Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: November 30, 2010
Publication Date: N/A
Technical Abstract: Enteric disease syndromes such as Poult Enteritis Complex (PEC) in young turkeys and Runting-Stunting Syndrome (RSS) in chickens are a continual economic burden for poultry producers worldwide. The only reliable method to reproduce these syndromes in experimental birds is oral inoculation with crude preparations of intestinal contents from naturally infected birds. Further, these syndromes are difficult to reproduce experimentally with isolated viruses. There remains a possibility that an unknown virus or combination of viruses may play a role in poultry enteric disease. Regional and national enteric virus surveys in the United States have revealed the ongoing presence of avian reoviruses, rotaviruses and astroviruses in turkey and chicken flocks, with combinations of these viruses often present in the poultry gut. A recent experimental approach utilizing a sequence-independent molecular screen of virus particle associated nucleic acid (PAN) in chicken and turkey enteric samples identified novel chicken and turkey parvoviruses (ChPV and TuPV). These parvoviruses are members of the Parvovirinae sub-family within the Parvoviridae, and a novel molecular diagnostic test that targets the parvovirus non-structural (NS) gene has revealed the presence of ChPV and TuPV in numerous turkey and chicken flocks in the United States. In order to further characterize the un-described viruses present in the turkey gut, we utilized the Roche/454 Life Sciences pyrosequencing platform to compile a ribonucleic acid (RNA) virus metagenome from turkeys experiencing enteric disease. Viral particles were isolated and concentrated from a pooled intestinal homogenate sample representing several affected turkey flocks reared in North Carolina, U.S.A. Viral RNA was isolated from this pool and complementary deoxyribonucleic acid (cDNA) was generated using the SuperScript Choice system (Invitrogen). Pyrosequencing was performed using the Roche/454 titanium chemistry and platform, and contigs were assembled using the gsAssembler software (454 Life Sciences). Using the assembled contigs as query sequences, the BLAST non-redundant (nr) protein database (GenBank) was searched using blastx. The blastx output was analyzed and contigs were assigned to taxa using Metagenome Analysis Software (MEGAN). This approach yielded numerous sequences homologous to viruses in the database, many of which have not been described in turkeys. This extensive dataset includes sequences from the dsRNA viruses (Reoviridae and Picobirnaviruses), and the ssRNA viruses (Caliciviridae, Leviviridae, Picornavirales, and Astroviridae). The majority of the assigned viral contigs showed similarity to database sequences from the Picornavirales order and from other picorna-like viruses, viruses that, as a group, contain a positive sense single-stranded RNA genome and a virion approximately 30nm in diameter. A novel calicivirus and a novel picobirnavirus were selected for further characterization, and new RT-PCR based diagnostic assays were used to detect theses viruses in archived gut samples stored at the Southeast Poultry Research Laboratory (USDA/ARS), and in field samples submitted by industry stakeholders. The generation of the initial metagenome and the subsequent validation of molecular diagnostic tests for novel viruses served as a proof-of-concept for this approach to viral discovery. Subsequently, a comparative metagenomic analysis was performed using a healthy teaching unit turkey flock reared each year at North Carolina State University, U.S.A. and a sister flock placed in the field that developed signs of enteric disease. cDNA produced from each flock was DNA-barcoded prior to 454 pyrosequencing. The direct comparison of the “healthy” vs. the “affected” gut viromes revealed that the affected flock contained numerous viral sequences not present in the healthy flock; in particular, numerous sequences from the Picornavirales order were noted in the affected flock and not in the healthy flock. In addition to implicating certain viruses or viral groups in enteric disease syndromes, this comparative metagenomic approach will prove useful in determining the effect of management decisions and geographic distribution of gut pathogens on overall flock health and performance.