Day et al. Virology Journal 2010, 7:313 http://www.virologyj.com/content/7/1/313 RESEARCH Open Access Metagenomic analysis of the turkey gut RNA virus community J Michael Day1*, Linda L Ballard2, Mary V Duke2, Brian E Scheffler2, Laszlo Zsak1 Abstract Viral enteric disease is an ongoing economic burden to poultry producers worldwide, and despite considerable research, no single virus has emerged as a likely causative agent and target for prevention and control efforts. Historically, electron microscopy has been used to identify suspect viruses, with many small, round viruses eluding classification based solely on morphology. National and regional surveys using molecular diagnostics have revealed that suspect viruses continuously circulate in United States poultry, with many viruses appearing concomitantly and in healthy birds. High-throughput nucleic acid pyrosequencing is a powerful diagnostic technology capable of determining the full genomic repertoire present in a complex environmental sample. We utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric dis- ease. This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys. Our analysis of this turkey gut RNA metagenome focuses in particular on the turkey-origin members of the Picornavirales, the Caliciviridae, and the turkey Picobirnaviruses. Introduction remains elusive, and many enteric viruses can be Enteric disease syndromes such as Poult Enteritis Com- detected in otherwise healthy turkey and chicken flocks plex (PEC) in young turkeys and Runting-Stunting Syn- [3,4]. Regional and national enteric virus surveys have drome (RSS) in chickens are a continual economic revealed the ongoing presence of avian reoviruses, rota- burden for poultry producers. The only reliable method viruses and astroviruses in turkey and chicken flocks, to reproduce the clinical signs of these syndromes in with combinations of viruses often present in the poul- experimental birds is oral inoculation with crude pre- try gut [3,4]. A non-biased, comprehensive approach to parations of intestinal contents from naturally infected virus discovery that would not require viral cultivation birds. Further, the full spectrum of the field signs would reveal a great deal about the complex viral com- observed associated with these syndromes is difficult to munity in the turkey gut. Further, a community-based reproduce experimentally with isolated viruses [1,2]. understanding of the viruses in the poultry gut will be Numerous viruses are known to be circulating in turkey an invaluable asset in ongoing studies of the enteric dis- flocks in the United States, with recent research efforts ease syndromes and would be welcome knowledge to targeting RNA viruses such as the turkey astroviruses, poultry producers who rely upon efficient conversion of novel turkey-origin reovirus, and avian rotavirus, and feed in the gut to produce an economically important DNA viruses such as the recently described turkey par- commodity. A recent study utilizing a sequence-inde- vovirus [3-6]. However, there remains a possibility that pendent molecular screen of virus particle associated an unidentified virus or combination of viruses may play nucleic acid (PAN) in chicken enteric samples identified a role in poultry enteric disease. Despite the isolation a novel chicken parvovirus (ChPV). This parvovirus is a and characterization of many of these suspect viruses, member of the Parvovirinae sub-family within the Par- the etiology of the poultry enteric disease syndromes voviridae, and a PCR-based diagnostic test has been developed that targets the ChPV non-structural (NS) * Correspondence: [email protected] gene [6,7].The success of this PAN procedure suggests 1Southeast Poultry Research Laboratory Agricultural Research Service United that similar approaches can be used for virus discovery States Department of Agriculture 934 College Station Road Athens, GA in the poultry gut [7]. Ultra high-throughput nucleic 30605 USA Full list of author information is available at the end of the article acid pyrosequencing has emerged as a powerful © 2010 Day et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Day et al. Virology Journal 2010, 7:313 Page 2 of 8 http://www.virologyj.com/content/7/1/313 diagnostic technology that can be applied to determine taxon tree similar to the one presented in Figure 1 and the full genomic repertoire present in a complex envir- revealed that many of the unassigned contigs were simi- onmental sample [8]. Viral metagenomics can be specifi- lar to avian sequences. cally utilized to analyze viral sequences in just about any The majority of the assigned viral contigs (620) sample type, and is a powerful tool for virus discovery showed similarity to database sequences from the Picor- [9-13]. Further, viral metagenomics can be specifically navirales order and other picorna-like viruses, viruses applied to the problem of determining etiology in dis- that, as a group, contain a positive sense single-stranded eases and disease syndromes with no known cause RNA genome and a virion approximately 30 nm in dia- [14-16]. In order to characterize the un-described meter [17]. Recently, a retrospective study of electron viruses present in the turkey gut, we utilized the Roche/ micrographs of enteric viruses from California turkeys 454 Life Sciences GS-FLX pyrosequencing platform to experiencing enteric disease revealed a large number of compile an RNA virus metagenome from turkeys “small round viruses” ranging in size from 15 to 30 nm, experiencing enteric disease. The present analysis like most members of the Picornavirales [18]. These focused on RNA viruses in the turkey gut due to the small round viruses are present in turkeys across a large number of RNA viruses that have been identified range of ages, but they have only been identified mor- to date as possibly contributing to enteric disease and phologically, making specific identification difficult. poultry production problems. This approach yielded Avian enterovirus-like viruses have been described for numerous sequences homologous to viruses in the years in domestic poultry; again this designation has National Center for Biotechnology Information (NCBI) historically been made based primarily upon morpholo- BLAST non-redundant (nr) protein database, many of gical characterization, and little is known about their which have not been described in turkeys. These results pathogenicity or their transmission characteristics[19]. It validate this metagenomic approach to identifying is unclear what role these picornaviruses and picorna- known and novel RNA viruses in the poultry gut. The like viruses may play in turkey enteric disease or in tur- sequence data generated via this approach will prove key performance in general, but the presence of picor- useful in the molecular characterization of the viral con- naviruses in other agricultural species has been closely stituency of the poultry gut, and will inform the selec- associated with enteric disease, namely in pigs and cat- tion of molecular diagnostic tests for enteric viruses. tle [20-23]. Members of the Picornavirales also infect This will facilitate the development of updated molecu- avian species, with the etiologic agents of duck hepatitis lar diagnostic tests, and a more thorough knowledge of (DH) in ducklings and avian encephalomyelitis (AE) in the viral constituency in the poultry gut will lead to a several poultry species both being picornaviruses better understanding of the role viruses play in enteric [24,25]. The present metagenomic analysis has identified disease and in the performance of poultry flocks in RNA sequences with homology to seven of the nine general. recognized picornavirus genera [26], with the largest proportion of the sequences bearing homology to the Results and Discussion Kobuvirus genus. The initial pyrosequencing runs produced in excess of The picobirnaviruses (PBVs) are a relatively recently 139,000,000 bases of high quality nucleotide sequence described group of viruses that contain dsRNA, bi-seg- with an average read length of 362. The sequence data mented genomes and have non-enveloped capsids gen- was used to assemble 6526 contigs ranging in size from erally around 35 nm in diameter [27]. Since their initial 97 to 2578 bp, with the majority of contigs falling in the description, the PBVs have been detected in enteric range of approximately 250 to 450 bp. 4563 contigs pro- samples from several mammalian hosts, including duced no hits in the nr protein database using the blastx humans [28-30]. A PBV has been described in chickens search parameters and the MEGAN default settings. 724 based upon morphological characterization and electro- contigs had similarity to sequences from cellular organ- pherotyping, along with a similar virus with an apparent isms, including bacteria, fungi and avian species. 788 tri-segmented genome [31]. The chicken PBV was not contigs had similarity to RNA viral sequences, including specifically associated with enteric disease. PBVs have sequences from the dsRNA viruses (Reoviridae and been associated with gastroenteritis in humans [32,33], Picobirnaviruses),