Location: Exotic & Emerging Avian Viral Diseases Research
2024 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
Significant progress was made on all three objectives despite the two senior PIs having to spend a significant amount of time in emergency response to the current H5 highly pathogenic avian influenza outbreak in the United States which has immediate devastating impact on the poultry and bovine industry.
Under Objective 1, both ARS researchers in Athens, Georgia, and Auburn University (AU) researchers, continued to make progress with next generation sequencing (NGS, both Illumina and Nanopore MinION technologies) to establish them as a front-line diagnostic and surveillance tool. ARS and AU researchers applied different but complimentary approaches, a random amplification NGS approach which can potentially identify any pathogen in clinical samples in addition to NDV, and specific priming approach which can specifically target and amplify low quantity of NDV genome in samples, respectively. ARS researchers have identified many important poultry pathogens, in addition to NDVs, from foreign-origin samples using the random amplification approach and ARS and AU researchers are detecting, sequencing, and analyzing NDV genomes in samples from Mexico, Ecuador, Colombia, and Chile where NDV is endemic and has a potential for introducing NDV into the United States. Fortunately, no NDV or avian orthoavulavirus 1 (AOAV-1) (previously known as low virulent NDV) has been detected from US poultry samples tested and only class I AOAV-1, which has not caused outbreak in commercial poultry, were detected from wild bird surveillance samples. ARS scientists are also progressively optimizing the multistep workflow by customizing non-target RNA depletion to improve both sensitivity and cost-effectiveness of NGS and have published protocols with updated reagents and assay parameters.
To ensure that the diagnostic test used by National Veterinary Service Laboratories (NVSL) and National Animal Health Laboratory Network (NAHLN) perform optimally, the official real-time RT-PCR assays continue to be validated for NDV detection with new isolates and sequences as they become available. In addition, ARS researchers developed an assay called molecular beacon (MB) - loop mediated isothermal amplification (LAMP) assay where nucleic acid extraction from the sample, detection and differentiation of virulent from avirulent strains of AOAV-1 viruses can be performed in a single tube without the need for expensive consumables and equipment. The MB-LAMP assay gives two distinct readouts, a color change to detect presence of NDV genomic material and a fluorescent readout for virulent strains. The sensitivity of the assay was similar to current RT-PCR assay that detect virulent NDV and the new assay can also detect NDV directly from clinical swab material without the need to go through RNA isolation. The MB-LAMP assay can accelerate field-level NDV detection and differentiation especially in low resource settings.
ARS researchers established enzyme linked lectin assay (ELLA) as a functional analysis tool to evaluate virological and immunological characteristics of AOAV-1. ELLA-based neuraminidase assay (ELLA-NA) was shown to be more sensitive than hemagglutination test typically used for NDV detection and the new assay can also be applied to characterize the hemagglutinin-neuraminidase (HN) protein activity of different AOAV-1 strains. ARS scientists further demonstrated that neuraminidase inhibition antibody titer (measured by ELLA) in sera collected from birds vaccinated with Newcastle disease vaccine correlates well with hemagglutination inhibition (HI) antibody titer (the most common assay for evaluation of immune response) with higher sensitivity. These studies show that the ELLA carries the advantage of two commonly used serologic tests, HI test and ELISA, and the newly established assay can serve as useful tool for monitoring the infection and response to vaccination.
Under Objective 2, AU and ARS scientists have collaborated on identifying mutations in the genome of low virulent NDV isolated from wild birds during adaptation to chickens. The isolates have been passaged 10 times in eggs and the passaged isolates showed approximately 300 single nucleotide polymorphisms (SNPs) and 20 INDEL mutations. The selected isolates were characterized in birds and the phenotype will be correlated with the sequence changes. AU researchers also made significant progress with developing ex vivo systems to evaluate NDV evolution and adaptation. A soluble tetrameric HN protein representing the highly virulent strain was successfully produced and purified in cells and the functionality of the expressed protein including the HA and NA activity and binding to chicken tissues were confirmed. The recombinant HN protein has also been used as an antigen in an ELISA and is able to detect antibodies recognizing HN from LaSota vaccinated chickens. Additional HN proteins representing different NDV strains are being constructed and binding ability of the representative recombinant proteins to different tissues will be evaluated to study and predict the pathogenic potential of circulating NDVs for chickens and turkeys.
Current NDV vaccines are based on genotype II viruses; however, NDVs (belonging to 21 different genotypes) continuously evolve and major outbreaks are occurring with NDVs belonging to different genotypes. To understand the effect of vaccine driven humoral immune pressure on evasion, evolution, and emergence of new variant, ARS researchers established an in ovo (embryonated egg) model to partially neutralize the virus with immune sera from vaccine strain to allow virus to escape the selection pressure. The procedure was repeated for 15 passages in triplicate and data shows a reduction in viral titer during the passage depending on the antisera concentration applied. RNA samples extracted from each passage have been processed for NGS (Illumina) and data were analyzed to identify changes in fusion (F), HN, and other regions on the NDV genome.
Significant progress was made in Objective 3 to understand the innate immune response to AOAV-1s and how the virus blocks the host immune response, especially interferon (IFN), and vaccine candidates that induce high levels of IFN were developed by reverse genetics. ARS researchers further characterized and confirmed the safety of the candidate vaccines in vitro and in vivo. All the vaccine candidates replicated to high titer in vitro which is critical for vaccine production and demonstrated further attenuation compared to parental (LaSota) vaccine strain. Some of the vaccine cadidates did not kill any eggs inoculated at 9 days of embryonation, which demonstrates the high degree of attenuation for these isolates and potential for in ovo vaccin application (typically done at 18-19 days of embryonation). A vaccination study in 3- week-old chickens were also conducted to compare all the vaccine candidates side-by-side with parental (LaSota) vaccine and both innate and humoral immune responses are being analyzed.
ARS researchers are identifying microRNAs (miRNAs) that exhibit anti-viral or pro-viral activities and, based on in vitro screening of 18 miRNAs (preselected in prior years) against vaccine and virulent NDV strains, four miRNAs (miR-26a-5p, miR-19b-3p, miR-27b and miR-17) were selected and new recombinant vaccine viruses are being constructed that express the selected miRNAs. At the same time, computational analysis of each of these miRNA targets was performed to shortlist host genes most likely to be targeted by these miRNAs in chicken (DF1) cells.
AU scientists conducted several animal studies to determine mucosal cell and humoral immune responses elicited by LaSota vaccination in SPF chickens and commercial chickens with maternally derived antibody (MDA) against NDV. The study showed that LaSota vaccination elicits a vigorous cell immune response in the Harderian gland (HG). Furthermore, unlike the interference shown by MDA on vaccine-induced serum antibody responses, MDA does not interfere with the mucosal immune response of the HG. AU researchers also identified more than 2000 genes by total RNA sequencing that reacted to the NDV vaccination depending on the bird’s age and involvement of MDA. Twenty differentially expressed genes (DEGs) consistently showed up- or down-regulation both in HG and trachea 24 and 48 hours after vaccination and those DEGs may be used as vaccine efficiency markers. The baseline values obtained from immune response and transcriptome studies will be used as reference for vaccination-challenge studies using different NDV vaccines (commercial and experimental) in collaboration with ARS scientists. Two additional related experiments are on-going, one experiment investigating the vaccination with different strains (B2 and V4) on early gene expression, miRNA expression and respiratory microbiome, and the other experiment investigating transcriptome in chicken embryos after inoculation with the wild bird isolate passage sample described in Objective 2.
Accomplishments
1. Non-targeted next generation sequencing (NGS) is widely applied to identify the diversity of pathogens in field samples. However, abundance of host RNA (especially rRNA) and other environmental nucleic acids can reduce the abundance of pathogen specific reads of interest, reduce depth of coverage and increase surveillance costs. ARS researchers in Athens, Georgia, have progressively optimized the multistep workflow for host depletion and demonstrated that replacing the kit specific buffer with a commercially sourced alternative buffer yields similar or better data at a significant cost advantage. In addition, ARS researchers further optimized the non-target RNA depletion by testing multiple DNA degrading conditions and identified a replacement enzyme that significantly improved the efficiency of target RNA preparation and yields of pathogen specific sequencing reads.
2. ARS researchers in Athens, Georgia, tested potential adjuvants (ODN-1826 and Imiquimod) targeting pathogen recognition receptors to enhance the efficacy of the current Newcastle disease vaccine. Activation of those receptors lead to antiviral responses including the induction of the type I interferons. Birds were vaccinated intranasally with live LaSota strain with or without Imiquimod or ODN-1826 (50 µg/bird). Two weeks after vaccination, birds were challenged with virulent NDV. Both the vaccine groups adjuvanted with Imiquimod or ODN-1826 induced higher and more uniform antibody titer among birds compared with the live vaccine alone group. In addition, adjuvanted vaccines demonstrated greater protective efficacy in terms of reduction in virus shedding titer and number of birds shedding the challenge virus. Differential expression of antiviral and immune related genes was observed among vaccine groups from tissues (Harderian gland, trachea, cecal tonsil, and spleen) collected 3 days after treatment. These results demonstrate the potential use of cellular receptor targeted adjuvants as mucosal vaccine enhancers and warrant further optimization for efficacy and practical application in chickens.
3. Understanding and establishing correlates of protection are key for vaccine development. While a plethora of studies have reported that systemic antibodies are associated with protective efficacy, limited information is available on mucosal immune response following NDV vaccination. Auburn University researchers conducted several animal studies to determine mucosal cell and humoral immune responses elicited by live NDV vaccination in specific-pathogen-free chickens and commercial chickens with maternally derived antibody (MDA) against NDV. The study showed that LaSota vaccination elicits vigorous humoral and cell immune responses in the Harderian gland (HG). Furthermore, unlike the interference shown by MDA on vaccine-induced serum antibody responses, MDA did not interfere with the mucosal immune response of the HG. Auburn University scientists also identified 20 differentially expressed genes (DEGs) consistently shown to up- or down-regulate both in HG and trachea, 24 and 48 hours after vaccination. The baseline values of mucosal immune response and selected DEGs identified in these studies will be instrumental as a reference for vaccine efficiency markers in future vaccine development studies that will be conducted in collaboration with ARS scientists.
Review Publications
Campler, M.R., Cheng, T., Lee, C.W., Hofacre, C.L., Lossie, G., Silva, G.S., El-Gazzar, M.M., Arruda, A.G. 2024. Investigating the uses of machine learning algorithms to inform risk factor analyses: the example of avian infectious bronchitis virus (IBV) in boiler chickens. Research in Veterinary Science. 171(2024):105201. https://doi.org/10.1016/j.rvsc.2024.105201.
Kariithi, H.M., Volkening, J.D., Chiwanga, G.H., Goraichuk, I.V., Olivier, T.L., Msoffe, P.M., Suarez, D.L. 2023. Virulent Newcastle disease virus genotypes V.3, VII.2 and XIII.1.1 and their coinfections with infectious bronchitis viruses and other avian pathogens in backyard chickens in Tanzania. Frontiers in Veterinary Science. 10:1272402. https://doi.org/10.3389/fvets.2023.1272402.
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.
Bakre, A.A., Kariithi, H.M., Suarez, D.L. 2023. Alternative probe hybridization buffers for target RNA depletion and viral sequence recovery in NGS for poultry samples. Journal of Virological Methods. 321:114793. https://doi.org/10.1016/j.jviromet.2023.114793.
Goraichuk, I.V., Msoffe, P.L., Chiwanga, G.H., Dimitrov, K.M., Afonso, C.L., Suarez, D.L. 2023. Complete genome sequence of seven virulent Newcastle disease virus isolates of sub-genotype XIII.1.1 from Tanzania. Microbiology Resource Announcements. e00405. https://doi.org/10.1128/MRA.00405-23.
Kariithi, H.M., Volkening, J.D., Chiwanga, G.H., Goraichuk, I.V., Msoffe, P.L., Suarez, D.L. 2023. Molecular characterization of complete genome sequence of an avian coronavirus identified in a backyard chicken from Tanzania. Genes. 14(10):1852. https://doi.org/10.3390/genes14101852.