Location: Endemic Poultry Viral Diseases Research
2023 Annual Report
Objectives
1. Elucidate host-pathogen interactions of avian reovirus and develop veterinary countermeasures to detect, prevent and control the poultry production viral disease.
1.1. Identify reovirus determinants of virulence associated with arthritis and tenosynovitis in poultry production.
1.2. Develop new diagnostic platforms for the early detection of avian reoviruses on poultry farms.
1.3. Develop new vaccine strategies to prevent and control avian reoviruses on poultry farms.
2. Elucidate host-pathogen interactions of infectious bursal disease virus and develop veterinary medical countermeasures to detect, prevent, and control the poultry production viral disease.
2.1. Apply systems biology approaches to characterize host-pathogen interactions associated with infectious bursal disease virus strain variation, immunosuppression, and pathogenesis.
2.2. Develop genomics and immune intervention strategies to prevent and control infectious bursal disease virus (IBDV), including emerging very virulent and variant IBDV strains.
3. Elucidate host-pathogen interactions of polymicrobial infections in broiler chicken production.[C4, PS4B].
3.1. Characterize immune responses to single or mixed infections with pathogens that are relevant to broiler production. (Auburn and EPVDRU).
3.2. Determine the pathological and physiological correlates of immunosuppression, including microbiota and metabolome, subsequent to single or mixed pathogen infection during broiler production. (Auburn and EPVDRU).
3.3. Evaluate control strategies, with an emphasis on providing protection across a wide variety of antigenically diverse pathogens. (Auburn and EPVDRU).
Approach
Avian reovirus (ARV) and infectious bursal disease virus (IBDV) are economically important pathogens of poultry that are endemic in the U.S. and threaten poultry production. ARVs cause viral arthritis syndrome/tenosynovitis in young chickens and turkeys, but the full extent of clinical disease is unclear. IBDV-infected flocks have high mortality, poor feed conversion ratio, and decreased meat production. There are major knowledge gaps for both viruses with respect to tools for control and prevention, as well as a lack of basic knowledge of the viral pathogenicity and host immune response address these gaps, our research on ARV will focus upon 1) developing an ARV whole-genome sequence database and an antigenic cross-reactivity database to be used for antigenic cartography and vaccine development; 2) exploring the ability of ARVs to suppress the host antiviral innate immune response; and 3) exploring the use of herpesvirus of turkeys (HVT) and Newcastle disease virus (NDV) as delivery vectors for multivalent ARV sigma C antigen-based vaccines. For IBDV, we plan to 1) develop a reverse genetic system to investigate the role of IBDV virus protein 2 (VP2) gene in virulence determination; 2) create an IBDV disease/challenge model to study disease pathogenesis and vaccine protective efficacy; 3) investigate host innate immunity and genetic resistance to IBDV; and, finally, 4) develop NDV vectored in ovo dual vaccines against NDV and IBDV. The outcome of this project will include 1) basic knowledge of the viral pathogenicity and innate immunity against ARV and IBDV; 2) knowledge to guide producers in breeding IBD-resistant chicken lines; 3) disease models to assist in pathogenesis studies and vaccine evaluation; and 4) new ARV and IBDV vaccines to benefit the poultry meat and egg production industries and the American consumer.
Progress Report
To achieve the research goals in Sub-objective 1.A-C, ARS researchers in Athens, Georgia, received avian reovirus (ARV) isolates from two different collaborator laboratories and established favorable and economically viable conditions for the growth, isolation, and propagation of ARVs. Over a dozen ARV isolates have been sequenced. Variants of the ARV sigma A protein have been identified and placed in expression vectors for testing in the type I interferon production assay. From some of these ARV isolates, ribonucleic acid (RNA) was isolated, and polymerase chain reaction (PCR) was optimized for amplifying individual genes of the virus. All ten virus genes were amplified and ready to clone into a plasmid vector for sequencing and transfection.
In support of Sub-objective 2.1.A., obtained two different infectious bursal disease virus (IBDV) isolates from California. These two isolates were propagated in chicken embryos. The viral ribonucleic acid (RNA) was isolated, and polymerase chain reaction (PCR) was optimized for amplifying individual genes of the virus. The viral genes of the IBDV isolates were amplified, cloned into a pGEMT easy vector, and confirmed by sequencing.
To accomplish the research goals in Sub-objective 2.1.B., 2.1.C., and 2.2.A., investigated the chicken genetic resistance to infectious bursal disease virus (IBDV) infection and analyzed the immune response of certain inbred chicken lines during infection with the classical IBDV strain STC. The results showed differences in immune response based on the dose of virus given, with lower doses yielding varying responses and higher doses causing consistent disease and immune responses. The overall data suggest that genetic resistance to the STC strain of IBDV is multifactorial, with the involvement of several factors in the chicken’s immune system as well as the dose of virus determining the overall immune response.
In support of Sub-objective 1.3.B. and 2.2.B., developed the Newcastle disease virus (NDV) TS09 strain as an in ovo vaccine vector to deliver antigenic proteins of avian reovirus (ARV) and infectious bursal disease virus (IBDV) as dual or multivalent vaccine candidates. The viral protein 2 (VP2) gene of an IBDV field strain was amplified and cloned into the TS09 vector. The resulting TS09/IBDV-VP2 plasmid was used to rescue an infectious TS09/IBDV-VP2 recombinant virus using reverse genetic technology. This rescued virus is ready for evaluation of its safety and suitability as an in ovo dual vaccine candidate.
Accomplishments
1. Attenuation of the Newcastle disease virus LaSota vaccine by codon pair deoptimization (CPD) of its virulence genes for in ovo vaccination. 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 the NDV LaSota (LS) vaccine strain for in ovo vaccination, ARS researchers in Athens, Georgia, deoptimized the codon usage of the LS vaccine virus fusion (F) and hemagglutinin-neuraminidase (HN) genes with approximately 44% suboptimal codon substitutions. Three NDV LS recombinant viruses vectoring codon deoptimized F, HN, or both genes were generated using reverse genetics technology. Biological assays and in ovo vaccination experiments showed that the CPD of the virulence genes of the LS strain at the current level of suboptimal codon substitutions could not sufficiently attenuate the virus for in ovo vaccine. Deoptimizing a greater proportion of the F and HN genes or additional gene(s) may be required to attenuate the LS strain adequately.
2. Rapid development of a reverse genetics system for distinct Newcastle disease virus genotypes. In the late 1990s, a new molecular technique, reverse genetics system (RGS), was developed to genetically modify the Newcastle disease virus (NDV) genome for vectored vaccines and cancer therapy. This RGS technology is involved in cloning a complete error-free genome of NDV into a plasmid vector, which is difficult and time-consuming due to the complexity and length of the NDV genome. To improve and simplify the NDV cloning procedure, ARS researchers in Athens, Georgia, in collaboration with scientists at Henan University of Science and Technology, China, developed a two-step ligation-independent cloning (LIC) method to assemble a full-length clone of different genotype NDV strains and rescue an infectious NDV within three weeks. This two-step LIC approach significantly reduced the number of cloning steps and saved researchers substantial time constructing NDV infectious clones. This novel cloning approach may apply to the rapid development of NDV-vectored vaccines against emerging animal diseases and the generation of different genotypes of recombinant NDVs for cancer therapy.
Review Publications
Eldemery, F., Ou, C., Kim, T.N., Spatz, S.J., Dunn, J.R., Silva, R.F., Yu, Q. 2022. Evaluation of Newcastle disease virus LaSota strain attenuated by codon pair deoptimization of the HN and F genes for in ovo vaccination. Veterinary Microbiology. 277:109625. https://doi.org/10.1016/j.vetmic.2022.109625.
Hearn, C.J., Cheng, H.H. 2023. Contribution of the TCR beta repertoire to Marek's disease genetic resistance in chicken. Viruses. 15(3):607. https://doi.org/10.3390/v15030607.
Mohanty, S.K., Donnelly, B., Temple, H., Mowery, S., Poling, H.M., Meller, J., Malik, A., Mcneal, M., Tiao, G. 2022. Rhesus rotavirus receptor-binding site affects high mobility group box 1 release, altering the pathogenesis of experimental biliary atresia. Hepatology Communications. 6(10):2702-2714. https://doi.org/10.1002/hep4.2024.
Yu, Z., Zhang, Y., Li, Z., Yu, Q., Jia, Y., Yu, C., Chen, J., Chen, S., He, L. 2023. Rapid construction of a reverse genetics rescue system for distinct Newcastle disease virus genotypes. Frontiers in Veterinary Science. 10. Article 1178801. https://doi.org/10.3389/fvets.2023.1178801.
