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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Exotic & Emerging Avian Viral Diseases Research » Research » Research Project #441214

Research Project: Intervention Strategies to Predict, Prevent, and Control Emerging Strains of Virulent Newcastle Disease Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

2022 Annual Report

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.

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
Progress was made on all three objectives and their sub-objectives despite the six months delay in a new researcher review and approval due to departure of the two primary ARS researchers and hiring of the two new researchers. Under Objective 1, the main goal is to identify and sequence circulating Newcastle disease virus (NDV) isolates that present a threat to the U.S. poultry industry. ARS researchers for the NDV project are using Next Generation Sequencing (NGS) as a primary diagnostic tool on clinical samples. Using the immense depth of sequencing from NGS, a random amplification approach was used to potentially identify any pathogen in the sample. In addition to many co-infecting respiratory pathogens identified from clinical samples collected from poultry, a NDV vaccine-like strain with an unusual F gene cleavage site sequence (110 GGGRQGR/F117) compared with typical low virulent NDV cleavage site sequences (110 GGGRQGR/L117) was identified and isolated. The mean death time (MDT) of the isolates in embryonated chicken eggs showed typical characteristics of low virulent NDV strains and Intracerebral Pathogenicity Index (ICPI) will be determined to confirm the virulence of the isolate. To assure that the National Animal Health Laboratory Network (NAHLN) real-time RT-PCR (rRT-PCR) assays continue to perform with high sensitivity and specificity, experiments utilizing F gene rRT-PCR, which specifically detects virulent Newcastle disease virus (vNDV) was conducted with this unique isolate and, interestingly, it showed weak, but consistent positive result. The researchers further confirmed this unexpected positive signal by conducting PCR with synthetic DNA template generated to match the specific sequence in the cleavage site of the isolate. Further evaluation of the current F gene rRT-PCR for its specificity to optimize/update will be conducted as needed. In addition to existing collaborations with many institutions/programs within the US, the NDV researchers are making substantial progress in establishing new contacts for the collection of clinical samples. Domestic collaborators have been contacted who will provide surveillance samples from poultry and wild birds. Internationally, we continue to collaborate with Boehringer Ingleheim Animal Health to evaluate clinical samples from Mexico using NGS. In addition, one of the scientists visited Ecuador and Chile and actively communicated with Argentina and Colombia to establish contacts in South American countries to obtain NDV viruses. An Animal and Plant Health Inspection Service (APHIS) permit (610-21-281-00620) was obtained by Auburn University to import and transport controlled materials, organisms and vectors from various countries outside the US. The permit specifically allows the importation of NDV RNA on FTA cards. Veterinarians and Agriculture officers in Chile and Ecuador have been provided FTA cards to collect samples. This project provides a unique surveillance opportunity to understand the pathogens circulating in the Central, South American and Mexico regions. In regard to NGS, considerable efforts to improve the sensitivity of detection of pathogens continues through the depletion of non-target sequence. The NDV project's existing RNase H based strategy to deplete host transcripts and improve yield of viral reads was efficient at removing a majority of host ribosomal RNA (rRNA), but several host transcripts still persisted in abundance in our NGS data across multiple sequencing runs. New probes were developed to add to the host depletion protocol that demonstrated that the depletion of transcripts were effectively improved. To optimize specific probes, a qPCR-based assay to determine exact copy number decrease for specific transcripts was developed that could gauge an accurate estimate of our RNAse H efficacy. In addition, to reducing the cost, a comparison was conducted of different probe hybridization buffers using qPCR assay. Two simpler and significantly more cost-effective buffers for RNAse H digestion were identified. These new buffers will be tested with field samples. If successful, they will allow us to save significant research dollars. Improvement to bioinformatics workflow was made. Existing analysis workflow at local Galaxy cluster only identifies viral and bacterial reads from samples subjected to Illumina NGS and rely on an older version of the chicken genome (version 6.0) and transcriptome (version 5.0) for analysis of host transcripts. To improve the efficacy of workflow, the workflow on the ARS owned SCINet Galaxy or the Ceres supercomputing cluster at Ames, Iowa was established. Old tools to obtain data were replaced by newer pipelines at SCINet Ceres Galaxy cluster. The advantage of adopting the workflows of this new environment has accelerated analysis of 196 regular computing nodes, 26 high memory nodes and one GPU node to a turnaround time from several days to several hours. The issue of uploading massive NGS data to the cluster has been resolved via the establishment of the high-band width 10GBps line between local (SEPRL) and the Ceres cluster by the SCINet Virtual Research Support Core (VRSC) and local IT support. All workflows now use the latest version (ver. 7.0) of the Gallus_gallus genome and transcriptome and ensures that our analysis outputs are current with the state of art sequence information. An alternative sequencing platform, MinIon sequencing, has also been used with data analysis and identification tools analyzed on the SCINet cluster. Objective 2, one of the goals is to identify mutations in the genome of low virulent NDV isolated from wild birds during adaptation to chickens and determine how certain mutations contribute to host and tissue specificity of NDV. Progress was made to select eight class II wild bird NDV isolates to be used for adaptation in chickens. The isolates are from various hosts and geographic location in the US and full genome sequences are available. Based on the sequences of the fusion genes, they also represent different genotypes. The development of ex vivo system to understand the mechanisms of NDV evolution and adaptation is underway. The goal is to develop and optimize expression system(s) for soluble tetrameric hemagglutinin-neuraminidase (HN) protein that binds to chicken respiratory epithelium in protein histochemistry assays. Progress was made in designing the codon-optimized synthetic construct for expression of soluble tetrameric HN protein in HEK293T cells. Progress was made in Objective 3 to understand the host specific innate immune response in relation to NDV infection. The NDV researchers used knockout (KO) chicken fibroblast cells (DF1) which lack two key pathogen recognition receptors (TLR3 and MDA5) known to mediate innate immune responses to virus infection. Treatment of cells with synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] resulted in significant upregulation of interferon (IFN) a and ß, and IFN stimulated genes (ISG), MX1, gene expression in wild type (WT) cells, but not in KO cells. KO cells supported significantly higher level of NDV vaccine virus replication compared with WT cells. However, there was no significant difference in type I IFN or ISG expression level between WT and KO cells after virus infection. Utilizing single TLR3 or MDA5 KO cells, the individual and coordinated roles of TLR3 and MDA5 in the replication of NDV and induction of innate immune responses upon NDV infection will be further evaluated. Progress was made in studying the identification of early immune genes involved (upregulated or downregulated) in innate responses induced by vaccination with the lentogenic NDV LaSota vaccine strain. Although the study was delayed due to recruiting PhD students, the two newly hired graduate students worked on characterization of cell immune responses and early immune gene detection, respectively, in initial trials using NDV and a small number of experimental animals and have established the procedures. A larger experiment using the established procedures will be initiated during the second half of 2022. Under Objective 3, one of the main goals is to develop improved NDV vaccine platforms and develop improved vaccine candidates by selecting for superior IFN inducing NDV vaccine virus subpopulations. Although biological type I IFN assay is highly sensitive and useful method in selecting high IFN inducing virus, the assay is rather time consuming. Thus, we investigated the practical value of commercial chicken IFNa ELISA in quantifying IFN level in comparison to biological IFN assay and confirmed a good correlation between two assays and potential use of ELISA in identifying high IFN inducers. To expedite the study, ELISA screening will be used to identify large number of vaccine candidates and selected candidates will be confirmed by the IFN biological assay. In another approach, research was conducted to identify and characterize pro- and anti-viral chicken microRNAs (miRNAs) that affect NDV replication with a goal to develop miRNA-based vaccines to improve vaccine yield and reduce reactogenicity. Literature and miRNA candidates were analyzed to shortlist a set of 65 miRNAs that showed most potential. Conditions of transfection, multiplicity of infection for the reported virus was optimized. Two initial screens to identify pro- and anti-viral miRNAs from our set was run. Several candidate miRNAs have been identified that inhibit or upregulate NDV replication and continue to assess viral replication kinetics, cytotoxicity, and immune responses to make final selection of candidate miRNAs.

1. Host rRNA depletion to improve sensitivity of next generation sequencing. The use of random amplification next generation sequencing (NGS) is increasingly being used for diagnostic testing because it can potentially identify any pathogen in the sample, but a major limitation of this technology is the limited sensitivity of the assay. When using random amplification NGS, a high percentage of the sequences identified are host and/or bacterial ribosomal RNA (rRNA) sequence. ARS researchers in Athens, Georgia, developed a simple and cost effective approach to remove the host and bacterial rRNA from a clinical sample, which effectively increases the detection and sensitivity of everything else in the sample. Employing this host depletion method provided a 100-1000 fold increase in the amount of pathogen sequence in the sample which allows for not only identification of pathogens but in-depth sequence analysis to allow for the determination of subtype, genotype, or pathotype of the virus to be identified. Advances in sequencing technology can provide better and more comprehensive diagnostics to identify more pathogens in samples which allows better disease control. This improved technology allows diagnostic laboratories better opportunities to detect pathogens in any species which can directly support the farmer’s efforts to control disease.

2. Next Generation Sequencing (NGS) identifies important poultry respiratory pathogens in Mexico. The use of NGS techniques to identify pathogens in clinical samples continues to improve. Often full viral genomes can be identified in samples and this sequence information can be used to not only identify what pathogens are in samples, but also determine the potential virulence and origin of the pathogen. This information can be used to predict which viruses are likely to cause severe disease and can help guide the use of which vaccines should be used for control efforts. The study conducted by ARS researchers in Athens, Georgia, examined samples from poultry farms in Mexico. One of the most common pathogens identified was infectious bronchitis virus (IBV), which is an important respiratory disease pathogen in chickens. The analysis of full genomes of over 20 viruses identified 5 different genotypes of IBVs circulating in Mexico in the last two years and allows for targeted vaccination to achieve full protection in flocks. IBV presents an ongoing problem to U.S. poultry industry and NGS provides a new approach to identify and characterize IBV to allow more targeted vaccination.

3. The thermal stability of Newcastle disease virus (NDV) was determined in poultry litter. Both disposal and decontamination of organic waste, such as poultry litter, after an outbreak of NDV can be expensive and difficult. Some, options such as composting, burial, or landfilling are not always available. Therefore, the duration that NDV could remain infectious in poultry litter at different temperatures was determined. In some cases, heating a house may be possible to help decontaminate material, if not data on viability at lower temperatures informs approaches where premises may remain fallow so the virus can deteriorate over time. ARS researchers in Athens, Georgia, found that at temperatures at or above 90°F quickly degrades the virus within a week. At lower temperatures (50-70°F) the virus remained infectious for weeks and even months.

4. The strong inflammatory response of chickens can cause more severe Newcastle disease. Newcastle disease virus (NDV) causes fatal illness especially in young poultry and can severely affect egg production in laying hens. Although the virus itself can damage tissues, sometimes the chickens’ strong immune response of producing inflammatory proteins (cytokines) can exacerbate the outcome of NDV infection. ARS researchers in Athens, Georgia, monitored the virus, cells, and inflammatory cytokines at the same time directly in affected tissues and identified key cells and cytokines involved which can be used as markers to predict how the disease progress. This study helps in understanding the role of the bird immune system in the disease caused by NDV and also developing preventive strategy targeting and modulating chicken’s immune system which can provide additional layer of protection.

5. Development of Newcastle disease virus expressing small protein (IL-4) important for cell signaling. ARS researchers in Athens, Georgia, generated recombinant Newcastle disease virus expressing IL-4. IL-4 is a biological protein and key regulator in humoral and adaptive immunity. The recombinant virus replicates efficiently in chickens and the production of IL-4 was successfully demonstrated in multiple tissues of infected chickens. IL-4 is known to enhance humoral immunity and recombinant Newcastle disease virus co-expressing IL-4 has a potential to be developed as a vaccine to enhance protection against virulent Newcastle disease virus infection.

6. Newcastle disease vaccines administered in the egg. Newcastle disease virus (NDV) remains a serious disease threat for poultry farmers, and vaccination is commonly used for control. Although live vaccines are available, they are difficult to administer to all birds in a flock and get a strong uniform immune response. One alternative method of administration is to vaccinate the chicken embryo inside the egg (in ovo) which allows robotic vaccination and potentially an early immune response. Current NDV vaccines cannot be used in ovo because it causes the chicks to either not hatch or be sick when they hatch. In order to improve the in ovo vaccination, ARS researchers in Athens, Georgia, developed a new NDV vaccine that is more attenuated and can be used safely in embryonating chicken eggs while providing good protection from virulent NDV challenge. The use of in ovo vaccination is commonly used in the broiler industry in the U.S., and this vaccine shows the possibility of use as a live vaccine given in ovo which can potentially improve our ability to protect poultry from this disease.

Review Publications
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.
Marcano, V.C., Cardenas-Garcia, S., Diel, D.G., Antoniassi Da Silva, L.H., Gogal Jr., R.M., Miller, P.J., Brown, C.C., Butt, S.L., Goraichuk, I.V., Dimitrov, K.M., Taylor, T.L., Williams Coplin, T.D., Olivier, T.L., Stanton, J.B., Afonso, C.L. 2021. A novel recombinant Newcastle disease vaccine improves post-in ovo vaccination survival with sustained protection against virulent challenge. Vaccines. 9(9):953.
Brown, C., Zhang, J., Pantin Jackwood, M.J., Dimitrov, K., Ferreira, H., Suarez, D.L. 2022. In situ gene expression in early stage of virulent Newcastle disease in chickens. Veterinary Pathology. 59(1):75-81.
Marcana, V., Susta, L., Diel, D.G., Cardena-Garcia, S., Miller, P.J., Afonso, C.L., Brown, C.C. 2021. Evaluation of chickens infected with a recombinant virulent NDV clone expressing chicken IL4. Microbial Pathogenesis. 159:105116.
Kariithi, H.M., Volkening, J.D., Leyson, C.M., Alfonso, C.L., Christy, N., Lucio, E.D., Lemiere, S., Suarez, D.L. 2022. Genome sequence variations of infectious bronchitis virus serotypes from commercial chickens in Mexico. Frontiers in Veterinary Science. 9:931272.
Parris, J.D., Kariithi, H., Suarez, D.L. 2022. Non-target RNA depletion strategy to improve sensitivity of next-generation sequencing for the detection of RNA viruses in poultry. Journal of Veterinary Diagnostic Investigation. 34(4):638-645.
Mo, J., Stephens, C.B., Spackman, E. 2022. The thermal stability of Newcastle disease virus in poultry litter. Avian Diseases. 66(2):1-4.