Location: Exotic & Emerging Avian Viral Diseases Research
2025 Annual Report
Objectives
1. Identify the emergence of new vNDV strains.
1.A. Identify NDV genetic changes important for transmission and pathogenicity in poultry and wild birds.
1.B. Develop rapid identification assays for variant vNDV strains.
1.C. Conduct prevalence studies in poultry from countries where vNDV strains are endemic to determine the presence of variant and emerging viruses in NDV vaccinated poultry and the prevalence of NDV in wild birds.
2. Develop predictive models for risk assessment of virus evolution.
2.A. Develop predictive models using NextGen sequencing to evaluate the rate of change in different virulent NDV strains from unvaccinated, sub-optimally vaccinated, and well-vaccinated poultry.
2.B. Develop in vivo and ex vivo systems to understand the mechanisms of NDV evolution and adaptation.
3. Develop improved NDV vaccines platforms.
3.A. Determine and compare mucosal, cell, and early immune responses associated with protection elicited by available NDV vaccines to predict protection conferred by vaccination.
3.B. Identify and evaluate effective and user friendly NDV vaccine platforms for in ovo administration in broiler chickens.
3.C. Identify and evaluate low-cost vaccines that produce minimal vaccine reactions to prevent decreased productivity.
3.D. Develop NDV vaccine platforms capable of preventing viral replication, shedding, and transmission in domestic poultry.
Approach
We will conduct Newcastle disease virus (NDV) surveillance from poultry and wild birds in the United States and foreign countries to better understand the prevalence of NDVs and to identify important genetic markers that could change virus more fatal to poultry and also make it easier to transmit among birds. We will use state of the art Next Generation Sequencing (NGS) technology and bioinformatics tools to analyze large amounts of genetic information. Novel viruses that display evidence of increased virulence will be further characterized in animals. In conjunction with surveillance effort, we will vigilantly evaluate and update NDV diagnostic assays to assure that the official diagnostic assays used by National Animal Health Laboratory Network continue to perform with high sensitivity and specificity. In addition, we will develop new NGS-based diagnostic assays as a practical tool for the detection of previously known and newly emerging NDVs, and also for differentiation of low and highly virulent viruses.
Like many other RNA viruses, NDVs continue to change and make them better fit to the environment. In this objective, we will study the complex interaction between virus and host. We will specifically assess: 1) how vaccine-induced immunity affect the evolution of NDV, 2) how NDV isolated from wild birds adapt in chickens, and 3) if specific gene or genetic marker determines how NDV replicates in birds or in specific tissues of the birds. The information obtained in this study will be used for risk assessment and applied to develop predictive model to improve control measures.
We will study different aspect of immunity (innate, mucosal, antibody and cell mediated immunity) to predict protection conferred by vaccination and to develop new vaccines or further improve current vaccines. Interferons (IFN) are proteins made and released by host cells in response to viral infection and vaccination. In this objective, we will develop vaccines that modulate the IFN responses and enhance both innate and adaptive immune responses. The safety and protective efficacy of new vaccines will be evaluated in birds in comparison to currently available vaccines.
Progress Report
Under Objective 1, ARS researchers in Athens, Georgia, and researchers at Auburn University, Auburn Alabama (AU) optimized several technologies for next generation sequencing (NGS). They applied NGS as a front-line diagnostic and surveillance tool for the detection and genetic characterization of type 1 avian orthoavulavirus (AOAV-1). As part of the optimization, ARS scientists developed a novel RT-PCR assay which is used to evaluate the quality of sample preparation before conducting NGS. In addition, ARS scientists applied an RNA sequencing methodology that does not require a pre-amplification step, therefore it is faster and less expensive. The sensitivity was high and sequence data for a nearly full-length AOAV-1 viral genome could be obtained. In addition to application as a diagnostic tool, these methods will aid studies into the viral epi-transcriptome, which will elucidate the viral infection and pathogenesis process.
Both ARS and AU scientists successfully utilized NGS for rapid genetic characterization of surveillance, clinical, and experimental samples. No Newcastle disease virus (NDV), the virulent form of AOAV-1, has been detected from US poultry surveillance samples. However, NGS was used to characterize the avian metapneumovirus subtype A (AMPV) for the first time in the US. The phylogenetic analysis revealed that the isolated AMPV strains were most closely related to recent Mexican strains also first identified by ARS scientists. Class I AOAV-1, which has not caused outbreaks in commercial poultry, were detected from wild bird surveillance samples in the U.S. which shows that viruses related to NDV are still carried by wild birds.
The considerable genetic diversity of AOAV-1 means that molecular (i.e., genetic) detection assays used by National Veterinary Service Laboratories and National Animal Health Laboratory Network must be periodically updated to maintain high sensitivity and specificity. To ensure that these diagnostic tests perform optimally, the official assays continue to be updated for NDV detection as genetic sequences from new contemporary isolates are produced (which is now more efficient because of the NGS methods described above). The current USDA real-time RT-PCR assay for NDV was originally designed for now out-of-date genotype class II AOAV-1 isolates with an emphasis on early-2000s US strains. In collaboration with Texas A&M University, efforts to improve the test’s sensitivity with current viral genotypes that are circulating globally additional primer and probes, which are the key elements of the tests that determine what is being detected, were incorporated into a multiplex assay. Using field clinical samples and experimental samples, both the specificity and sensitivity of the updated assay were improved. As viruses change rapidly the tests will be monitored for performance using data from surveillance samples obtained from studies such as the one below.
Scientists at AU have collected NDV samples from several countries where NDV is endemic to monitor the virus because it has a potential to be introduced into the U.S. Vaccine-like viruses (genotype V) was detected from commercial poultry samples in Colombia. This genogroup is important because they were previously detected in Mexico and were the genogroup associated with the 2002 and 2018 NDV outbreaks in the U.S. In addition, a genotype often referred to as pigeon paramyxoviruse 1, was detected in wild bird samples from Chile. Recently, PPMV-1 related NDV outbreaks occurred in two pigeon farms in Canada and enhanced surveillance efforts both in wild bird and poultry is warranted. These surveillance studies are on-going.
Under Objective 2, ARS and AU scientists have collaborated to improve vaccines by comparing vaccine virus growth system to find which is most efficient. They evaluated genetic changes in the genomes of low virulence AOAV-1 isolates from wild birds by passaging the viruses in embryonating chicken eggs (ECE) and in different cells types in vitro. The goal is to find a method with as few changes as possible. Overall, AOAV-1 viruses adapt differently depending on the host system (ECE or cells). In comparison to ECE and avian-origin cells, significantly more genetic changes were observed, especially in the HN and L proteins, when virus was passaged in a cell type called a Vero cell. Embryonating chicken eggs supported efficient replication compared with passage in Vero and avian cells. In addition, Vero cell passaged viruses showed lowered replication and reduced ability to induce antibodies in chickens. As expected, exposing day old chickens to the vaccine viruses did not cause disease. One of the ECE passaged wild bird isolates induced immune responses, which included induction of mucosal IgA in lacrimal fluid, that were comparable to the widely used, but old, LaSota vaccine virus, supporting potential use for this virus and system to be used as an updated live vaccine.
Auburn University researchers made significant progress with developing ex vivo systems to evaluate host-pathogen interaction of NDV in poultry. Ex vivo systems provide a safer and less expensive method for studying viruses. To accomplish this, a soluble tetrameric hemagglutinin-neuraminidase (HN) protein from an isolate with a fusion protein cleavage site indicating virulence but which has been shown to have less virulence for chickens than other virulent NDV isolates, was successfully constructed, purified in cells and had hemagglutinating and neuraminidase (NA) activity (i.e., was functional). The reconstructed HN protein was further characterized by testing it’s binding to chicken tissues, and in comparison to an HN protein from a virulent isolate and no detectable difference in binding was observed. To examine the authenticity of the reconstructed HN protein, ARS researchers utilized newly established test, enzyme linked lectin assay (ELLA). The ELLA test serves as a functional analysis tool to evaluate virological and immunological characteristics of reconstructed HN proteins. Properties such as the optimal pH for NA activity of the reconstructed HN proteins and the original viruses were compared and found to be the same. Both reconstructed and original HN proteins showed the peak of NA activity at 37°C, which further shows the correct function of the reconstructed protein. The antigenicity of the reconstructed HN protein was also compared with the original virus by using it to determine the neuraminidase inhibition (NI) titers of reference sera and showed that the reconstructed HN protein and original virus were almost identical with a Spearman correlation coefficient of 0.91 (P<0.0001). In addition to serving as a tool for ex vivo pathobiological studies, recombinant HN proteins can be used to evaluate immunity to virulent NDV more safely by using non-infectious materials to conduct the tests.
Significant progress was made in Objective 3 to understand the immune responses elicited by commercially available NDV vaccine strains and the development of potential vaccine candidates through different approaches. Auburn University scientists continued an earlier study comparing three common vaccine strains (B1, V4 and LaSota) for their ability to modulate immune gene expression in day old chickens post vaccination. All three vaccines induced unique immune-related gene expression or differentially expressed genes (DEGs). The DEGs were noted particularly in the Harderian gland and lungs. The B1 and V4 vaccine strains induced broader immune responses. However, B1 affected innate immunity more, and the V4 vaccine affected genes in both the innate and adaptive immunity pathways. The LaSota vaccine showed a more focused upregulation of antiviral and inflammatory genes. The identification of common DEGs across strains provides potential biomarkers for early immune activation and understanding difference in immune activation by different vaccines will drive development of vaccines which induce an optimal response.
Live attenuated vaccine candidates were characterized for induction of interferon (IFN) by ARS scientists. Good induction of the IFN response is critical to an effective vaccine. Through a reverse-genetics approach two vaccine candidates were identified that induce higher IFN and more IFN stimulating genes. These vaccines also induce similar or higher level of serum antibody response compared with the LaSota vaccine strain, indicating effective activation of the chicken immune system. In addition, the two live vaccine candidates may potentially be administered in ovo because they don’t cause excessive mortality in ECE, therefore is not expected to adversely affect hatch. To further enhance the safety of NDV vaccine candidates that could be administered in ovo, antibodies specific for the vaccine were used to delay vaccine virus replication and control its infectivity. The optimal ratio of each candidate vaccine virus and antibody concentrations were determined so the vaccines could be applied in ovo as vaccine-antibody complexes which enhances safety and vaccine efficacy.
ARS researchers also made significant progress in developing microRNA (miRNA)-based vaccines. microRNAs are small, naturally expressed molecules that can enhance vaccine efficacy by controlling virus replication. Seven miRNAs were selected based on antiviral and proviral activity against NDV strain during in vitro testing. These molecules have been used to make a vaccine for testing.
An overarching result for objectives 2 and 3 is that the immunological (NI-ELLA, in vitro expressed reconstructed HN protein) and genetic tests (DEG, quantitative real-time RT-PCR) developed here, will be applied to ongoing work to improve vaccines and to improve prevention and control strategies for NDV in US poultry.
Accomplishments
1. Optimization of next generation sequencing as diagnostic and surveillance tool for Newcastle disease virus. ARS researchers in Athens, Georgia further optimized a new highly efficient method for genetic sequencing called next generation sequencing (NGS) as a as a front-line diagnostic and surveillance tool. ARS scientists developed a novel 28S ribosomal RNA RT-qPCR assay which evaluates the quality of sample preparation before conducting costly NGS for the detection and genetic characterization of avian RNA viruses. In addition, ARS scientists applied a direct sequencing methodology for the virus genetic material that eliminates some processing steps but is still very effective; they characterized nearly the full-length of the viral genome. This new method enables the determination of the genetic sequence of Newcastle disease virus very efficiently and can be used for rapid virus epidemiology during outbreaks.
2. Development of new Newcastle disease virus assay. ARS researchers in Athens, Georgia, developed an assay called a molecular beacon-loop mediated isothermal amplification (MB-LAMP) assay that uses genetic material from a diagnostic sample to detect and differentiate virulent from non-virulent strains of avian orthoavulavirus 1 (AOAV-1). This test can be performed in a single tube without the need for expensive consumables or equipment, and does not require isolation of genetic material from the sample as a separate step. The sensitivity of the assay is similar to the current RT-PCR assay that detects virulent NDV. The MB-LAMP assay can accelerate field-level virulent AOAV-1 detection and costs less.
3. Established immunologocal tools for evaluation of Newcastle disease vaccine-induced immunity. Researchers at Auburn University in Auburn, Alabama and ARS researchers in Athens, Georgia have collaboratively established immunological tools to better evaluate Newcastle disease (ND) vaccine induced immunity and tools to identify correlates of protection which are critical for vaccine development and monitoring of vaccine efficacy. A new serologic assay that determines the level of antibodies to the vaccine was established using proteins made to simulate the real virus but are safer and cheaper. The assay showed great potential to evaluate vaccine induced antibody response with high sensitivity and has high-throughput screening capability. In addition, different immune correlates of protection and genetic markers for vaccine efficacy were identified. These developments improve the safety, cost, and accuracy of in vitro testing for adequate immunity from vaccination.
4. Determining vaccine candidates from wild bird origin avian orthoavulavirus 1 (AOAV-1) isolates. Auburn University researchers in Auburn, Alabama and ARS researchers in Athens, Georgia collaboratively examined the replication and adaptation of wild bird origin avian orthoavulavirus 1 (AOAV-1) isolates and assessed the potential as vaccine candidates for chickens. All isolates grew efficiently in embryonating eggs with limited genetic changes in contrast to virus grown in cell culture which showed lower replication efficiency with higher genetic variation. One-day-old chickens inoculated with isolates passaged in eggs developed serum antibody levels similar to those elicited by a vaccine strain that is widely used but is not closely related to current field strains. In contrast, isolates grown in cell culture showed limited replication in chickens and didn’t elicit antibodies as well as the egg-grown strain. The study shows that recent wild bird origin AOAV-1 could be grown in embryonating chicken eggs and could induce an overall immune response comparable to current vaccine strains virus supporting their potential use as updated vaccines for poultry.
5. Determined the complete genome of subtype A avian metapneumovirus (AMPV). Avian metapneumovirus (AMPV) poses a significant threat to the U.S. poultry industry by impacting broilers, layers, layer and broiler breeders, and turkeys. ARS scientists in Athens, Georgia participated in AMPV surveillance efforts and were first to report the complete genome of subtype A AMPV (AMPV-A) when it was detected in U.S. poultry. Genetic analysis revealed close relatedness between U.S. isolates collected from turkeys in 2023 and Mexican strains from chickens collected 2020–2022, suggesting that the AMPV strains were related. The genetic data was additionally used to update the AMPV subtype-specific RT-PCR test and validated in comparison to two commercial kits. Genetic data provided epidemiological information on the origin of the viruses in U.S. poultry and the updated diagnostics tests will now provide accurate results for samples from U.S. poultry.
Review Publications
Bakre, A.A., Mears, M.C. 2024. Characterizing host microRNA: virus interactions of Orthoavulavirus javaense. Viruses. 16(11):1748. https://doi.org/10.3390/v16111748.
Khatiwada, S., Ngunjiri, J., Boley, P.A., Yadav, K.K., Ghorbani, A., Abundo, M., Lee, C.M., Poelstra, J., Lee, C.W., Kenney, S.P., Gharaibeh, S., Rajashekara, G. 2025. Age-based host response to turkey arthritis reovirus in commercial turkeys in the presence of maternally derived antibodies. BMC Veterinary Research. https://doi.org/10.1186/s12917-025-04525-1.
Chaves, M., Hashish, A., Goraichuk, I., Caserta, L.C., Mears, M.C., Gadu, E., Bakre, A.A., Alexander, M.E., Shelkamy, M.M., Nadendla, S., El-Gazzar, M. 2025. Nanopore Sequencing in Veterinary Medicine: From Concepts to Clinical Application. Frontiers in Cellular and Infection Microbiology. 15:1545032. https://doi.org/10.3389/fcimb.2025.1545032.
Alexander Morris, E., Schroeder, M.E., Anderson, P.N., Schroeder, L.J., Monday, N., Gabriel, S., Ficken, M., Ferro, P.J., Suarez, D.L., Dimitrov, K.M. 2025. Optimization and validation of a universal real-time RT-PCR assay to distinguish between virulent Newcastle disease viruses and viruses of low virulence. Viruses. Viruses 2025, 17(5), 670. https://doi.org/10.3390/v17050670.
Mears, M.C., Olivier, T.L., Williams Coplin, T.D., Espinoza, E., Bakre, A.A. 2024. Detection and differentiation of low virulence and virulent avian orthoavulavirus javaense using a molecular beacon with RT-LAMP. Scientific Reports. 2024(14):18047. https://doi.org/10.1038/s41598-024-68816-7.
Goraichuk, I.V., Harden, M., Spackman, E., Suarez, D.L. 2023. The 28S rRNA RT-qPCR assay for host depletion evaluation to enhance avian virus detection in illumina and nanopore sequencing. Frontiers in Microbiology. Volume 15. https://doi.org/10.3389/fmicb.2024.1328987.
Goraichuk, I.V., Torchetti, M., Killian, M., Kapczynski, D.R., Kulkarni, A., Sary, K., Suarez, D.L. 2024. Introduction of avian metapneumovirus subtype A to the United States: molecular insights and implications. Frontiers in Microbiology. 15. https://doi.org/10.3389/fmicb.2024.1428248.