2012 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.
Research and analysis is ongoing to characterize the multiple enteric viruses that have been revealed via a metagenomic analysis and a comparative metagenomic analysis of the turkey gut viral community. This approach includes the design 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 picobirnavirus, calicivirus, picornavirus, and turkey enteric coronavirus. An in-house bioinformatic analysis pipeline has been devised to ensure reproducible, consistent handling of enteric samples that are selected for metagenomic analysis. This pipeline includes.
1)initial sample handling and processing,.
2)nucleic acid isolation and associated protocols,.
3)the generation and amplification of complementary deoxyribonucleic acid (cDNA),.
4)the selection and use of pertinent high-throughput nucleic acid technology, and.
5)bioinformatic analysis of sequence data in order to identify disease-associated viruses and/or genes that can serve as targets for diagnostic assays and the construction of viral vectors.
Institutional effluent decontamination system (EDS). In order to validate an institutional effluent decontamination system (EDS) in use at our facility, determined the time and temperature necessary for the complete inactivation of common poultry enteric viruses: avian rotavirus, avian reovirus, and avian astrovirus. This investigation determined the thermal inactivation curve for rotavirus, reovirus and astrovirus in a buffered solution in a benchtop thermal cycler, as well as the endpoint thermal inactivation of these non-enveloped viruses in the EDS, which decontaminates effluent prior to disposal in the municipal sewer. This study confirmed the full inactivation of these viruses by the EDS. Determining the efficacy of our effluent decontamination system is a critical step in ensuring the biosecurity of our facility, and this report has provided data on the thermal inactivation of common poultry pathogens.
Turkey gut viral metagenome from a flock affected by enteric disease. Based upon the success of an earlier turkey gut viral metagenome from a flock affected by enteric disease, a comparative metagenome comparing the ribonucleic acid (RNA) viral metagenomes from a healthy flock and a “sister flock” affected by enteric disease was further analyzed. This analysis revealed numerous picornaviruses that were determined to be homologous to the avian turdiviruses and the turkey and duck hepatitis viruses. Further, molecular diagnostic assays targeting the picornavirus capsid protein gene were designed and tested using archived and field enteric samples. These novel picornaviruses are un-described in turkey and chicken flocks in the United States, and this assay has allowed an initial phylogenetic assay of these viruses.