Location: Endemic Poultry Viral Diseases Research
2019 Annual Report
Objectives
Objective 1: Characterize the evolution of avian tumor viruses in poultry production systems, including characterizing the effect of vaccination on the evolution of Marek’s disease virus field strains.
Sub-objective 1.1: Characterize the effect of vaccination on the evolution of Marek’s disease virus field strains.
Sub-objective 1.2: Surveillance for virulent strains of avian tumor viruses in field flocks and development of improved diagnostics for new strains.
Objective 2: Identify host-pathogen interactions that drive the transmission of avian herpesviruses, including identifying viral determinants that drive transmission and determining host genetic effects on virus transmission.
Sub-objective 2.1: Host and virus gene expression patterns in the skin cells facilitate production of cell-free enveloped infectious virus particles.
Sub-objective 2.2: Determine host genetic effect on virus transmission.
Objective 3: Elucidate the genetic and biological mechanisms that drive Marek’s disease resistance, including characterizing and defining innate defense mechanisms that contribute to Marek’s disease resistance.
Sub-objective 3.1: Role of the innate defense mechanisms that drive Marek’s disease resistance, including defining and characterizing innate defense mechanisms that contribute to Marek’s disease resistance.
Sub-objective 3.2: Define innate defense mechanisms that contribute to Marek’s disease vaccinal synergy.
Objective 4: Discover safe and highly effective vaccine platforms that convey protection against avian herpesviruses, including developing a vaccine strain of Marek’s disease virus serotype 1 that is cell free and does not require liquid nitrogen for storage and shipment, and discovering novel Infectious laryngotracheitis virus (ILTV) vaccine platforms that are safe, efficacious, and cost-effective.
Sub-objective 4.1: Develop cell-free Marek’s disease vaccine.
Sub-objective 4.2: Generate novel infectious laryngotracheitis virus vaccines.
Approach
Marek’s disease (MD) and infectious laryngotracheitis (ILT) are agronomically-important diseases of chickens caused by two alphaherpesviruses, Marek’s disease virus (MDV) and infectious laryngotracheitis virus (ILTV), respectively. Although chickens have been vaccinated against these diseases for decades and though highly successful, the vaccines fail to protect against reinfection and transmission. One significant consequence has been the evolution of more virulent MDV field strains in MD-vaccinated flocks. This vicious cycle of virus evolution followed by introduction of new expensive vaccines is not sustainable in the large, expanding, and highly concentrated chicken meat and egg industries. Another shortcoming of MD vaccination is the requirement for storage and transportation of viable vaccine virus in liquid nitrogen. These vaccines are prone to breaks in vaccine control due to improper handling and have restricted usage on a global basis due to the limits of cold chain processes in developing countries. Since current vaccines fail to induce complete immunity, we plan on investigate the role of innate immunity in preventing MDV infection, identify host and virus determinants involved in transmission that undoubtedly play a role in virus evolution, and define the mechanism by which MDV vaccine strains act synergistically in protective immunity. ILTV vaccines are also imperfect and recent research suggests that not only can they revert to virulence by simple bird-to-bird transmission, but also vaccine strains can recombine to generate new virulent strains. There is a need to engineer better modified-live ILT vaccines incapable of reversion to virulence and subunit vaccines incapable of recombination.
Progress Report
Significant progress has been made to assess how vaccination with a leaky vaccine affects pathogen transmission and subsequent disease development in infected contact individuals. We used a shedder-sentinel challenge model to determine when, how much, and how long Marek's disease virus (MDV) was transmitted. We performed 16 biological replicates with shedder birds that were unvaccinated or herepsvirus of turkey (HVT) vaccinated, then challenged with MDV. After challenge, shedder birds were transferred to new isolators of naïve sentinel birds on days 13 and 20. Sentinel birds were monitored for 8 weeks and necropsied to determine if they developed Marek's disease (MD). Each shedder bird was sampled at each transfer and sentinel birds were bled and feathers collected at 14 days post-exposure to shedder birds. Shedder vaccination did not block transmission, but dramatically reduced the negative impacts of infection in sentinels. Infected sentinels were much less likely to show visible disease symptoms at necropsy after contact with vaccinated (232 out of 437 sentinels; 53%) than sham-vaccinated (558 out of 569; 98%) shedders. Development of disease symptoms in infected sentinels was also more likely in the 20 DPI than 13 DPI contact groups (p < 0.0001), but this effect was smaller when shedders were sham-vaccinated (p < 0.05).
Significant progress has been made in our quest to generate recombinant infectious laryngotracheitis viruses (ILTV) using non mammalian (e.g. bacteria and yeast). Initially we had great success in generating ILTV recombinants using a collection of cosmids and hybrid yeast/E.coli constructs [yeast centromere plasmids (ycp) and bacterial artificial chromosomes (Bac) containing various fragments of the ILTV genome. Addition of these constructs reconstituted the virus upon transfection of LMH cells and generate a platform for the creation of multivalent vaccines. However, it appears that some of the large vectors containing ILTV fragments are genetically unstable in E. coli. DNA sequencing of these recombinant vectors have indicated that those containing the origins of replication (OriL and OriS) in cosmid 34 and cosmid 52/ModKLO, respectively, contain large deletions. To correct for this, we have repaired the deleted regions using recombineering in yeast. Any attempt to propagate the yeast-repaired constructs in three genetically stable strains of E. coli (DH10Beta, Top10 and Scarab 6787) resulted in the generation of various origin of replication deletion mutants. Although this was unfortunate, it was not surprising since it has been long reported that large palindromic sequences, such as those of the origins of replication within the ILTV genomes, are unstable in bacteria. Therefore, future manipulations of these constructs which are instrumental in order to generate ILTV recombinants, will solely involve genetic engineering in yeast.
Vaccine efficacy studies of a non-oncogenic Gallid herpesvirus 3 (GaHV-3) strain known as 301B/1, which was reconstituted from a recombinant bacterial artificial chromosome (Bac), showed significant protection against a very virulent Marek’s disease (strain Md5) challenge. Comparably, the reconstituted recombinant was just as protective as the wild-type non recombinant GaHV-3 301B/1 virus against Marek’s disease virus challenge. Since foreign DNA sequences necessary for propagation in bacteria were still present in the reconstituted virus and might affect the vaccine performance, the 301/B BAC was reengineered to contain genetic elements (FRT sites) facilitating the removal of the foreign DNA sequences with FLP recombinases. It is hoped that removal of these sequence will increase the protective index of the GaHV-3 301B/1 BAC vaccine.
Accomplishments
1. The role of vaccination on transmission of Marek's disease virus in poultry. To assess how vaccination with a leaky vaccine affects pathogen transmission and subsequent disease development, ARS researchers in East Lansing, Michigan, used a shedder-sentinel challenge model to determine when, how much, and how long Marek's disease virus (MDV) was transmitted. Shedder chickens were unvaccinated or herpesvirus turkey (HVT) vaccinated, then challenged with MDV and transferred to new isolators of naïve sentinel birds on days 13 and 20. Sentinel birds were monitored for 8 weeks and necropsied to determine if they developed Marek's disease (MD). Shedder vaccination did not block transmission, but dramatically reduced the negative impacts of infection in sentinels. Infected sentinels were much less likely to show visible disease symptoms at necropsy after contact with vaccinated than sham-vaccinated shedders. The results revealed that shedder vaccination did not block infection of unvaccinated sentinel birds, but a reduction in virus exposure dose with shedder vaccination lessened the negative impacts on sentinels. Current vaccines against Marek’s disease, although protect against clinical signs and tumor formation, do not protect against reinfection and shedding. Vaccination protocols that reduce shedding should dampen the capacity of field viruses to evolve to greater levels of virulence.
2. The generation of a battery of recombinant clones containing large fragments of the ILTV genome with intact origins of replication. Previously, four overlapping cosmid clones and a yeast centromere plasmid (ycp) clone that contain large fragments of the infectious laryngotracheitis virus (ILTV) genome were generated and upon reconstitution in LMH cells, viable virus was achieved. However, these clones were shown to be genetically unstable in E. coli. ARS researchers in Athens, Georgia, re-engineered all recombinant clones needed to reconstitute ILTV in yeast to contain intact origins of replication. In doing so, two large recombinants were created containing a partial ILTV genome (135 kB) and the complete genome. These two vectors can be easily manipulated in yeast to generate vaccine strains as well as vaccines containing multiple antigens. Since current modified live vaccines used to control infectious laryngotracheitis virus (ILTV) can revert to virulence there is a need to develop a molecular clone of ILTV for the production of vaccine seed stocks.
3. The generation of a vaccine platform for Marek’s disease and other avian pathogens using the non-oncogenic avian herpesvirus Gallid herpesvirus 3 (GaHV-3). To develop an efficacious vaccine, the complete genome of Gallid herpesvirus 3 (GaHV-3) strain 301B/1 was successfully cloned by ARS researchers in Athens, Georgia, into a bacterial artificial chromosome (Bac) plasmid. The protective efficacy of the reconstituted 301B/1 virus was evaluated in birds following very virulent Marek’s disease virus challenge and shown to have a comparable protective index to that of wild-type non recombinant 301B/1 virus. This BAC clone and the recombineering technology developed will provide the capability to create efficacious vector vaccines against Marek’s disease and other important poultry diseases.
4. The generation of a cell-free vectored vaccine against Marek’s disease. In order to generate a Marek’s disease vaccine that does not require manufacturing, storage and shipping in liquid nitrogen, the genes encoding the major antigens of Gallid herpesvirus 2 (GaHV-2) were cloned by ARS researchers in Athens, Georgia, into an infectious clone of an avian virus. Recombinant viruses were generated containing different GaHV-2 antigens and evaluated in protective efficacy studies with virulent GaHV-2 challenge. One vaccine candidate was selected which possessed the necessary characteristics for further examinations. Over the last 4 decades Marek’s disease (MD) vaccines have been very successful in the control MD, however they are expensive. It is estimated that utilization of a cell-free MD vaccine would save an estimated 73 million USD annually.
Review Publications
Dar, M.A., Urwat, U., Ahmad, S.M., Ahemd, R., Kushoo, Z.A., Dar, T.A., Shah, R.A., Heidari, M. 2018. Gene expression and antibody response in chicken against Salmonella Typhimurium challenge. Poultry Science. 98(5):2008-2013. https://doi.org/10.3382/ps/pey560.
Spatz, S.J., Garcia, M., Riblet, S., Ross, T.A., Volkening, J.D., Taylor, T.L., Kim, T.N., Afonso, C.L. 2019. MinION sequencing to genotype US strains of Infectious Laryngotracheitis Virus. Avian Pathology. 48(3):255-269. https://doi.org/10.1080/03079457.2019.1579298.
Kim, T.N., Hunt, H.D., Parcells, M.S., Van Santen, V., Ewald, S.J. 2018. Two class I genes of the chicken MHC have different functions: BF1 is recognized by NK cells while BF2 is recognized by CTLs. Immunogenetics. 70(9):599-611. https://doi.org/10.1007/s00251-018-1066-2.
Loncoman, C.A., Hartley, C.A., Coppo, M.C., Vaz, P.K., Diaz-Mendez, A., Browning, G.F., Garcia, M., Spatz, S.J., Devlin, J.M. 2017. Genetic diversity of infectious laryngotracheitis virus during in vivo coinfection parallels viral replication and arises from recombination hot spots within the genome. Applied and Environmental Microbiology. 83(23):e01532-17. https://doi.org/10.1128/AEM.01532-17.
Dunn, J.R., Black Pyrkosz, A., Steep, A., Cheng, H.H. 2019. Identification of Marek’s disease virus genes associated with virulence of US strains. Journal of General Virology. 100(7):1132-1139. https://doi.org/10.1099/jgv.0.001288.
Dunn, J.R., Dimitrov, K.M., Miller, P.J., Garcia, M., Turner-Alston, K., Brown, A., Hartman, A. 2018. Evaluation of protective efficacy when combining HVT vector vaccines. Avian Diseases. 63(1):75-83. https://doi.org/10.1637/11979-092818-Reg.1.
