|Day, James - Michael|
Submitted to: International Poultry Scientific Forum
Publication Type: Abstract Only
Publication Acceptance Date: 10/21/2009
Publication Date: 12/22/2009
Citation: Day, J.M. and Zsak, L. 2009. Identification of RNA viruses in the turkey gut using metagenomics (abstract). International Poultry Scientific Forum, Southern Conference on Avian Disease. January 25-26, 2010, Atlanta, Georgia. CDROM. Interpretive Summary:
Technical Abstract: Poultry enteric disease is marked by diarrhea, stunting, increased time to market, immune dysfunction and increased mortality. Numerous viruses have been detected in the intestinal tract of poultry, and have subsequently been implicated in enteric disease. Knowledge of the complete viral flora present in the poultry gut would facilitate the development of molecular diagnostic tests, and would lead to a better understanding of viral enteric disease and the effect viruses have on poultry performance in general. This study aimed to identify the turkey gut ribonucleic acid (RNA) virus community—an RNA virus metagenome. Intestinal tracts from turkey farms with histories of enteric disease were processed into 20% homogenates in sterile phosphate-buffered saline (PBS). A stepwise centrifugation and filtration process was used to remove large particles and bacteria. This was followed by ultracentrifugation to pellet virus-sized particles. The pellet was resuspended and treated to remove non-viral nucleic acid. RNA was extracted from the treated pellet and complementary deoxyribonucleic acid (cDNA) was generated using the Invitrogen SuperScript Choice System. This cDNA was used in high-throughput pyrosequencing using Genome Sequencer FLX Titanium technology (Roche). Contigs were assembled using the gsAssembler software (454 Life Sciences). Using the contigs as queries, the Basic Local Alignment Search Tool (BLAST) non-redundant (nr) protein database (GenBank) was searched using the blastx program. The blastx output was analyzed and contigs were assigned to taxa using Metagenomic Analysis (MEGAN) software. The analysis produced 6526 contigs ranging in size from 97 to 2578 base pairs. 788 contigs were similar to RNA viral sequences, including the double stranded RNA viruses (Reoviridae and Picobirnaviruses), and the single stranded RNA viruses (Caliciviridae, Leviviridae, Picornavirales, and Astroviridae). The majority of the viral contigs (620) were similar to database sequences from the Picornavirales order. The data was used to design novel reverse transcriptase polymerase chain reaction (RT-PCR) primers targeting the turkey picobirnaviruses and caliciviruses, and the sequences were identified in archived enteric material. These results validate this metagenomic approach to identifying known and novel RNA viruses in the poultry gut.