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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF AVIAN TUMOR VIRUS SUSCEPTIBILITY Title: Research update: Avian Disease and Oncology Laboratory avian tumor viruses

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 4, 2011
Publication Date: July 30, 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. In: Proceedings for the 115th Annual Meeting of the United States Animal Health Association, September 29-October 5, 2011, Buffalo, New York. 462-464.

Technical Abstract: Genomics and Immunogenetics 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. Our combined approaches of (1) sequencing of MD resistant and susceptible chicken lines to identify genomic regions under selection for MD incidence, (2) chromatin immunoprecipitation followed by sequencing (ChIP seq) to identify MDV Meq and chicken c-Jun binding sites, (3) gene expression profiling between cell lines to identify genes and pathways regulated by MDV Meq, and (4) allele-specific expression screens by RNA sequencing to identify genes with differential allele response to MDV infection have identified 97 high-confidence candidate genes that are directly regulated by MDV Meq and help explain differences in genetic resistance to MD. If confirmed, these genes and their associated genetic markers would be ideal candidate for genomic selection. Genetics Effect on Vaccine Efficacy. Since their invention, vaccines have proven to be the most effective and economical method to combat infectious diseases in humans as well as in livestock. Efforts to improve vaccine protective efficiency have continued and expanded. Host genetics differences were investigated for the influence on MD vaccine efficacy using unique genetic lines of chickens. Our data suggests that host genetics play an important role influencing MD vaccine protection efficiency. Continuous analyses of our research data further suggested that different genetic lines of chickens respond to the same one vaccine with different protective efficiency. 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. Thus, we predict the best way to reduce the chronic problem of MD incidence in commercial chickens is to rotate the placement of MHC haplotypes similar to the simple method of crop rotation used to control pests in the field. Incorporation of this method into modern poultry production may greatly reduce future virus evolution resulting in substantial savings to the poultry industry. Marek’s Disease Transient Paralysis (TP). A neurological disorder associated with MDV infection. Highly pathogenic strains of MDV are capable of inducing TP even in the resistant line 6-3. This year, we investigated the mechanism of TP in both resistant and susceptible chicken lines inoculated with a vv+ strain of MDV. Gene expression profiling indicated that IL-1', Il 4, and IL-8 were down regulated in the brain tissues of birds from line 7-2 exhibiting TP symptom. Vaccination prior to challenge prevented the suppression of these cytokines. IL-10, an anti-inflammatory cytokine was significantly up-regulated in the infected line 7-2 birds with or without TP. This strong activity of IL-10 had resulted in severe suppression of MHCII transcripts. Over all immunity measured in the brain tissues of birds from both resistant and susceptible lines with and without TP indicated that the immune response to MDV infection is much more vigorous in line 6-3 than line 7-2. Differential expression of immune-related gene provides insight into possible modulation of the immune system toward an effective T cell mediated immune response against MDV infection using cytokine and chemokines as genetic adjuvant. This information is important for understanding the pathogenesis of TP. Surveys and Pathotyping. In our attempts to survey field flocks for MDV of unusual pathogenicity, we have received blood samples from Rispens-vaccinated layer and broiler breeder flocks experiencing high Marek’s disease mortality in Pennsylvania and Iowa. Two Pennsylvania virus isolates from 2010 pathotyped as v and vv+MDV and interestingly shared a specific mutation in the MDV pp38 gene similar to Pennsylvania isolates from 2007 and 2009. Although the pathotyped strains were not unusually virulent, this unique mutation in combination with problems in several surrounding flocks indicate there may be a mutated MDV strain circulating in Pennsylvania. MD remains a problem in this area and we have recently isolated virus from additional layer flocks in this same area of Pennsylvania in 2011. In addition 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. This study will determine if the mortality in the affected flocks can be attributed to virus evolution and the presence of the virus mutation in the pp38 gene may be useful for understanding the epidemiology of mutated virus strains. Vaccines. Although deletion of Meq gene of MDV rendered the virus non oncogenic and was shown through experimental and field trials to be an efficacious vaccine, it still induces lymphoid organ atrophy like that of the parental virus, rMd5, in maternal antibody negative chickens. This year, we developed a method to rid this most effective vaccine against MD from a serious side effect, namely immunosuppression. We have generated 80 cell culture passages of rMd5' meq viruses and found no significant lymphoid organ atrophy beginning at 35th passage onward when compared with un-inoculated control chickens; this development will assist vaccine manufacturers to proceed with their plans for commercializing the vaccine. In other experiments, we also found that the ability of a virus to induce thymic atrophy directly correlated with the virus’s capacity to replicate to high titers in the thymus, suggesting that ability of MDV to induce tumors and disease is separate from its ability to induce atrophy. Avian Leukosis. Chickens from Avian Disease and Oncology Laboratory (ADOL) line alv6 that is known to be resistant to infection with subgroups A and E avian leukosis virus (ALV) were vaccinated at hatch with a trivalent MD vaccine containing serotypes 1, 2 and 3 MD viruses, and were maintained under specific-pathogen free (SPF) conditions from the day of hatch until 56 weeks of age. Lymphoid leukosis tumors were detected in several chickens that died after 20 weeks of age. Chickens tested negative for a

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