Title: Determination of the turkey gut RNA virus metagenome Authors
Submitted to: World Veterinary Poultry Association Congress Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: September 4, 2009
Publication Date: November 8, 2009
Citation: Day, J.M., Zsak, L. 2009. Determination of the turkey gut RNA virus metagenome. World Veterinary Poultry Association Congress Proceedings, p. 203. November 8-12, 2009, Marrakesh, Morocco. Technical Abstract: Introduction. Enteric disease syndromes are a continual economic burden for the United States poultry industry. Poultry enteric disease is marked by diarrhea, stunting, and increased time to market; the more severe forms of enteric disease are characterized by immune dysfunction and increased mortality. Numerous viruses have been detected in or isolated from the intestinal tract of poultry (both diseased and healthy), and have subsequently been implicated in poultry enteric disease. An important tool in ongoing research into poultry enteric disease would be knowledge of the viral intestinal flora present in the normal poultry gut and in poultry experiencing enteric signs. This would facilitate the development of updated molecular diagnostic tests, plus a more thorough knowledge of the viral constituency in the poultry gut would lead to a better understanding of the role viruses play in enteric disease and in the performance of poultry flocks in general. This study aimed to identify the ribonucleic acid (RNA) virus community present in the turkey gut—an RNA virus metagenome. Materials and Methods. Intestinal tracts from turkey farms in North Carolina, United States of America with histories of enteric disease problems were received at the Southeast Poultry Research Laboratory in October 2008 and were pooled and processed promptly into ~20% homogenates in sterile phosphate buffered saline. After an initial 5000 rpm centrifugation step, a stepwise filtration process involving 0.8µm, 0.45µm, and 0.2µm cutoff filters was used to remove large particles and bacteria. This was followed by ultracentrifugation (5 hours., 113,000 X G, 4°C) to pellet virus-sized particles. The pellet was resuspended in Tris-hydrochloric acid buffer and treated with RNAse and DNAse to remove unencapsidated (non-viral) nucleic acid. The Qiagen QIAmp Minelute Virus Spin Kit was used to extract RNA from the treated pellet. Complementary deoxyribonucleis acid (cDNA) was generated using the Invitrogen SuperScript Choice System. This cDNA was utilized in high-throughput nucleic acid sequencing using Genome Sequencer Titanium pyrosequencing technology and reagents (Roche). Contigs were assembled using the gsAssembler software (454 Life Sciences) using stringent parameters (50bp overlap with 95% identity). Using the assembled contigs as query sequences, the 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 Metagenome Analysis (MEGAN) software. Results and Discussion. The initial pyrosequencing runs were used to assemble 6526 contigs ranging in size from 97 to 2578bp. 4563 contigs produced no hits in the nr protein database. 724 contigs had similarity to sequences from cellular organisms, including bacteria, fungi and vertebrates. 788 contigs had similarity to RNA viral sequences, including sequences from the double-stranded RNA viruses (Reoviridae and Picobirnaviruses), and the single-stranded RNA viruses (Caliciviridae, Leviviridae, Picornavirales, and Astroviridae). The majority of the assigned viral contigs (620) showed similarity to database sequences from the Picornavirales order. These results validate this metagenomic approach to identifying known and novel RNA viruses in the poultry gut. The sequence data generated via this approach will prove useful in the molecular characterization of the viral constituency of the poultry gut, and will inform the selection of molecular diagnostic tests for enteric viruses.