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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

2020 Annual Report

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.

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
In Objective 1, we made significant progress in determining the influence of imperfect vaccines and dilution on transmission and evolution to higher virulence. Marek's disease virus (MDV) was 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 transmit 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). Following back-passages, the phenotype of the starting virus was compared with virus isolates stored following five passages to determine the effect of vaccine dilution. In Objective 2, virus transmission studies, we aimed to determine to what extent poultry host genetics affects pathogen transmission and subsequent disease development in infected contact individuals. We repeated in vivo experiments comparing virus transmission from MD-susceptible (Line 7) or MD-resistant (Line 6) shedder birds challenged with MDV. In addition, we also included a highly resistant commercial layer chicken in our studies. Shedder birds were transferred to new isolators of naïve recipient birds on days 10, 12, 14, 16, 18, and 20. Recipient birds were monitored for eight weeks and necropsied to determine if they developed Marek's disease. Each donor bird was sampled weekly for eight weeks and at each transfer, and each recipient bird was bled at 14 days post-exposure to donor birds. Results demonstrated higher virus load in feathers of susceptible chickens, with minimal downstream effect in contact birds. In another part of Objective 2, defining the proteome or "protein profile" of cells infected with Marek's disease virus, we aimed to determine whether there are differences in the types of proteins and their abundance in infected cultured cells, where the production of cell-free virions is severely restricted, versus infected feather follicle epithelium, where cell-free virions are released as dander from infected chicken. It has been demonstrated by sequencing the genome of a transmission incompetent strain of MDV1 that two genes encoding an attachment receptor, glycoprotein C, and one encoding a regulatory protein, protein kinase UL13, are essential for the virus's ability to infect other chickens. By repairing mutations within these genes, restoration of transmission was demonstrated. To further understand the role of the UL13 protein kinase in transmission, additional mutations were introduced within the MDV1 genome. A single amino acid residue, the invariant lysine 170 (K170), was identified to be essential for the spread of the virus. These newly generated viruses allowed us to define better the cellular pathways necessary for the transmission of Marek's disease virus among chickens. These results suggest that proteins expressed during the late phase of virus infection are severely affected in cells (feather follicles), which are the sites for virus release. Overall, the UL13 protein kinase of MDV1 is not required to infect neighboring cells, nor is it essential for disease induction and cancer; however, it is necessary for interindividual spread. These results are significant to not only herpesviruses that infect chickens, but other animal and human herpesviruses since UL13 orthologs are conserved in all herpesvirus subfamilies (alpha, beta, and gamma). In Objective 3, utilization of the recently created bacterial artificial chromosome (BAC) clone of a strain of Gallid alphaherpesvirus (GaHV-3) 301B/1, we made significant progress in constructing a Marek's disease vaccine candidate that expresses a critical regulatory protein (IL-15) essential in innate immunity against Marek's disease virus. It has been shown that the cytokine interleukin 15 (IL-15), which is produced by many types of immune cells (i.e., dendritic cells, monocytes, and macrophages), stimulates cells important in cell-mediated immunity. This cytokine is necessary to maintain a special kind of T cells (CD8+ T cells) involved in immunological memory and activating (NK) cells to seek out kill cells infected with Marek's disease virus. The chicken IL-15 gene was inserted into the 301B-clone within a nonessential locus using standard genetic engineering techniques. Recombinant 301B virus containing the IL-15 gene was successfully reconstituted from cells transfected with the modified 301B-BAC DNA. Furthermore, the expression of the inserted IL-15 gene was verified by indirect immunofluorescent assay (IFA) using an anti-chicken IL-15 monoclonal antibody. To reduce the potential systemic toxicity of IL-15, the recombinant protein was engineered to contain a membrane anchor for cell surface expression, rather than secretion. The retargeted cell surface expression of IL-15 was also confirmed using an IFA. Vaccine efficacy studies of recombinant 301B-IL-15 viruses expressing membrane-bound and secreted IL-15 isoforms are planned for the 4th quarter of 2020. In Objective 4, a 301B-BAC clone containing the entire GaHV-3 genome was modified to express the multiple glycoprotein antigens, glycoprotein D (gD), glycoprotein I (gI) and partial glycoprotein E (gE) of infectious laryngotracheitis virus (ILTV), an important avian respiratory pathogen. The ILTV glycoprotein genes were inserted into the 301B genome using BAC recombineering. Several recombinant 301B/1 viruses with ILTV gD, gI, partial gE were successfully reconstituted from the modified 301B-BAC clones. The expression of inserted ILTV glycoproteins in recombinant 301B-BAC-ILT virus-infected chicken embryo fibroblasts was confirmed by IFA using chicken sera specific for ILTV antigens.

1. The role of host genetics on the transmission of Marek's disease virus (MDV) in poultry. To assess how host genetics 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 MDV was transmitted. Shedder birds were from 2 laboratory chicken lines and 1 commercial layer line of differing MD susceptibility. After challenge with MDV, shedder birds were exposed to naïve sentinel birds between days 10-20 post-infection. Laboratory MD-susceptible chickens had higher virus load in feathers compared to MD-resistant shedder chickens, but minimal downstream effect in sentinel chickens. The commercial layer line had significantly better survival for shedder chickens compared to the laboratory lines following challenge, but there was no correlation with feather virus load in shedders. Only marginal differences were seen for sentinel feather virus load, infection status, disease status, and survival at each time point based on host genetics of the three shedder chicken lines. The results revealed that host genetics is not nearly as significant compared to vaccination on the reduction of virus transmission and the resulting effect of disease symptoms in naïve unvaccinated sentinel chickens.

2. Newcastle disease virus (NDV) LaSota strain-based recombinant virus expressing the glycoprotein B (gB) of MDV as a dual vaccine (rLS/MDV-gB) against Marek's disease (MD) and Newcastle disease (ND). To overcome the costs and problems associated with the storage and shipping of commonly used MD vaccines in liquid nitrogen, a novel MDV vaccine platform was developed by ARS researchers in Athens, Georgia, using reverse genetics technology. The NDV LaSota strain, a naturally-occurring low virulence NDV strain, is routinely used as a live vaccine to prevent Newcastle disease. This vaccine induces protective immunity both locally and systemically and can be readily administered through drinking water supplies or by directly spraying the birds. Furthermore, the LaSota vaccine is completely safe and stable, and there are no reports of virulence reversion or recombination in the field to generate new virulent strains. This NDV recombinant vaccine expressing the antigen gB of Marek's disease virus represents a bivalent vaccine that provides dual protection against MDV and NDV. More importantly, the NDV vectored recombinant vaccine has been proven to be safe and stable. It can be readily administered en mass without requiring expensive cold-chain for the storage and transportation as required for current commercial Marek's disease vaccines. This new vaccine will directly impact the worldwide poultry industry and make their products (meat and eggs) even more inexpensive to the consumers.

3. Cloning the complete genome of infectious laryngotracheitis virus (ILTV). The modified live vaccines against infectious laryngotracheitis are protective and can reduce virus shedding. However, these vaccines can revert to virulence and infect unvaccinated birds. To prevent this, ARS researchers in Athens, Georgia, sought to develop a molecular clone of ILTV. Previous research developed a two-vector system to reconstitute ILTV, which can be manipulated in vitro to generate vaccine strains as well as vaccines containing multiple antigens. The new research has created a single infectious clone containing the complete genome of ILTV. The new clone will facilitate the rapid generation of recombinants for pathogenicity and gene function studies.

4. Development of an efficacious vector vaccine platform against multiple poultry diseases. Marek's disease vaccines have recently been used as vectors that include genes from other poultry viruses that allow protection against multiple poultry pathogens. However, mixing multiple vectors can lead to vaccine interference. ARS researchers in Athens, Georgia, developed a new vector vaccine candidate based on a vaccine platform of another avian pathogen, infectious laryngotracheitis virus (ILTV). This combined vaccine contains transgenes that were verified in a recombinant virus-infected chicken embryo fibroblasts (CEF). The genetic stability of the transgenes in the viral recombinants was also confirmed during seven in vitro blind passages in CEF cultures. The results indicate that our vaccine platform is ideal for the expression of multiple antigens of poultry pathogen and other immunostimulatory genes, which, under certain situations, may be necessary to boost vaccine efficacy.

Review Publications
Umthong, S., Dunn, J.R., Cheng, H.H. 2019. Towards a mechanistic understanding of the synergistic response induced by bivalent Marek’s disease vaccines to prevent lymphomas. Vaccine. 37(43):6397-6404.
Kim, T.N., Spatz, S.J., Dunn, J.R. 2020. Vaccinal efficacy of molecularly cloned Gallid alphaherpesvirus 3 strain 301B/1 against very virulent Marek’s disease virus challenge. Journal of General Virology. 101(5):542-552.
Kim, T.N., Volkening, J.D., Spatz, S.J. 2020. Comparative molecular characterization of three Gallid alphaherpesvirus type 3 strains 301B/1, HPRS24, and SB-1. Avian Diseases. 64(2):174-182.
Yu, Q., Li, Y., Dimitrov, K., Afonso, C.L., Spatz, S.J., Zsak, L. 2020. Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos. Vaccine. 38(4):925-932.
Bailey, R.I., Cheng, H.H., Chase-Topping, M., Mays, J.K., Anacleto, O., Dunn, J.R., Doeschl-Wilson, A. 2020. Pathogen transmission from vaccinated hosts can cause dose-dependent reduction in virulence. PLoS Biology. 18(3):e3000619.
Sadigh, Y., Tahiri-Alaoui, A., Spatz, S.J., Nair, V., Ribeca, P. 2020. Pervasive differential splicing in Marek’s Disease Virus can discriminate CVI-988 vaccine strain from RB-1B virulent strain in chicken embryonic fibroblasts. Viruses. 12(3), 329.