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).
All objectives have been met, with a higher number of epidemiological objectives due to the development of new collaborations with international organizations. During 2016 over 300 isolates have been characterized, including viruses from Nigeria, Bulgaria, Ukraine, Indonesia, Pakistan, Egypt, and the United States. We have sequenced 336 samples by Next Generation Sequencing (NGS) and of these we have obtained 126 nearly complete genomes. Since July 2016 we have submitted to GenBank 78 complete fusion gene sequences, 22 complete genomes and 2 avian infectious bronchitis virus (IBV) complete genomes. International collaborations are essential to the study of Newcastle disease virus (NDV). Virulent NDV (VNDv) is present in the United States (U.S.), however it is only present in cormorants and pigeons, and is not normally found in domestic poultry. However, virulent NDV is endemic in many countries commercially linked to the U.S., including Mexico. Whether in domestic poultry or wild birds, VNDv strains remain a threat to all producers of poultry. The longstanding collaboration between the Agricultural Research Service (ARS) Southeast Poultry Research Laboratory (SEPRL) and Mexico, Ukraine, Egypt, Indonesia and Pakistan has been maintained as a result of those agreements, and the U.S. has obtained important epidemiological information on the movement and evolution of the Newcastle disease virus in Asia. We have also performed training with collaborators that have improved their capabilities to rapidly detect, identify, and characterize new strains of viruses. To address the issue of endemicity and circulation of virulent Newcastle disease virus epidemiological, studies were conducted on backyard poultry, wild birds and pet birds. It was found that backyard poultry, pets and wild birds in other countries can carry virulent strains of NDV. In contrast evaluation of wild birds in the U.S. demonstrate that with the exception of pigeon and cormorants many other types of waterfowl and shorebirds do not carry virulent NDV. In the area of epidemiology and diagnostics our laboratory has contributed to the better characterization of Newcastle disease viruses. As a part of an international team we have contributed to the latest (2016) update on the Taxonomy of the order Mononegavirales as accepted by the International Committee on Taxonomy of Viruses (ICTV). In order to clarify the confusing origin and classification of new Newcastle disease virus isolates we have reviewed the most recent epidemiological and evolutionary studies on Newcastle disease viruses. ARS scientists in Athens, Georgia have further enhanced our knowledge of the pathogenic potential of new isolates and described the pathogenesis of new strains of Newcastle disease virus from the U.S. and abroad. We recently published an article entitled “Natural Infections With Pigeon Paramyxovirus Serotype 1: Pathologic Changes in Eurasian Collared-Doves (Streptopelia decaocto) and Rock Pigeons (Columba livia) in the United States”. To address objective 4, development of new vaccine friendly platforms and in ovo vaccine containing an interleukin 4 gene is being evaluated. Results suggest significant attenuation in comparison to the current LaSota vaccine.
1. Identification of epidemiological connections between vaccinated poultry and wild birds. To identify new epidemiological links between Newcastle disease virus (NDV) from vaccinated poultry and synantropic birds ARS researchers in Athens, Georgia, conducted field studies in Atlanta pigeons and performed mega data analysis of GenBank genetic databases. These studies demonstrated that vaccine viruses may co-circulate between synatropic birds and poultry in North America and across the globe. The presence of vaccine-derived Newcastle disease viruses in wild birds was identified on 17 different species of wild birds across four continents from 1997 through 2014. Furthermore, ARS scientists in Athens, Georgia, demonstrated that the most commonly used vaccine like viruses were detected in pigeons (n = 23), and ducks (n = 13). Vaccine like viruses, corresponding to the most widely used vaccine types, were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). These results and the ubiquitous nature of wild pigeons highlighted their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The demonstration of spillover of live vaccines from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines is an underappreciated and poorly studied effect of human activity on wildlife and our data suggest that further studies are needed.
2. Identification of previously unrecognized genetic diversity in avian paramyxovirus serotype 1 (APMV-1) of low virulence isolated from wild birds. To determine the presence of variant and emerging viruses in wild birds we collected swabs from multiple wild bird species in North America. ARS researchers in Athens, Georgia obtained the genetic sequence of 58 APMV-1 isolates recovered from thirteen species of wild birds sampled throughout the USA during 2007-2014. The work conducted in collaboration with the United States Geological Service in Alaska and the University of Georgia identified previously unknown genetic diversity of APMV-1 in wild birds in North America. Because more recent viruses were related to viruses circulating in North America during previous years, we determined that diversity is likely a function of continued viral evolution in reservoir hosts. We did not, however, find support for the emergence or maintenance of APMV-1 strains predicted to be pathogenic to poultry in wild birds of North America outside of the order Suliformes (i.e., cormorants) and Columbiformes (pigeons).
3. Demonstration of the existence of previously unrecognized genetic diversity of highly virulent viruses in Eastern Europe. ARS researchers in Athens, Georgia, demonstrated the repeated isolation of virulent Newcastle disease viruses (VNDv) of sub-genotype VIId from backyard chickens in Bulgaria and Ukraine between 2002 and 2013, confirming the existence of Newcastle disease virus variants in virulent viruses in backyard poultry in Eastern Europe. The epidemiology suggests the possibility of a "domestic" or "urban" cycle of maintenance. The identification of previous unrecognized genetic diversity of virulent avian paramyxovirus serotype 1 in poultry will be used for the development of more specific diagnostics and vaccines that match the circulating viruses.
4. Development of effective identification assays for variant Newcastle disease virus (NDV) strains. To address the need for rapid identification assays for variant NDV strains, ARS researchers in Athens, Georgia have developed a technique that allows ultra-deep sequencing of nucleic acids without using target-specific primers. We produced a robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses used by our laboratory as well as the sequencing of unknown agents. We demonstrated that the new technique allows detection of unsuspected variants and co-infecting agents. The combination of multiplexing next generation sequencing (NGS) technology with the publically available bioinformatics platforms resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length avian paramyxovirus (APMV) genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some samples. Development of robust cost and effective NGS identification assays for variant NDV strains will lead to more specific diagnosis of causes of vaccine failures and to the development of more specific diagnostic agents.
5. Characterization of variant Newcastle disease virus (NDV) from recent outbreaks in countries in which the disease is endemic. To address the need to identify variants that may cause NDV vaccine failures in endemic countries, ARS researchers in Athens, Georgia conducted isolation and characterization of recent NDV variants from farms that reported vaccine failures and nearby areas in Pakistan. Highly virulent Newcastle disease viruses were found in poultry and captive non-poultry avian species in Pakistan from 2011 to 2016 demonstrating that virulent viruses of the sub-genotype VIIi in vaccinated poultry are epidemiologically linked to viruses present in pets, backyard chickens and wild birds. We also demonstrated the presence of highly virulent viruses in a clinically healthy duck (Anas platyrhynchos domesticus) and in pigeons in Pakistan.
6. Identification of co-infecting agents. ARS researchers in Athens, Georgia, determined that birds infected with Newcastle disease were often co-infected with H9N2 low pathogenic avian influenza in Pakistan. As part of the same objective we identified and characterized new avian paramyxoviruses (APMV). The characterization of multiple isolates of APMV of serotypes 10 and 13 was done utilizing next generation sequencing and the complete genomic sequence was made publically available. The awareness of mixed infection with multiple agents and the Newcastle disease virus variants will lead to more specific diagnostics and control strategies.
7. Effect of multiple challenges on vaccine performance. To understand the causes of vaccine failures ARS researchers in Athens, Georgia demonstrated that vaccine failures in the field are not caused by multiple successive challenges with virulent virus or by high challenge doses that may be capable of overwhelming the immune system. We conducted repeated challenges with high doses of virulent Newcastle disease virus and demonstrated that repeated challenges do not decrease the efficacy of currently used vaccines. The work is important because it demonstrates that additional factors are yet to be identified as being responsible for field vaccine failures while re-assuring the producer on the efficacy of current vaccines.
8. Role of codon usage on Newcastle disease virus (NDV) evolution. To develop predictive biology strategies for risk assessment of virus evolution ARS researchers in Athens, Georgia, compared codon usage and codon adaptation indexes among groups of NDV that differ in biological, ecological, and genetic characteristics. We found distinctive codon usage for the two avian paramyxovirus serotype 1 (APMV-1) classes and for different transcriptional regions. The data suggested that codon usage has changed significantly since the two APMV-1 classes diverged, however codon adaptation in APMV-1 occurs through a slow evolutionary process and is not likely to have a significant role in the increased virulence of new genotypes. The identification of genomic regions with faster rates of codon usage adaptation may lead to the development of more focused predicted studies.
9. Development of cell lines to grow vaccine viruses. To address the need to develop new technologies that may lead to the production of improved Newcastle disease virus (NDV) vaccines, ARS researchers in Athens, Georia, developed chicken induced pluripotent stem cells that are tolerant to Newcastle disease virus and therefore more likely to produce higher titers of vaccine viruses. This was accomplished by creating induced pluripotent stem cell (iPSC) to reprogram the adult cells into an embryonic stem cell-like state that were permissive to infection with NDV and then by selecting for increased resistance through multiple rounds of infection. Since ciPSCs that survived infection demonstrated the ability to recover quickly and to become re-infected these have potential to be used to produce NDV vaccines. These new technologies may facilitate the production of high titer vaccines in cell culture.
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Goraichuk, I.V., Dimitrov, K.M., Sharma, P., Miller, P.J., Swayne, D.E., Suarez, D.L., Afonso, C.L. 2017. Complete genome sequences of four avian paramyxoviruses of serotype 10 isolated from Rockhopper Penguins on the Falkland Islands. Genome Announcements. 5:e000472-17. doi:org/10.1128/genomeA.00472-17.
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