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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Research Project #432152

Research Project: Genetic and Biological Determinants of Avian Herpesviruses Pathogenicity, Transmission, and Evolution to Inform the Development of Effective Control Strategies

Location: Endemic Poultry Viral Diseases Research

2021 Annual Report

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. 1.1. Characterize the effect of vaccination on the evolution of Marek’s disease virus field strains. 1.2. Surveillance for virulent strains of avian tumor viruses in field flocks and development of improved diagnostics for new strains. 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. 2.1. Host and virus gene expression patterns in the skin cells facilitate production of cell-free enveloped infectious virus particles. 2.2. Determine host genetic effect on virus transmission. 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. 3.1. Define innate defense mechanisms that contribute to Marek’s disease vaccinal synergy. 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. 4.1. Develop cell-free Marek’s disease vaccine. 4.2. Generate novel infectious laryngotracheitis virus vaccines.

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
This is the final report for this project which will be replaced by a bridging project pending the completion of the research review. In Objective 1.1, ARS researchers in Athens, Georgia, and East Lansing, Michigan, evaluated the effects of imperfect vaccines and vaccine dilution on Marek’s disease virus (MDV) evolution. Progress was made in analyzing samples generated from virus back-passaged naturally through 5 successive generations. Each group consisted of 10 birds kept in an individual isolator and replicated at least three times to provide sufficient power for the statistical analyses. For each bird, feathers were sampled at least three different times to determine viral load and shedding, as well as the ability for viral genomic sequencing. To get information on viral replication and transmission, shedder (donor) birds that transmit infectious virions needed to be sampled before, at, and following co-housing with the contact (recipient) birds. Birds infected in Passage 1 transmitted the virus to recipients in Passage 2, and so on. At hatch, birds were either fully vaccinated (V) or vaccinated at 1:10 dilution (1:10V). The experimental design did not result in the successful recovery of the virus after five back-passages. In Objective 1.2, the ARS researchers continued surveillance of Marek’s disease virus with unusually high pathogenicity. Multiple samples from outbreaks were received and evaluated for diagnosis and virus isolation. Results were shared with submitting laboratories to aid in disease control. The ARS scientists were successful in isolating the virus and developed working stocks in preparation for pathotyping assay in chickens. In Objective 2, virus transmission studies, research was directed at determining to what extent poultry host genetics affects pathogen transmission and subsequent disease development in infected contact individuals. This year marked the completion of all experiments and analyses. A comparison was made of virus transmission from Marek’s disease (MD) virus-challenged shedder birds that were either MD-susceptible (Line 7), MD-resistant (Line 6), or from a highly resistant commercial layer chicken. There was a highly significant difference between the three donor lines in proportion positive for MD clinical signs, and the MD-susceptible donor line had a higher virus load in feathers at each sampling time point. However, there were no significant differences in survival or disease severity in contact birds based on the shedder host genetics at any time point. In Objective 3, the research focused on defining the transcriptome and proteome of MDV-infected feather follicle epithelium and infected splenocytes and chicken embryo fibroblasts. Using the K170 mutant and wildtype MD virus, the proteomic data suggest subtle differences in the phosphoproteome of infected feather follicle epithelia (FFE) vs. infected cells (i.e., chicken embryo fibroblasts (CEF) and splenocytes) where the production of cell-free virions is severely restricted. In the transcriptomic experiments mapping long reads, it was determined that the majority of the MDV transcripts are polycistronic and group according to their temporal expression. To map these transcripts to the MDV genome, ARS scientists at Athens, Georgia, and East Lansing, Michigan, have recently embarked on studies to determine the prevalence of the four isomers of the class-E genome of MDV. The results suggest that there are at least two isomers in the infected cells. Using DNA purified from MD nucleocapsids and pulse-field gel electrophoresis, the ARS scientists identified bands corresponding to the four isomers of the MDV genomes. Surprisingly, defective interfering (DI) genomes in these purified nucleocapsid DNA preparations have been identified. The significance of the defective genome in the pathobiology of MDV is ongoing. In Objective 4, individual recombinant 301B/1 viruses expressing two differing forms of chicken interleukin -15 (IL-15) protein, one recombinant expressed as a secreted IL-15 protein, and the other expressed as a membrane-anchored form. Both recombinants were successfully propagated in chicken embryo fibroblasts and their virus growth kinetics determined. In addition, the expression of inserted chicken IL-15 gene was visualized using immunocytological staining with IL-15 specific antisera. Vaccine efficacy studies of recombinant 301B/1-IL-15 viruses expressing membrane-bound and secreted IL-15 isoforms are ongoing in the 4th quarter of 2021.

1. Deletion of thymidine kinase from Marek's disease virus (MDV) vaccine candidate. The history of MDV evolution creates the need for improved vaccines. ARS researchers in East Lansing, Michigan, previously developed a vaccine candidate with superior protection compared to commercially available vaccines but with safety issues due to residual virulence in specific-pathogen-free chickens. To improve safety, the thymidine kinase gene was removed from this vaccine candidate, which reduced replication of the live-attenuated vaccine and resulted in the elimination of residual virulence. This change also reduced protection, which suggests that lowering vaccine replication may be an unsuitable method for improving vaccine candidates while maintaining superior protection.

2. Determine host genetic effect on virus transmission. To assess how host genetics affects pathogen transmission and subsequent disease development, ARS researchers in Athens, Georgia, and East Lansing, Michigan, used a shedder-sentinel challenge model to determine when, how much, and how long Marek’s disease virus was transmitted. While selection on genetic resistance to disease symptoms has not yet led to full flock-level protection and is currently outperformed by vaccination, differences between chicken lines in viral shedding combined with partial correlations between genetic resistance to symptoms and feather viral load indicate that joint selection on both traits could be effective. Furthermore, shedder feather viral load proved to be a sufficiently accurate bioindicator of the exposure dose of contact birds, meaning that joint selection could be done quickly and cheaply, without the need for expensive and time-consuming transmission experiments. The threshold exposure dose required to reduce symptoms remains unclear and requires further testing. The ARS researchers suggest that a focus on selecting host genomic variants affecting viral replication and load would represent a rapid and efficient means to increase flock-level protection through promoting both increased resistance and reduced infectivity.

3. To develop an efficacious, multivalent vector vaccine platform against poultry diseases. To develop a vaccine platform in which one platform (a vector) can be used to accommodate antigens that can protect against other poultry pathogens, ARS researchers in East Lansing, Michigan, modified the genome of an MD vaccine strains (301B/1) to express an immunostimulating factor. This factor or cytokine [interleukin-15 (IL-15)] is postulated to increase the immunological potential of the vaccine strain. Experiments have been completed to demonstrate the addition of the cytokine gene did not hamper the recombinant’s replicative capacity in cultured cells. Furthermore, serial passage studies suggest that this recombinant is genetically stable and can be used in downstream experiments to demonstrate its vaccinal efficacy in animal studies.

Review Publications
Chuard, A., Courvoisier-Guyader, K., Remy, S., Spatz, S.J., Denesvre, C., Pasdeloup, D. 2020. The tegument protein pUL47 of Marek’s disease virus is necessary for horizontal transmission and is important for expression of glycoprotein gC. Journal of Virology. 95(2):1-19.
Vega-Rodriguez, W., Xu, H., Ponnuraj, N., Akbar, H., Kim, T.N., Jarosinski, K.W. 2021. The requirement of glycoprotein C (gC) for interindividual spread is a conserved function of gC for avian herpesviruses. Scientific Reports. 11:7753.
Umthong, S., Dunn, J.R., Cheng, H.H. 2020. Depletion of CD8aß+ T cells in chickens demonstrates their involvement in protective immunity towards Marek’s disease with respect to tumor incidence and vaccinal protection. Vaccines. 8(4):557.
Hauck, R., Mays, J.K., Dunn, J.R., Shivaprasad, H.L. 2020. Two cases of Marek's disease in backyard turkeys. Avian Diseases. 64(3):347-351.
Conrad, S.J., Hearn, C.J., Silva, R.F., Dunn, J.R. 2020. Codon deoptimization of UL54 in meq-deleted Marek's disease vaccine candidate eliminates lymphoid atrophy but reduces vaccinal protection. Avian Diseases. 64(3):243-246.