Location: Endemic Poultry Viral Diseases Research
2019 Annual Report
Objectives
1. Characterize the intestinal virome associated with poultry enteric diseases, including assessing the intestinal microbiome of poultry for the presence of novel enteric pathogens, and developing molecular tools to study the epidemiology, ecology, and evolution of enteric pathogens.
2. Investigate the role of the poultry gut microbiome in promoting overall health and performance gains, including defining the interactions between the gut microbiome and the host immune system that contribute to enteric diseases and performance problems and developing the microbiome as a poultry health phenotype.
3. Develop vaccine platforms that will lead to highly efficacious vaccines that have been rationally designed to control enteric diseases of poultry, including developing vaccines targeting specific enteric pathogens early during the poultry production cycle.
Approach
Viral infections of the avian gastrointestinal tract negatively impact poultry production; however, determining the complex etiologies of the viral enteric diseases in poultry has been difficult. Research in our Unit over the past five+ years has focused in part on the characterization of the poultry gut virus community and initial characterizations of novel viruses. The research proposed in Objective 1 will continue and expand upon this line of investigation. As a logical extension of our viral metagenomic work, we have further performed comparative metagenomic analyses of healthy and enteric disease-affected poultry flocks, leading to descriptions of potential disease-associated viruses such as the enteric picornaviruses. Objective 2 again continues and expands upon these investigations, proposing extensive flock comparisons using powerful next-generation sequencing techniques, pathogenesis work with viruses, and defining the immune response of poultry suffering from enteric maladies. Finally, the discovery of disease-associated genes and infectious agents in Objective 2 will directly inform the design of targeted interventions in Objective 3, which will use our established, efficacious recombinant vectored vaccine platforms to produce vaccines targeting enteric viruses early during the poultry production cycle.
Progress Report
Turkey clinical enteric disease caused by turkey coronavirus (TCoV) results in significant economic losses to the turkey industry in the United States and abroad. To date, there is no commercial vaccine available to prevent the disease because TCoV does not readily grow in cell cultures or chicken embryos, which hampers conventional vaccine development. To overcome this barrier to vaccine development, ARS scientists in Athens, Georgia, developed a novel approach by using an enteric Newcastle disease virus (NDV) vaccine as a vector to express TCoV antigens as vaccine candidates. Four NDV vectored TCoV vaccine candidates have been generated. Currently, these vaccine candidates are being evaluated in cell cultures or chicken embryos for their safety, genetic stability, growth kinetics, and the expression of the TCoV antigens.
To evaluate the protective efficacies of TCoV vaccine candidates, a pathogenic TCoV strain in turkeys is required for use as a challenge virus in the vaccination/challenge animal experiment. Because TCoV does not readily grow in cell cultures, ARS scientists in Athens, Georgia, investigated the possibility of using turkey embryos to propagate virulent TCoV. The results showed that the TCoV grew in turkey embryo intestinal tissue to a high titer. Infection of turkey poults with the turkey embryo propagated TCoV can reproduce enteric disease signs, demonstrating the propagated TCoV maintained its virulent pathogenicity. Currently, the turkey embryo propagated TCoV is being used as a challenge virus to evaluate the protective efficacies of NDV vectored TCoV vaccine candidates in turkeys.
Accomplishments
1. Newcastle disease (ND) recombinant expressing the infectious laryngotracheitis virus (ILTV) glycoprotein D(gD) has been demonstrated to confer protection against both virulent NDV and ILTV challenges. However, there is a concern on the genetic stability of the recombinant vaccine. ARS scientists in Athens, Georgia, evaluated the genetic stability of the recombinant vaccine after eight serial passages in embryonated chicken eggs (ECE) to mimic Newcastle disease virus (NDV) vaccine production. Next-generation sequencing analysis of the vaccine master seed and the egg-passaged virus stocks confirmed their genome integrity and revealed a total of thirteen single-nucleotide polymorphisms (SNPs). However, none of these SNPs was located in the ILTV gD insert or any of the known critical biological determinant positions. Virological and immunofluorescent assays provided additional evidence that the egg-passaged virus stocks retained their growth kinetics, low pathogenicity, and robust level of gD expression comparable to that of the vaccine master seed virus. These results demonstrated that the insertion of ILTV gD gene into the NDV LaSota backbone did not significantly affect the genetic stability of the recombinant virus, and that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE.
