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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Research Project #422701

Research Project: Intervention Strategies to Control and Prevent Enteric Viral Diseases of Poultry

Location: Endemic Poultry Viral Diseases Research

2015 Annual Report


1a. Objectives (from AD-416):
1. Determine the virus microbiome of poultry from different geographical regions in the United States and complete the full genome sequence of poultry enteric viruses using metagenomics approaches. 2. Develop molecular diagnostic tests to determine the prevalence of novel poultry enteric viruses on poultry farms and their geographical distribution. 3. Identify host and/or viral genetic determinants that control pathogenicity, transmission, and drive the evolution of novel poultry enteric viruses. 4. Develop molecular vaccine platforms that will lead to highly efficacious vaccines that have been rationally designed to control enteric diseases of poultry, including mass delivery capability and companion diagnostics to differentiate naturally infected from vaccinated birds.


1b. Approach (from AD-416):
There is a pressing need to identify the novel viruses present in the poultry gut—an important first step in determining their role(s) in enteric disease and production losses. Recent efforts in our laboratory using the next generation of nucleic acid sequencing and related techniques to discover and characterize novel viruses in the poultry gut have been very successful, and suggest that our knowledge of the poultry gut virus community is incomplete. It is possible that unidentified viruses or viral communities may play specific roles in enteric disease syndromes and can act as predictors of enteric disease. Therefore, the use of high-throughput, sequence-independent pyrosequencing technology (next generation sequencing and associated technologies) and metagenomic analysis techniques will allow the discovery and characterization of novel RNA and DNA poultry enteric viruses and viral communities, allowing the association of certain etiologic agents with the poultry enteric disease syndromes noted in the field. Proper and effective management of poultry enteric disease will require novel, up-to-date diagnostic assays in order to determine the prevalence of enteric viruses on farms and to characterize the pathology caused by enteric viral infection. Based upon the full genome and/or gene sequences of novel poultry enteric viruses discovered and initially characterized using high-throughput pyrosequencing and metagenomic analyses, conserved sequences will be identified as targets for molecular diagnostic tests. These tests will be designed and validated in our laboratory and made available to industry partners and the research community at large. Finally, a successful control strategy for poultry enteric disease must include novel vaccine platforms that have been specifically designed to improve flock performance, lessen disease severity, and reduce viral transmission in the field. A targeted approach will be used to design recombinant (viral) vaccines using live turkey/chicken enterotropic viruses as expression vectors for specific enteric virus proteins deemed to be disease-associated using bioinformatic approaches to analyze enteric viral nucleic acid.


3. Progress Report:
Enteric viral diseases of poultry are responsible for substantial economic losses to the poultry industry in the United States and abroad. To discover novel vaccine platforms and vaccination strategies that can be integrated into effective prevention and control programs of poultry enteric diseases (and in line with Objective 3 of the current project), project scientists are developing enterotropic recombinant vaccine vectors using a Newcastle disease virus (NDV) vaccine strain. Infectious clones containing targeted enteric viral antigens, such as chicken parvovirus VP2 gene and turkey corona virus S gene, have been constructed. The recombinant viruses rescued from these infectious clones are currently being evaluated as vaccine candidates in cell cultures and animals. Further, there is a concern that the maternally derived antibodies (MDA) against Newcastle disease virus (NDV) in chicks may compromise the efficacy of NDV vectored vaccines. To address this concern, project scientists are evaluating NDV LaSota vaccine strain-based recombinant viruses, expressing the glycoproteins B (gB) or D (gD) of infectious laryngotracheitis virus (ILTV) in broiler chickens in the presence of MDA against NDV. The results suggest that the NDV MDA moderately suppressed the immune response to the NDV vector. However, the ILTV antigens expressed from the NDV vector were still sufficient to confer a significant protection against infectious laryngotracheitis (ILT) clinical disease. In line with Objectives 1 and 2 of the current project, project scientists have investigated the species specificity, molecular phylogenetics, and pathogenesis of novel viruses initially discovered in the poultry gut using a metagenomic approach. These investigations have been facilitated by poultry enteric virus molecular diagnostic assays designed and validated by project scientists. Specifically, analysis of the full-length genomes of turkey and chicken enteric parvoviruses suggests that these viruses have diverged from a common ancestor and subsequently experienced host-specific adaptation. Further, the species specificity observed in the turkey and chicken parvoviruses is primarily influenced by the parvovirus VP1 structural protein. Comparative metagenomic analyses of the intestinal viral community in broiler chickens, along with experimental work in commercial turkeys, is revealing a role for the poultry enteric picornaviruses in the performance problems and enteric syndromes noted in both turkeys and chickens in the field. Specifically, turkeys experimentally infected with picornavirus-positive intestinal homogenates experienced significant reduction in weight gain over the course of three weeks and continually shed picornavirus in feces during that time.


4. Accomplishments
1. A novel approach for foreign gene expression by Newcastle disease virus (NDV). This approach involves expression of a foreign gene from a modified Newcastle disease virus NDV backbone in order to improve the efficacy of NDV-vectored vaccines. Six NDV LaSota strain-based recombinant viruses were engineered in the laboratory using reverse genetics technology. Quantitative measurements were used to test the expression of foreign genes in the laboratory, and results suggested that the level of foreign gene expression could be regulated using this approach to maximize the efficacy of vaccine and gene therapy.

2. Investigating thermostable recombinant Newcastle disease virus vaccines. Thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease (ND) for village flocks, since they do not rely on cold chains for delivery and storage. To explore the potential use of a thermostable NDV as a vaccine vector, the thermostable NDV vaccine strain TS09-C vectoring a green fluorescent protein (GFP) gene was generated using reverse genetics technology. The recombinant virus, rTS-GFP/M, retained the same thermostability as its parental virus. Vaccination of specific pathogen free (SPF) chickens with the rTS-GFP/M virus conferred complete protection against virulent NDV challenge. The results suggested that the rTS09-C virus could be used as a vaccine vector to develop bivalent thermostable vaccines against ND and the target avian diseases for village chickens, especially in the developing and least-developed countries.

3. Comparative metagenomic analysis of the bacterial and viral communities in the chicken gut. This investigation provided insight into the colonization of specific pathogen free (SPF) chickens by intestinal microorganisms under field conditions and compared the pre-contact intestinal bacterial and viral communities (the gut “microbiome”) with the altered gut microbiome following contact with poultry raised in the field. Analysis of the intestinal virus community from contact birds (“sentinels”) placed on farms revealed colonization of the gut by viruses that were not present in pre-contact birds or were present in proportionally lower numbers. Analysis of the sentinel gut bacterial community revealed an altered community in the post-contact birds, notably by members of the Lachnospiracea/Clostridium and Lactobacillus groups of bacteria. This investigation continues our analysis of novel viruses (picornaviruses and picobirnaviruses) that have only recently been described in the poultry and avian gut; certain members of these groups of viruses are associated with production losses in poultry, and it is important to characterize these novel viruses—including their interactions with intestinal bacteria—in order to inform targeted interventions in the field, and in order to understand their role(s) in avian health and disease.

4. Investigating the role of the novel enteric turkey picornaviruses in performance problems in young turkeys. Previous investigations of the intestinal viral community in the poultry gut have provided some insight into the geographical distribution and the rapidly evolving taxonomy of the avian enteric picornaviruses. The present investigation involved a comparative metagenomic analysis of the gut virome from a healthy turkey flock versus a flock placed in the field and experienced signs of enteric disease. This investigation revealed a number of enteric picornavirus sequences that were present in the commercial birds in the field but that were completely absent in the healthy flock. A novel molecular diagnostic assay was designed and used to track the shedding of field strains of turkey enteric picornavirus in commercial poults inoculated with picornavirus-positive intestinal homogenates prepared from turkeys that were experiencing moderate enteric disease. The propagation of this novel enteric picornavirus in commercial poults, resulted in significant reduction in weight gain, and suggests that this common inhabitant of the turkey gut may result in performance problems or enteric disease in the field.

5. Phylogenetic analysis of novel chicken and turkey parvoviruses. Parvoviruses have been identified in chickens and turkeys exhibiting enteric disease characterized by stunting, diarrhea, and mortality. Previous phylogenetic analysis comparing the nonstructural (NS) parvovirus gene sequences revealed a strong similarity between the chicken and turkey parvoviruses. It was also evident that most of the chicken and turkey parvovirus strains formed distinct phylogenetic groups, suggesting that these viruses might have diverged from a common ancestor and subsequently experienced host-specific adaptation. In our current studies we further characterized the evolutionary and biological relationships of chicken and turkey parvoviruses with the use of comparative full-length genome sequence analyses and studied host-specific virus replication after infection of day-old chickens and turkeys. Our results suggest that both the chicken and turkey parvoviruses are highly adapted to their respective host species, and the viral structural VP1 gene is primarily responsible for this host specificity. Based on the strong species specificity, it is not likely that potential heterologous, cross-species infection with either of these parvoviruses would play an important role in the epidemiology of the parvovirus-induced enteric diseases in chickens or turkeys.


Review Publications
Zhao, W., Zhang, Z., Zsak, L., Yu, Q. 2015. P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression. Journal of General Virology. 96:40-45.
Day, J.M., Oakley, B., Seal, B.S., Zsak, L. 2015. Comparative analysis of the intestinal bacterial and RNA viral communities from sentinel birds placed on selected broiler chicken farms. PLoS One. 10(1):e0117210. doi: 10.1371/journal.pone.0117210.
Yu, Q. 2015. Have we found an optimal insertion site in a Newcastle disease virus vector to express a foreign gene for vaccine and gene therapy purposes. British Journal of Virology. 2(2):15-18.
Zsak, L., Cha, R., Li, F., Day, J.M. 2014. Host specificity and phylogenetic relationships of chicken and turkey parvoviruses. Avian Diseases. 59(1):157-161. doi: 10.1637/10939-092414-ResNote.
Day, J.M., Zsak, L. 2015. Investigating turkey enteric picornavirus and its association with enteric disease in poults. Avian Diseases. 59(1):138-142. doi: 10.1637/10940-092414-RegR.
Zhao, W., Spatz, S.J., Zhang, Z., Wen, G., Garcia, M., Zsak, L., Yu, Q. 2014. Newcastle disease virus (NDV) recombinants expressing infectious laryngotracheitis virus (ILTV) glycoproteins gB and gD protect chickens against ILTV and NDV challenges. Journal of Virology. 88:8397-8406. doi: 10.1128/JVI.01321-14.
Sun, J., Wei, Y., Rauf, A., Zhang, Y., Ma, Y., Zhang, X., Shilo, K., Yu, Q., Saif, Y.M., Lu, X., Yu, L., Li, J. 2014. Methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with the homologous Colorado strain and the heterologous Minnesota strain. Journal of Virology. 88(21):12348-12363. doi: 10.1128/JVI.01095-14.
Bai, F., Yu, Y., Tian, H., Ren, G., Wang, H., Zhou, B., Han, X., Yu, Q., Li, D. 2014. Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy. Cancer Biology & Therapy. 15(9):1226-1238. doi: 10.4161/cbt.29686.
Cardenas-Garcia, S., Diel, D., Susta, L., Lucio-Decanini, E., Yu, Q., Brown, C.C., Miller, P.J., Afonso, C.L. 2014. Development of an improved vaccine evaluation protocol to compare the efficacy of Newcastle disease vaccines. Biologicals. 43:136-145. doi: 10.1016/j.biologicals.2014.11.003.
Wen, G., Chen, C., Guo, J., Zhang, Z., Shang, Y., Shao, H., Luo, Q., Yang, J., Wang, H., Wang, H., Zhang, T., Zhang, R., Cheng, G., Yu, Q. 2015. Development of a novel thermostable Newcastle disease virus vaccine vector for expression of a heterologous gene. Journal of General Virology. 96:1219-1228. doi: 10.1099/vir.0.000067.