Location: Endemic Poultry Viral Diseases Research
2024 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 1.1.A-C, we obtained avian reovirus (ARV) isolates from various collaborating laboratories. We then established optimal and cost-effective conditions for growing, isolating, and propagating the ARVs. Furthermore, we successfully sequenced several ARV isolates. Subsequently, ribonucleic acid (RNA) was extracted from some of these isolates, and polymerase chain reaction (PCR) was fine-tuned to amplify the individual genes of the virus. All ten virus genes were then inserted into a plasmid vector for sequencing and transfection, establishing a reverse genetics system.
In support of Milestone 5, "Generate a recombinant herpes virus of turkey (HVT) containing two or more avian reovirus (ARV) sigma C variants", we have created a recombinant HVT that contains sigma C genes from two strains of ARV. These strains are strain s1133 (a vaccine strain) and strain AVS-HMM, which is a virulent strain of ARV obtained from Milos Markis at AviServe. The biological properties of the HVT/ARV sigma Cs recombinant are currently being evaluated.
To achieve the research goal in Sub-objective 1.3.B, we have developed a bivalent vaccine candidate using an avian paramyxovirus type 1 (APMV-1) TS09 vaccine strain-based recombinant virus containing the sigma C gene of avian reovirus (ARV). We assessed the characteristics of the recombinant virus in day-old chicks and embryonated chicken eggs and verified the expression of the ARV sigma C protein by an immunofluorescent assay. The data indicates that this TS09/ARV-sigma C recombinant is a safe vaccine candidate. It should be further tested through in-ovo vaccination trials to evaluate its protective efficacy against ARV and Newcastle disease virus challenges.
In support of Sub-objective 2.1.A. we isolated viral ribonucleic acid (RNA) from the infectious bursal disease virus (IBDV) rA strain. The polymerase chain reaction (PCR) was optimized to amplify individual genes of IBDV. The PCR product was cloned into a vector, the sequence was confirmed, and the clones were used for transfection to develop the reverse genetics system.
To accomplish the research goals in Sub-objective 2.1.B. and 2.1.C. we utilized their previously designed infectious bursal disease virus (IBDV) challenge model to assess the genetic resistance of Southeast Poultry specific-pathogen-free broiler chickens to the very virulent IBDV strain rA or the standard challenge(STC) strain. The results showed susceptibility to the rA strain of IBDV based on bursal score comparisons to the previous B congenic challenge studies. Results from the STC challenge agreed with previous observations, highlighting the importance of STC virus dose in the disease progression observed post-challenge.
To achieve the research goals in Sub-objective 2.2.A. we studied two lines of B congenic chickens that were previously identified as resistant and susceptible to investigate the early mortality observed after rA inoculation. We found no significant differences between the two chicken lines in the IBDV viral loads in the bursa or in bursal damage as determined by the histological bursal lesion score. However, we did observe significant differences in macrophage and CD4+ T cell infiltration at 2 days post-inoculation and in CD8a+ T cells, T cell receptor ¿d+ T cells, and natural killer cells at 3 days post-inoculation using flow cytometry. These findings shed light on the role of the adaptive and innate immune systems in response to viral infection and suggest the need for further research to explore the complexity of early viral infection and immune response interactions.
In support of Sub-objective 2.2.B, we created a recombinant virus using the avian paramyxovirus type 1 (APMV-1) TS09 vaccine and incorporating the viral protein 2 (VP2) gene of infectious bursal disease virus (IBDV). We assessed the characteristics of the recombinant virus in day-old chicks and embryonated chicken eggs and confirmed the expression of the IBDV VP2 protein by an immunofluorescent assay. The results suggest that this TS09/IBDV-VP2 recombinant is a safe vaccine candidate. Further testing is recommended through in-ovo vaccination trials to evaluate its protective efficacy against ARV and Newcastle disease virus challenges.
Accomplishments
1. ARS researchers in Athens, Georgia, developed four recombinant viruses were developed that protect against both Marek’s disease virus (MDV) and Newcastle disease virus (NDV). Marek's disease (MD) is a contagious viral tumor disease in chickens caused by Marek's disease virus (MDV), leading to economic losses in the poultry industry. The commonly used live and/or vectored MDV vaccines are expensive to produce and difficult to handle due to the requirement of liquid nitrogen for manufacturing and delivering frozen infected cells that are viable. This study aimed to create a Newcastle disease virus (NDV) vectored MDV vaccine that can be stored at ambient temperature.All four recombinants conferred complete protection against the velogenic NDV challenge. These results show that the rLS/MDV-gB virus is a safe and effective dual vaccine candidate that can be mass-administered to large chicken populations at a low cost.
2. ARS researchers in Athens, Georiga, published an approach and protocol that allows laboratories to sequence large numbers of avian reovirus strains rapidly and affordably. The avian reoviruses have become major pathogens of farmed poultry within the last 15-20 years. Infected animals exhibit lameness and fail to gain weight. The disease caused by avian reoviruses is highly contagious and can be transmitted both from individual to individual within a flock and from hens to progeny. Vaccines based upon older strains of avian reoviruses are no longer protective against the newer strains now found on poultry farms. We are focused on sequencing new avian reovirus isolates to determine their immunological characteristics and create new vaccines.
Review Publications
Nour, I., Blakey, J.R., Alvarez Narvaez, S., Mohanty, S.K. 2023. Whole genome sequencing of infectious bursal disease viruses isolated from a Californian outbreak unravels the underlying virulence markers and highlights positive selection incidence. Viruses. 15(10):2044. https://doi.org/10.3390/v15102044.
Nour, I., Alvarez Narvaez, S., Harrell, T.L., Conrad, S.J., Mohanty, S.K. 2023. Whole genomic constellation of avian reovirus strains isolated from broilers with arthritis in North Carolina, USA. Viruses. 15:2191. https://doi.org/10.3390/v15112191.
He, L., Spatz, S.J., Dunn, J.R., Yu, Q. 2023. Newcastle disease virus (NDV) recombinant expressing Marek’s disease virus (MDV) glycoprotein B protects chickens against MDV and NDV challenges. Vaccine. 41(40):5884-5891. https://doi.org/10.1016/j.vaccine.2023.08.038.
Alvarez Narvaez, S., Harrell, T.L., Oluwayinka, O., Sellers, H.S., Khalid, Z., Hauck, R., Chowdhury, E.U., Conrad, S.J. 2023. Optimizing the conditions for whole-genome sequencing of avian reoviruses. Viruses. 15(9):1938. https://doi.org/10.3390/v15091938.
Lee, C.W., Bakre, A.A., Olivier, T.L., Alvarez Narvaez, S., Harrell, T.L., Conrad, S.J. 2023. Toll-like receptor ligands enhance vaccine efficacy against a virulent Newcastle disease virus challenge in chickens. Pathogens. 12(10):1230. https://doi.org/10.3390/pathogens12101230.
