ARS | Publication request: Initial identification, detection, and analysis of novel RNA viruses in the turkey gut using a metagenomic approach

Submitted to: American Association of Avian Pathologists
Publication Type: Abstract Only
Publication Acceptance Date: December 30, 2009
Publication Date: August 1, 2010
Citation: Day, J.M., Zsak, L. 2010. Initial identification, detection, and analysis of novel RNA viruses in the turkey gut using a metagenomic approach. American Association of Avian Pathologists Annual Meeting in Atlanta, Georgia, August 1-4, 2010.

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 entire 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. 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 with enzymes 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. From this RNA pool, cDNA was produced and used in high-throughput pyrosequencing using Genome Sequencer FLX Titanium technology (Roche). The sequences were used to assemble 6526 contigs ranging in size from 97 to 2578bp. 788 contigs had similarity to RNA viral sequences, including sequences from the double stranded RNA viruses (Reoviridae and Picobirnaviruses), and the ssRNA viruses (Caliciviridae, Leviviridae, Picornavirales, and Astroviridae). The majority of the viral contigs showed similarity to database sequences from the Picornavirales order, including representative viral sequences from members of the Cardiovirus, Kobuvirus, and Teschovirus genera. A specific molecular diagnostic test was developed targeting the RNA dependent RNA polymerase of a novel turkey-origin picobirnavirus. This test was used to identify the presence of turkey picobirnavirus in United States turkey flocks experiencing enteric disease signs.