2013 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. 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.
Viral enteric diseases of poultry are responsible for substantial economic losses to the poultry industry in the United States and abroad. Research and analysis is ongoing to characterize the multiple enteric viruses that have been revealed via analyses of the turkey and chicken gut viral communities. This approach includes the design, updating, and validation of diagnostic assays for novel and known enteric viruses, and the use of these novel assays to survey archived samples and field samples for enteric viruses such as avian enteric picornavirus and re-emerging turkey enteric coronavirus. Consistent with the milestones for the current fiscal year as outlined in the Project Plan, and in cooperation with industry stakeholders, a functional metagenomic analysis of sentinel specific-pathogen free (SPF) chickens placed on broiler farms with historic enteric disease problems revealed numerous suspect enteric viruses that may be involved in enteric disease and performance problems. In response to the re-emergence of turkey enteric coronaviruses (TCoVs) in the Southeastern United States, and with the cooperation of industry stakeholders, numerous regional enteric samples from affected turkey producing areas were tested for the presence of TCoV, and variant TCoV capsid gene sequences were identified as possible recombinant vaccine candidates. Despite decades of research studying the roles that individual viruses play in poultry enteric diseases, vaccines targeting the known enteric viruses have not been developed. To discover novel vaccine platforms and vaccination strategies that can be integrated into effective prevention and control programs of poultry enteric diseases, project scientists are developing an intestinal recombinant vaccine vector using a Newcastle disease virus (NDV) vaccine strain. Infectious clones containing targeted enteric disease-associated viral proteins, such as chicken parvovirus VP2 gene and TCoV 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. The continued progress of this project is supported by a recent grant (3/29/2013) from the United States Poultry and Egg Association awarded to the Lead Scientist for the “Molecular characterization of novel enteric viruses circulating in poultry in the United States”.
Novel intestinal picornaviruses described in turkeys. ARS researchers in Athens, Georgia performed nucleic acid sequence analysis of a novel avian enteric picornavirus originally detected in the intestinal contents of a North Carolina turkey and revealed a protein sequence similar to the capsid protein of previously described novel avian picornaviruses. Further analysis with similar protein sequences revealed that the capsid protein was very similar to a novel picornavirus (Turkey gallivirus) recently described from Hungary. This analysis also revealed that these novel turkey enteric picornaviruses from the United States and Hungary are similar to but genetically distinct from the members of the Turdidae family of viruses that infect birds like robins, with the turkey-origin enteric picornaviruses forming a distinct group with turdivirus 1. The large proportion of novel picornaviruses detected in the turkey gut via a community analysis suggests that this family of viruses plays an as-yet-undescribed role in the complex gut viral community and may play a role in the poultry enteric syndromes.
Avian parvoviruses studied in broiler chickens. ARS researchers in Athens, Georgia completed pathogenesis experiments in young broiler chickens using novel parvoviruses isolated from the intestines of chickens that were affected by enteric diseases. Experimental infection of 2-day-old broiler chickens resulted in the characteristic signs of enteric disease in the birds, including signs consistent with runting-stunting syndrome (RSS) in four of five experimental groups with significant growth retardation. Shedding of virus in the feces was detected via a molecular assay at early times following inoculation of virus, which was followed by the presence of virus in the blood and generalization of infection. Chicken parvovirus could be detected in most of the major tissues for weeks after inoculation, and parvovirus could be detected in cells in the intestines of inoculated birds at 7 and 14 days following inoculation. Multiple enteric viruses have been implicated in the enteric disease syndromes noted in poultry in the United States; these data indicate that chicken parvovirus alone can induce signs consistent with RSS in broilers and may be an important determinant in the complex etiology of the enteric disease syndromes of poultry.
Li, J., Hu, H., Yu, Q., Diel, D.G., Li, D., Miller, P.J. 2012. Generation and characterization of a recombinant Newcastle disease virus expressing the red fluorescent protein for use in co-infection studies. Virology Journal. 9:227. DOI 10.1186/1743-422X-9-227.
Cornax, I., Diel, D.G., Rue, C.A., Estevez, C., Yu, Q., Miller, P.J., Afonso, C.L. 2013. Newcastle disease virus fusion and haemagglutinin-neuraminidase proteins contribute to its macrophage host range. Journal of General Virology. 94:1189-1194.