Location: Endemic Poultry Viral Diseases Research
2022 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
This project (6040-32000-073-000D) was terminated on 01/02/2022. The new Project Title is: Systems Biology Approaches to Develop Medical Countermeasures to Detect, Prevent, and Control Poultry Production Viral Diseases, with a new Project Number: 6040-32000-084-000D.
Fiscal Year 2022 Progress. In ovo vaccination is an attractive immunization approach for the poultry industry. However, commonly used Newcastle disease virus (NDV) vaccines cannot be administered in ovo because of the reduced hatchability and embryo mortality. To attenuate NDV LaSota (LS) vaccine strain as an in ovo vaccine vector, ARS scientists in Athens, Georgia, manipulated the LS vaccine by codon pair deoptimization (CPD) of the major surface glycoprotein genes, HN and F, using reverse genetics technology. The CPD of the HN gene slightly attenuated the virus, whereas the CPD of the F gene marginally increased the virus pathogenicity compared to the parental LS. Nevertheless, all CPD rLS viruses were still lethal to 10-day-old specific-pathogen-free (SPF) chicken embryos. In ovo injection of 18-day-old SPF chicken embryos with the CPD viruses severely reduced chicken’s hatch and survival rates. These results suggested that the CPD of the HN and F genes of the LS strain at the current level of suboptimal codon substitutions could not sufficiently attenuate the virus for the use as an in ovo vaccine, and codon deoptimizing a greater proportion of the F and HN genes or additional gene(s) may be required for sufficient attenuation of the LS vaccine.
5 Year Summary of the project. Enteric viral infections in poultry have caused a great deal of economic loss in the poultry industry worldwide. Several enteric viruses have been identified from the intestine of poultry either alone or in combination, suggesting the multicausal etiology of this disease. From our archived samples collected from field cases of diarrheic turkeys, ARS researchers in Athens, Georgia, analyzed 3 picornavirus- and 5 coronavirus-positive fecal samples by Illumina MiSeq next generation sequencing (NGS) to determine viral community sequences and to investigate the presence of other concomitant enteric viruses. In the archived samples determined to be positive for picornavirus, metagenomic analysis of sequence reads revealed that 98, 10, and 83% of the virus reads consisted of Picornaviridae virus sequences, among which the most commonly indicated piconaviruses were turkey hepatitis virus (genus Megrivirus) and turkey gallivirus (genus Gallivirus). Other than the picornaviruses, astroviruses were the only other enteric virus reads with 0.69, 49.1, and 12.4%, respectively. In the coronavirus-positive samples, 96, 66, 97, 81, and 85% of the virus reads were identified as coronavirus, with turkey coronavirus comprising the majority of virus sequences and infectious bronchitis virus being a minor constituent. Other known enteric viruses were not indicated in 3 out of 5 coronavirus-positive samples, while a small percentage (less than 7%) of astrovirus and picornaviruses sequence reads were detected in the other 2 samples. NGS and metagenomics analyses in this study demonstrate the diversity of enteric viruses in turkey flocks with enteric problems in the U.S.
Turkey enteric coronavirus (TCoV) causes clinical enteric disease in turkeys, resulting in significant economic losses to the turkey industry in the United States and abroad. To date, no commercial vaccine is available to prevent the disease because TCoV does not readily grow in cell culture, which hampers conventional vaccine development. To overcome this barrier to vaccine development, ARS scientists in Athens, Georgia, developed a novel approach using an enteric Newcastle disease virus (NDV) LMV vaccine as a vector. TCoV spike glycoprotein (S) subunit 1 (S1) and subunit 2 (S2), and nucleocapsid (N) protein genes were cloned into the enteric NDV vaccine vector. Several NDV/TCoV recombinant viruses have been generated using reverse genetics technology. Biological assessments of these recombinant viruses showed that they retained a similar growth ability in cell cultures and were slightly attenuated in chicken embryonated eggs and day-old chickens compared to the LMV vaccine vector. The TCoV S or N protein expression was detected from the virus-infected cells by immune fluorescence assay (IFA).
For evaluation of 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 experiments. ARS researchers in Athens, Georgia, isolated a field strain (NC1743) of TCoV and evaluated its pathogenicity in specific-pathogen-free (SPF) turkey poults. The results showed that this TCoV strain was highly pathogenic and able to reproduce a typical enteric disease in day-old turkey poults with a standard minimal infectious dose. As the age increases, turkey poults became less sensitive to TCoV infection. The overall data suggest that young turkeys infected with the TCoV NC1743 strain could be used as a TCoV disease model to study the disease pathogenesis, and the minimal infectious dose of TCoV NC1743 could be used as a challenge virus to evaluate a vaccine protective efficacy.
Accomplishments
1. Recombinant vaccine against chicken infectious bronchitis. Chicken infectious bronchitis, caused by infectious bronchitis virus (IBV), is a major cause of economic losses in the poultry industry worldwide. Vaccination with a serotype-specific attenuated live IBV vaccine is a common practice to control the disease. However, the IBV vaccine viruses are genetically unstable, and some mutated vaccine subpopulations revert virulence, contributing to the infectious bronchitis outbreaks. To overcome the drawbacks of these commercial vaccines, ARS researchers in Athens, Georgia, in collaboration with the scientists at Auburn University, developed a Newcastle disease virus vaccine strain-based recombinant virus co-expressing the spike protein of Ark-serotype IBV and the granulocyte-macrophage colony-stimulating factor (a chicken cytokine). This recombinant virus was safe for one-day-old chickens and genetically stable after four passages in chicken embryos. Vaccination of chickens with this recombinant vaccine significantly reduced viral load and tracheal lesions after the IBV challenge. Chickens primed with this recombinant vaccine and boosted with the commonly used IBV Mass-type vaccine conferred significant cross-protection against the IBV Ark-type challenge. These results suggest that this recombinant virus is a safe and genetically stable vaccine candidate and can be combined with the live Mass vaccine to improve protection against Ark-type infectious bronchitis.
Review Publications
Zhao, W., Zhang, P., Bai, S., Lv, M., Wang, J., Chen, W., Yu, Q., Wu, J. 2021. Heterologous prime-boost regimens with Ad5 and NDV vectors elicit stronger immune responses to Ebola virus than homologous regimens in mice. Archives of Virology. https://doi.org/10.1007/s00705-021-05234-4.
Kang, K., Day, J.M., Eldemery, F., Yu, Q. 2021. Pathogenic evaluation of a turkey coronavirus isolate (TCoV NC1743) in turkey poults for establishing a TCoV disease model. Veterinary Microbiology. 259:109155. https://doi.org/10.1016/j.vetmic.2021.109155.
Khalid, Z., He, L., Yu, Q., Breedlove, C., Joiner, K., Toro, H. 2021. Enhanced protection by recombinant newcastle disease virus expressing infectious bronchitis virus spike ectodomain and chicken granulocyte-macrophage colony-stimulating factor. Avian Diseases. 65(3):364–372. https://doi.org/10.1637/aviandiseases-D-21-00032.
Dimitrov, K.M., Taylor, T.L., Marcano, V.C., Williams Coplin, T.D., Olivier, T.L., Yu, Q., Gogal Jr., R.M., Suarez, D.L., Afonso, C.L. 2021. Novel recombinant Newcastle disease virus-based in Ovo vaccines bypass maternal immunity to provide full protection from early virulent challenge. Vaccines. 9(10):1189. https://doi.org/10.3390/vaccines9101189.
