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

Location: Exotic & Emerging Avian Viral Diseases Research

2023 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 U.S. which has immediate devastating impact on the poultry industry. Under Objective 1, in addition to existing collaborations with many institutions/programs within the U.S., the Newcastle Disease Virus (NDV) project researchers are making substantial progress in establishing new contacts for the collection of both poultry and wild bird samples. The Auburn University research team has received wild bird samples from New Jersey, Georgia, and South Carolina and has identified avian orthoavulavirus 1 (AOAV-1) (previously known as low virulence NDV) and sequence analysis is being done. ARS researchers continue to collaborate with Boehringer Ingelheim Animal Health to evaluate clinical samples from Mexico using next generation sequencing (NGS). In addition, the Auburn group has established research collaboration agreements with the Departments of Agriculture of Ecuador and Colombia for the transfer of samples collected from NDV outbreaks. Samples from Colombia and Chile are currently being analyzed. Using protocols previously developed by ARS scientists, samples are collected on FTA cards, which inactivate the live virus and preserve genetic material, so the genes can be analyzed. This project provides a unique surveillance opportunity to understand the pathogens circulating in Central America, South America, and Mexico where NDV is endemic and historically has served as a threat for introducing NDV into the U.S. To support surveillance efforts and ensure that the US National Animal Health Laboratory Network (NAHLN) diagnostic assays perform with high sensitivity and specificity, 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, continued progress is being made with NGS (both Illumina and Nanopore MinION technologies) with a goal to establish them as a front-line diagnostic and surveillance tool. Using a random amplification NGS approach which can potentially identify any pathogen in clinical samples in addition to NDV, ARS scientists identified other important poultry pathogens from foreign-origin samples such as avian metapneumovirus, avian coronaviruses, avian astroviruses, and avian nephritis virus. In addition, while maintaining the efficacy of the current approach, sequencing costs were lowered by identifying a significantly more economical buffer to complete the process. ARS scientists also demonstrated the feasibility of a portable sequencer, MinION, for identifying disease agents in clinical respiratory samples. The results demonstrated that MinION sequencing can provide rapid, and sensitive detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the more complex Illumina technology. The Auburn group also applied MinION sequencing to establish a rapid identification assay for NDV. Using this platform, sample processing for RNA extraction, one-step RT-PCR of Fusion gene amplification, and sequencing can all be done within a day. As a new diagnostic tool, ARS researchers in Athens, Georgia, are developing a reverse transcription loop mediated isothermal amplification (RT-LAMP) assay that can amplify viral RNA in a single step, at a single temperature to detect and differentiate virulent from avirulent strains of AOAV based on genetic markers (the fusion gene). The preliminary data show that the new RT-LAMP assay can detect fusion gene RNA from in vitro transcription reactions (a control test) and NDV RNA from allantoic fluid in as little as 25 minutes. Testing also showed that the assay was able to detect NDV RNA from all 20 class II genotypes and approaches the detection limit of real-time RT-PCR. Under Objective 2, to identify mutations in the genome of low virulent NDV isolated from wild birds during adaptation to chickens, 9 isolates from various hosts and geographic location in the U.S. were selected by ARS researchers and transferred to the Auburn group after the isolates were confirmed to be safe to perform experiments in BSL-2 according to World Organization of Animal Health guideline. The Auburn researchers have passaged the isolates 10 times in eggs and the passaged isolates are being sequenced to identify mutations related to egg and host adaptation. Auburn researchers also made progress with developing ex vivo systems to understand the mechanisms of NDV evolution and adaptation. A soluble tetrameric protein, called HN, was expressed in cells to develop a protein histochemistry assay. Once functionality of the expressed protein is confirmed, it will be used to evaluate binding to different tissues and predict the pathogenic potential of circulating NDVs for chickens and turkeys. To identify whether humoral immune pressure can drive mutations in AOAV-1s, ARS researchers established an in ovo model to characterize viral evolution. A procedure was first set up with the LaSota vaccine strain of AOAV-1 (genotype II) which was incubated with homologous antisera then potential immune escapees are recovered in eggs. Serum treatments and rescues were repeated for 15 passages. Samples from each passage have been collected and will be subjected to next generation sequencing to identify mutations in the genome. The same procedure for passaging the viruses in the presence of antibody is being repeated with a virulent strain that belongs to a different genotype (genotype V). 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 to develop vaccines that induce high levels of IFN which improves efficacy. ARS researchers used knockout chicken and quail fibroblast cells which lack two key pathogen recognition receptors (TLR3 and MDA5) known to mediate innate immune responses to virus infection. Both low and high virulence AOAV-1s caused strong inhibition of the type I IFN response in avian fibroblast cells and did not induce a type I IFN response even in wild type cells. Interestingly, AOAV-1 induced a type III IFN response (which is known to have similar antiviral effect as type I IFNs that were originally targeted) indicating that the type III IFN response is not tightly associated with TLR3 and MDA5 expression. In addition, a recently isolated AOAV-1 strain was passaged five times in both wild type and the knockout cells to study, for the first time, the effect of the innate immune response on the evolution of AOAV-1s. The sequence analysis of the entire genome is pending. AOAV-1s strongly inhibit the IFN response through the V protein. Therefore, using reverse genetics to introduce mutations in the P gene that controls the expression of the V protein, ARS scientists knocked out the V protein function of the virus. Then, a gene that was previously identified to induce a high IFN response while also attenuating the virus, was incorporated into the AOAV-1 genome in its place. The resulting virus, with a disrupted V protein and a high interferon phenotype gene induced significant levels of type I and type III IFN. The newly generated high interferon inducing AOAV strains will be evaluated for safety and efficacy as a new vaccine candidate. To establish immunological methods to predict protection conferred by vaccination against NDV (i.e., correlates of protection), Auburn researchers conducted two animal trials with vaccinated and non-vaccinated specific pathogen free (SPF) and commercial white leghorn chickens with maternal antibody to the virus. A large number of differentially expressed genes (DEGs) were observed especially 48 hours after vaccination both in Harderian glands and tracheas. Immune-related DEGs that were significantly upregulated included: toll-like receptor and type I interferon signaling and response genes. Vaccinated birds also showed increased relative abundance of B cells, CD8, and CD4 lymphocytes compared to non-vaccinated birds. Maximum abundance of all cell types was detected on day 10. The relative abundance of T cells decreased thereafter while B cells maintained similar levels. ARS researchers are also developing microRNA (miRNA) based vaccines to improve vaccine yield to make vaccine production more efficient and to reduce mild disease that can occur from the current widely used live vaccines. Two initial screening steps were performed with 65 miRNAs and 10 miRNAs showed anti-viral roles and 8 miRNAs were pro-viral in function. Initial screening of miRNAs with more diverse AOAVs (genotype V and VII NDV) is in progress using multiple end point criteria such as viral plaque assays and quantitative RT-PCR for virus and host genes.

Accomplishments
1. Next-generation sequencing identifies important poultry pathogen in Mexico which is not found in the U.S. The use of next-generation sequencing (NGS) to identify pathogens in clinical samples is continually improving, providing information to identify the viral agents likely to cause disease and guide what vaccines to use for control efforts. As part of a project to improve NGS for diagnostics, ARS researchers in Athens, Georgia, tested samples collected from commercial chicken farms in Mexico and identified avian metapneumovirus, an important upper respiratory pathogen in poultry. For the first time, complete genomes of seven subtype A viruses, a subtype not found in the U.S., were analyzed and based on the sequence information, real-time reverse-transcriptase polymerase chain reaction test which is routinely used in diagnostic lab was evaluated and updated to quickly detect the virus in poultry samples.

2. An avian Orthoavulavirus-1 (AOAV-1) with a unique amino acid signature normally found in virulent virus was identified. Newcastle disease is one of the most important infectious diseases of poultry because of its potential for devastating loses. Not all AOAV-1 cause Newcastle disease, but the virus has ability to change their genetic makeup and become more virulent while adapting to new hosts and environments. ARS researchers in Athens, Georgia, identified a low virulent AOAV-1 isolate with a unique amino acid signature that is usually found in virulent viruses (i.e., NDV). Although the study confirmed that the virus is of low virulence, it is important to monitor whether viruses with a similar genetic makeup are circulating in poultry and any additional changes are occurring toward the more virulent form. It is also important to note that the new virus was detected by the official diagnostic test that was designed to specifically identifies virulent AOAV-1 (i.e., NDV). Thus, in addition to concern for potential pathogenic shift of the virus through additional genetic change, the finding warrants increased awareness of diagnosticians of potential false positive tests.

3. Knockout of immune regulatory gene function from avian cells enhance replication of AOAV-1, a Newcastle disease vaccine virus. With the recent advances in genetic modification techniques called the “CRISPR/Cas9” system, it is possible to produce cells that lack function of certain genes (gene knockout) more efficiently. Using the technique, ARS researchers in Athens, Georgia, in collaboration with the Ohio State University developed a chicken cell line that lost the function of two genes (TLR3 and MDA5) known to recognize viral pathogens and mediate the antiviral response. The newly developed chicken knockout cells showed better replication of Newcastle disease vaccine virus. In addition to its practical applicability in growing viruses, the knockout cells will be useful in studying the roles and relevance of those genes in viral replication and pathogenesis in avian species.

4. Genetically diverse Newcastle disease viruses circulate in wild and synanthropic birds in Ukraine. Newcastle disease virus (NDV) infects a wide range of bird species worldwide and is of importance to the poultry industry. Although certain virus genotypes are clearly associated with wild bird species, the role of those species in the movement of viruses and the migratory routes they follow is still unclear. ARS researchers in Athens, Georgia, analyzed 21,924 samples collected from wild and synanthropic birds in Ukraine from 2006 to 2015 and identified nineteen NDV sequences. Sequence analysis showed that synanthropic birds may play a role in viral transmission from vaccinated poultry to wild birds, which may also lead to the further spreading of vaccine viruses into other regions during wild bird migration. The study also highlights the possible exchange of NDV strains between wild waterfowl from the Azov-Black Sea region of Ukraine and waterfowl from different continents, including Europe, Asia, and Africa.

5. A portable genome sequencer can detect multiple pathogens in poultry clinical samples simultaneously. It is important to simultaneously detect multiple pathogens in clinical samples to manage diseases in poultry. ARS researchers in Athens, Georgia, demonstrated the feasibility of a portable sequencer (MinION) for identifying disease agents in clinical respiratory samples. NDV and various bacterial respiratory agents were detected in chicken samples collected from Kenyan live bird markets. The MinION platform also provided a rapid and accurate characterization of the co-infecting viral and bacterial pathogens in swab samples from experimentally infected birds. The MinION-based diagnostic approach provides a rapid, multiplexed, and cost-effective platform for the detection of viral and bacterial pathogens in clinical samples with sufficient sensitivity and represents an alternative for diagnostic laboratories that cannot afford more expensive equipment for next generation diagnostics.

Review Publications
Kariithi, H.M., Christy, N., Decanini, E.L., Lemiere, S., Volkening, J.D., Afonso, C.L., Suarez, D.L. 2022. Detection and genome sequence analysis of avian metapneumovirus subtype A viruses circulating in commercial chicken flocks in Mexico. Veterinary Sciences. 9(10):579. https://doi.org/10.3390/vetsci9100579.
Lee, C.W., Mahesh, K.C., Ngunjiri, J.M., Ghorbani, A., Lee, K. 2023. TLR3 and MDA5 knockout DF-1 cells enhance replication of avian orthoavulavirus 1. Avian Diseases. 67(1):94-101. https://doi.org/10.1637/aviandiseases-D-22-00065.
Kariithi, H.M., Suarez, D.L., Davis, J.F., Dufour-Zavala, L., Olivier, T.L., Williams Coplin, T.D., Bakre, A.A., Lee, C.W. 2023. Genome sequencing and characterization of an avian orthoavulavirus 1 VG/GA-like isolate with a unique fusion cleavage site motif. Avian Diseases. 67(1):33-41. https://doi.org/10.1637/aviandiseases-D-22-00064.
Butt, S.L., Kariithi, H.M., Volkening, J.D., Taylor, T.L., Leyson, C., Pantin Jackwood, M.J., Suarez, D.L., Stanton, J.B., Afonso, C.L. 2022. Comparable outcomes from long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory samples. Frontiers in Veterinary Science. 9:1073919. https://doi.org/10.3389/fvets.2022.1073919.
Goraichuck, I.V., Gerilovych, A., Bolotin, V., Solodiankin, O., Dimitrov, K.M., Rula, O., Muzyka, N., Mezinov, O., Stegniy, B., Kolesnyk, O., Pantin Jackwood, M.J., Miller, P.J., Afonso, C.L., Muzyka, D. 2023. Genetic diversity of Newcastle disease viruses circulating in wild and synanthropic birds in Ukraine between 2007 and 2015. Frontiers in Veterinary Science. 10:1026296. https://doi.org/10.3389/fvets.2023.1026296.
Goraichuk, I.V., Muzyka, D., Gaidash, O., Gerilovych, A., Stegniy, B., Pantin Jackwood, M.J., Miller, P.J., Afonso, C.L., Suarez, D.L. 2023. Complete genome sequence of an avian orthoavulavirus 13 strain detected in Ukraine. Microbiology Resource Announcements. 23. Article e00197. https://doi.org/10.1128/mra.00197-23.
Kariithi, H.M., Volkening, J.D., Chiwanga, G.H., Pantin Jackwood, M.J., Msoffe, P., Suarez, D.L. 2023. Genome sequences and characterization of chicken astrovirus and avian nephritis virus from Tanzanian live bird markets. Viruses. 15(6):1247. https://doi.org/10.3390/v15061247.
Kariithi, H.M., Volkening, J.D., Goraichuk, I.V., Ateya, L.O., Williams Coplin, T.D., Binepal, Y.S., Afonso, C.L., Suarez, D.L. 2023. Unique variants of avian coronaviruses from indigenous chickens in Kenya. Viruses. 15(2):264. https://doi.org/10.3390/v15020264.
Kariithi, H.M., Volkening, J.D., Alves, V.V., Reis-Cunha, J.L., Arantes, L.C., Fernando, F.S., Filho, T.F., Martins, N.R., Lemiere, S., Neto, O.C., Decanini, E.L., Afonso, C.L., Suarez, D.L. 2023. Complete genome sequences of avian metapneumovirus subtype B vaccine strains from Brazil. Microbiology Resource Announcements. 12(6):e00235-23. https://doi.org/10.1128/JVI.01567-16.