Objective 1. Identification of circulating and emerging Newcastle disease viruses, including conducting prevalence studies for NDV in poultry and in synanthropic birds from countries where virulent NDV strains are endemic to determine the presence of variant and emerging viruses in vaccinated poultry and in wild birds, and developing rapid identification assays for variant NDV strains. Objective 2. Identify agents that may cause NDV vaccine failures in endemic countries, including NDV variants and co-infecting agents that may immuno-compromise animals or enhance disease in vaccinated poultry flocks. Objective 3. Develop predictive biology strategies for risk assessment of virus evolution, including developing predictive biology strategies using NexGen (next generation) sequencing to evaluate the rate of change in different virulent NDV strains from unvaccinated, sub-optimally vaccinated, and well-vaccinated poultry. Objective 4. Develop improved NDV vaccines platforms, including identifying and evaluating effective and user friendly NDV vaccine platforms for in ovo or one-day old broilers, and identifying and characterizing protective immune responses for new vaccines platforms that are effective in ovo or in one-day old broilers.
Identification and characterization of new variants will be addressed by conducting active surveillance, and characterization of new isolates, and by developing rapid diagnostic assays that assures appropriate detection of these exotic samples (objective 1). Identifying immune suppressing agents and Newcastle disease viruses (NDV) variants that may cause disease in vaccinated animals will address the inadequate efficacy of commercial vaccines in endemic countries (Objective 2). Predictive strategies for identifying vaccines and vaccination practices that cause emergence of variant viruses will be addressed by Next Generation (NexGen) sequencing of viruses that emerge under different vaccination regimes (Objective 3). Reduced efficacy of commercial vaccines in young chickens with maternal antibodies will be addressed by developing an improved vaccine platform based in vector that is unknown to chickens (Objective 4).
This is the final report for this project, which will be replaced with a bridging project pending completion of research review. The research review was delayed 6 months due to the two vacant scientist positions. The goal was to identify circulating and emerging Newcastle disease viruses from countries where virulent Newcastle disease virus (NDV) strains are endemic to determine the presence of variant and emerging viruses in vaccinated poultry and in wild birds and developing rapid identification assays for variant NDV strains. As part of a Department of Defense supported project, samples were obtained from poultry and synanthropic birds from Kenya farms and live bird markets. Virulent Newcastle disease virus was isolated, sequenced, and the representative isolates were tested in animal studies. A new and unique genotype, V.3, was characterized. Additional samples were also tested from Tanzania as part of a separate study with 3 different genotypes of virus identified. The second major activity was the use of 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. Efforts to improve the sensitivity of this method through the depletion of non-target sequence proved successful. Multiple co-infecting pathogens including chicken parvoviruses, infectious bronchitis virus, fowl aviadenovirus D viruses, sicinivirus and other viruses were sequenced. The goal was to develop improved NDV vaccines platforms, including identifying and evaluating effective and user friendly NDV vaccine platforms for in ovo or one-day old vaccinated broilers. Several separate studies were conducted that examined new vaccine approaches for the control of Newcastle disease virus. Previous studies conducted at the Southeast Poultry Research Laboratory in Athens, Georgia, have documented that optimal vaccine protection is achieved when the vaccine being administered is closely matched to the virus strain circulating in the field. Most vaccines in the U.S. and around the world use live attenuated or inactivated virus that are genetically distinctly different from the virulent circulating strains and use of these vaccines can result in reduced protection and contribute to vaccine failures. Research was conducted on an Adenovirus viral-vectored vaccine that only expressed the fusion protein of NDV, a sub-unit vaccine. The results demonstrated that matching the vaccine to the challenge strain provided better protection. Thus, protection can be achieved after vaccination from various NDV proteins, however, matching of the fusion protein is important for optimal protection. In a separate set of studies, the use of a reverse genetics Newcastle disease vaccine virus that was expressing the IL-4 gene in reverse orientation was used with in ovo vaccination studies. Using typical NDV vaccine strains in ovo resulted in high mortality in embryos when administered at 18 days of incubation. The new vaccine virus was shown to be more attenuated in embryonating chicken eggs, and that vaccinated chicks would develop early protection when challenged from virulent virus. Studies were also performed demonstrating that eggs with the maternal antibody to NDV could still be protected using this vaccine strain. This data shows a promising approach to vaccinating in ovo, which is a convenient mass vaccination approach in broiler chickens. Progress was made on a new vectored vaccine for Newcastle disease virus using the avian paramyxovirus serotype 10 (APMV-10) virus. Using a reverse genetics approach, the APMV-10 vector was modified to express the fusion protein of Newcastle disease virus. This vaccine virus was shown to provide protective antibody after vaccination and was protective against virulent challenge. The APMV-10 vector originally comes from penguins, and poultry are not naturally exposed to it. Therefore, this new vaccine has the potential for use as a mass vaccination approach in the field. Substantial progress was made on all four objectives of the project in spite of departure of the two primary scientists working on the project. In Objective 1, the primary achievement of the project was response to the virulent NDV outbreak that occurred in 2018-2020 in California. The research team mobilized to characterize the new viruses and showed that they were similar to the 2002-2003 outbreak viruses from California and that the research data from that outbreak could be applied to this outbreak. Vaccines studies were also performed to show that both live attenuated NDV vaccines and HVT-viral vectored NDV vaccines were protective for the outbreak strain. Other foreign and domestic Newcastle disease viruses were evaluated in animal studies. One highlight was the determination that pigeon and cormorant adapted Newcastle disease viruses did not replicate or transmit well in chickens, which suggests a reduced risk of these viruses to cause poultry outbreaks. Considerable progress was made to develop Next Generation Sequencing into a useful and practical tool to identify viral and bacterial sequences from poultry samples. The use of NGS as both a research and diagnostic tool were demonstrated on clinical samples, and the improvements made to the sensitivity of the technique greatly increased the amount of usable sequence. In Objective 2 progress was made on identifying co-infections that likely led to increased clinical disease in flocks infected with virulent NDV. Using NGS technology and a random amplification approach, multiple viruses were fully sequenced and the information about the viruses were publicly released in GenBank. The identification and full genome sequencing identified for the first time viruses in the United States, i.e. sicinivirus or detection for the first time of viruses in different parts of the country, i.e. The NGS technology used in Objective 2 was also used in Objective 3 and because of the depth of coverage of the sequence, unique variants could be identified as subpopulations in a viral sample. Considerable progress was made in Objective 4 to look for new and improved ways to vaccinate poultry for Newcastle disease virus. A new vectored-vaccine was developed using the avian paramyxovirus 10 virus as the vector and it was demonstrated to protect chickens in laboratory studies. A new attenuated NDV isolate was developed that could be used in ovo to protect young chickens from virulent challenge. This was shown in both specific pathogen free chickens and commercial chickens that had maternal antibody to NDV. Finally, we evaluated a new adenovirus-vectored system and showed that matching the vaccine sequence to that of the field strain provided the best protection. The idea of matching the vaccine to the field strain continues to be championed by the laboratory as the best way to protect chickens from clinical disease in the field.
1. Diagnostic development of Next Generation Sequencing (NGS) to identify disease pathogens. The use of NGS as a diagnostic tool to identify and fully sequence any pathogen in a sample has continued to improve and allows for a more complete picture for poultry health. As part of an effort to improve the sensitivity of NGS from clinical samples, ARS researchers in Athens, Georgia, have identified and sequenced the full genomes of fowl aviadenovirus D, chicken parvoviruses, sicinivirus and infectious bronchitis virus. Having the full sequence information provides a better epidemiologic assessment of viruses circulating in poultry and improves our understanding of how co-infection of different pathogens may influence clinical disease. The sequence information, with information on the source of the samples, has been provided to the public through the GenBank and allows greater access to all poultry scientists to better understand disease co-infections and improve diagnostic testing.
2. An adenovirus-vectored vaccine to understand protection for Newcastle disease virus. The use of live virus-vectored vaccines for Newcastle disease virus and other pathogens is increasing in poultry because of proven protection and reduced vaccine reactions. ARS researchers in Athens, Georgia, used an adenovirus to express the fusion protein of Newcastle disease virus, demonstrated that the immune response to the fusion protein was effective by itself and that matching the sequence of the vaccine to the challenge virus provided better protection. Previous studies have shown that whole virus vaccines, either live or inactivated, had better protection when the vaccines were closely matched to the challenge strain. This study provides evidence that matching just part of the virus, the fusion protein, also has better protection when matched to the challenge virus. Most commercial vaccines use genotype 1 or 2 Newcastle disease viruses, but virulent circulating strains are other genotypes that differ in sequence by 10-20% and current vaccines are not providing optimal immunity. This study shows the adenovirus system to be a useful vaccine vector and that matching the vaccine to the challenge virus, even with sub-unit vaccines will provide the best protection.
3. Surveillance in Kenya shows that virulent Newcastle disease is endemic in the live bird markets in the country. Virulent Newcastle disease virus is normally a foreign animal disease to the United States, but the virus is endemic in many parts of the world and can be introduced into the United States as occurred in 2002 and 2018. The Kenya study conducted by ARS researchers in Athens, Georgia, examined samples from farms and live bird markets. The study demonstrated a high prevalence of virulent Newcastle disease virus in the market system due to the constant mixing of birds. The viruses detected were sequenced and shown to be a unique variant, V.3, that has only been found in the Kenya region of Africa. This project was funded through a U.S. Defense Department project and provided the opportunity to collect, study, isolate and sequence Newcastle disease viruses from Kenya. These viruses could present a problem to the United States should they be introduced to U.S. poultry. Ongoing research and efforts to manage the spread of virulent Newcastle disease viruses circulating around the world is critical for assuring effective diagnostic tests and vaccine availability to combat new virus strains if introduced into the United States.
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Goraichuk, I.V., Davis, J.F., Parris, D.J., Kariithi, H.M., Afonso, C.L., Suarez, D.L. 2021. Near-complete genome sequences of five sicinivirus from North America. Microbiology Resource Announcements. 10(19):e00364-21. https://doi.org/10.1128/MRA.00364-21.