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Title: Metagenomics and the poultry gut: using the next generation of nucleic acid sequencing to identify enteric viruses

Author
item Day, James
item Zsak, Laszlo

Submitted to: International Symposium on Animal Genomics for Animal Health
Publication Type: Abstract Only
Publication Acceptance Date: 3/16/2010
Publication Date: 5/31/2010
Citation: Day, J.M., Zsak, L. 2010. Metagenomics and the poultry gut: using the next generation of nucleic acid sequencing to identify enteric viruses [abstract}. 2nd International Symposium on Animal Genomics for Animal Health, Paris, France. p.62.

Interpretive Summary:

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. 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. In order to characterize the un-described viruses present in the turkey gut, we utilized the Roche/454 Life Sciences Genome Sequencer-FLX (GS-FLX) pyrosequencing platform to compile a ribonucleic acid (RNA) virus metagenome from turkeys experiencing enteric disease. Viral particles were isolated from a pooled intestinal content sample representing several affected turkey flocks. 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 GS-FLX titanium chemistry 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. A novel calicivirus and a novel picobirnavirus were identified, and new reverse transcriptase polymerase chain reaction (RT-PCR) based diagnostic assays were used to detect theses viruses in archived gut samples and in field samples submitted by industry stakeholders.