which found very little difference in lymphoid atrophy between groups and mild differences between replication rates by pathotype. The current trial evaluated differences using maternal antibody negative chickens. We found a significant increase in viral load in brain, bursa and lung tissue at days 9 and 11 post challenge for vvMDV and vv+MDV strains compared to vMDV strains. No significant difference was seen between vvMDV and vv+MDV strains. Similar results were seen comparing lymphoid atrophy between pathotype groups. Using these results, it may be possible to determine a replication rate threshold as a preliminary screen to separate vMDV from vv/vv+MDV strains. Role of macrophages in MDV infection. We investigated the specific role of macrophages (MQ) in the control or exacerbation of MD by depletion of these phagocytic cells using a chemical called clodronate (Cl2MBP) 48 hours prior to exposure to shedder birds. Our preliminary studies indicate that combination of intra-tracheal and intra-venous treatment of chickens with clodronate reduces the number of macrophages in the spleen and lungs significantly and this reduction in phagocytic cell population will likely influence the number of virus particles being transmitted from the lungs to the lymphoid organs. Macrophages in addition to the speculated role of virus dissemination play an essential role in viral replication and infection by production of nitric oxide and interferon gamma. This information is important in understanding the immunological responses to MD and development of immunomodulatory measures to prevent MDV infection and spread. Diagnosis. PCR was used in diagnosis of MD and reticuloendotheliosis (RE) in formalin-fixed, paraffin-embedded (FFPE) tumorous tissues that have been stored for periods varied from 5-244 months. In another experiment, PCR was also u

Last Modified: 12/17/2014
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