Location: Endemic Poultry Viral Diseases Research2014 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:
To design and develop novel vaccine platforms and vaccination strategies that can be integrated into effective prevention, intervention and control programs of poultry enteric diseases, project scientists are developing an enterotropic recombinant vaccine vector using a Newcastle disease virus (NDV) vaccine strain. A systematic evaluation of insertion sites in the NDV vaccine vector has demonstrated that the noncoding region between the P and M genes is the optimal insertion site for foreign genes expression. Infectious clones containing targeted enteric viral antigens, such as chicken enteric parvovirus VP2 gene and turkey enteric coronavirus S gene, in the optimal insertion site have been constructed. The recombinant viruses rescued from these infectious clones are currently being evaluated as vaccine candidates in cell cultures and animals. The construction of these infectious clones that can be used to express foreign genes from multiple viruses implicated in enteric disease or identified as novel enteric viruses is consistent with the milestones laid out in the project for the development of successful control strategies. The turkey enteric picornaviruses initially identified by project scientists during comparative metagenomic analyses are truly novel viruses, and very little is known about their growth and shedding in young turkeys and chicks. Further, propagation platforms for the novel enteric viruses do not exist, complicating their characterization. In line with the milestone projections for the project, project scientists have been utilizing their newly developed diagnostic assays for the novel turkey enteric picornaviruses and picobirnaviruses to experimentally track the propagation and shedding of these viruses in young commercial turkeys over time. These experiments have also allowed the generation of hyper-immune sera to the enteric picornaviruses. Finally, the generation of hyper-immune sera in young turkeys to re-emerging and novel turkey enteric coronaviruses has allowed the characterization of infectious clones expressing the turkey enteric coronavirus S glycoprotein gene. The continued progress of this project is supported by a grant.
1. Molecular diagnostics and characterization of an enteric picobirnavirus. A fully validated molecular diagnostic assay targeting the novel turkey picobirnaviruses (PBV) has been developed. The novel PBVs were first detected via metagenomic analyses of the turkey gut virome by project scientists, resulting in the first avian PBV sequence deposited in the public databases. Phylogenetic analysis comparing novel North Carolina turkey-origin PBV RNA-dependent RNA-polymerase partial coding regions, with genogroup I and II PBVs detected in several mammal species, revealed the turkey-origin PBVs are unique among the available sequences. Interestingly, the turkey origin PBVs could be genotyped as genogroup I PBVs, but do not appear to group closely with representative members of the recognized genogroup I or II PBVs, suggesting the turkey PBVs have relatively recently diverged from a genogroup I PBV.
2. Characterization of re-emerging turkey enteric coronaviruses circulating in the United States. In cooperation with industry stakeholders, an ongoing outbreak of turkey enteric coronavirus (TCoV) was investigated by project scientists, and the re-emerging TCoVs were characterized phylogenetically. In response to requests from several industry stakeholders, and in the absence of public laboratories able to provide rapid diagnostic services, numerous field samples were characterized via an in-house real-time reverse transcription polymerase chain reaction assay for TCoV. The results of the real-time assay allowed industry veterinarians to make management decisions in a timely manner, and proved to be a good indicator of subsequent seroconversion by TCoV positive flocks. Project scientists further completed metagenomic analysis, sequencing, and phylogenetic analysis of these re-emerging TCoV isolates, demonstrating that they are unique when compared to past isolates of TCoV and demonstrating their geographical distribution.
3. Characterization of enterotropic Newcastle disease virus (NDV) for use in recombinant vaccines. Many low-virulent enteric NDV strains have a larger hemagglutinin-neuraminidase (HN) protein with additional 39 amino acids (aa) at its C-terminus when compared with that of virulent respirotropic NDV strains. Therefore, it has been suspected that the HN C-terminal extension may contribute to the enteric tropism and reduced virulence. To develop a safe and enterotropic NDV vaccine vector, project scientists investigated the role of the HN C-terminal extension in tissue tropism and virulence using reverse genetics technology. The results showed that the HN C-terminal extension slightly attenuated the virus pathogenicity in embryonated eggs and in day-old chicks, but did not alter virus tissue tropism. Thus, the enteric NDV vaccine strains, such as V4 and PHY LMV42 which bear the HN C-terminal extension, can be used as an enteric vaccine vector to deliver an enteric virus antigen as a bivalent vaccine.
4. Characterization of the LaSota Newcastle disease virus vaccine strain for use as a recombinant, bivalent vaccine in chickens. Chicken infectious laryngotracheitis (ILT) and Newcastle disease (ND) are two of the most economically important infectious diseases of poultry. The current commercial ILT vaccines are either not safe or less effective. To develop safer and more efficacious ILT vaccines, project scientists generated Newcastle disease virus (NDV) LaSota vaccine strain-based recombinant viruses expressing the glycoproteins B (gB) and D (gD) of infectious laryngotracheitis virus (ILTV) using reverse genetics technology. These recombinant viruses were safe, stable and immunogenic, and replicated efficiently in birds. Vaccination of chickens with these recombinant viruses conferred complete protection against ILTV and NDV challenge. These novel bivalent vaccines can be mass-administered via aerosol or drinking water to large chicken populations at low cost, which will have a direct impact on poultry health, fitness and performance.
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Zhao, W., Hu, H., Zsak, L., Yu, Q., Yang, Z. 2013. HN gene c-terminal extension of Newcastle disease virus is not the determinant of the enteric tropism. Virus Genes. 47:27-33.
Zhao, W., Hu, H., Zsak, L., Yu, Q., Yang, Z. 2013. Application of the ligation-independent cloning (LIC) method for rapid construction of a minigenome rescue system for Newcastle disease virus VG/GA strain. Plasmid Journal. 70:314-320.
Zhao, W., Zhang, Z., Zsak, L., Yu, Q., Yang, Z. 2013. Effects of the HN gene c-terminal extensions on the Newcastle disease virus virulence. Virus Genes. 47(3):498-504.
Wu, Y., Yan, S., Lv, Z., Chen, L., Geng, J., He, J., Yu, Q., Yin, J., Ren, G., Li, D. 2014. Recombinant Newcastle disease virus Anhinga Strain (NDV/Anh-EGFP) for Hepatoma Therapy. Technology in Cancer Research and Treatment. 13:169-175.
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Zsak, L. 2013. Enteric parvovirus infections of chickens and turkeys. In: Swayne, D.E., Glisson, J.R., McDougald, L.R., Nolan, L.K., Suarez, D.L., Nair, V.L., editors. Diseases of Poultry. 13th edition. Ames, IA:Wiley-Blackwell in partnership with the American Association of Avian Pathologists. p. 399-405.