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Metagenomic analysis of the turkey gut RNA virus community.

Day JM, Ballard LL, Duke MV, Scheffler BE, Zsak L - Virol. J. (2010)

Bottom Line: High-throughput nucleic acid pyrosequencing is a powerful diagnostic technology capable of determining the full genomic repertoire present in a complex environmental sample.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Southeast Poultry Research Laboratory Agricultural Research Service United States Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA. michael.day@ars.usda.gov

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 disease. 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.

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Turkey picobirnavirus and calicivirus diagnostic agarose gel. 1% agarose gel with RT-PCR amplicons from the turkey calicivirus ORF1 polyprotein region (1369 bp, lane 2) and the turkey picobirnavirus RdRp gene (1135 bp, lane 3). RNA was isolated from the intestinal contents collected from commercial North Carolina turkeys in 2009 (calicivirus positive) and 2010 (picornavirus positive).
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Figure 4: Turkey picobirnavirus and calicivirus diagnostic agarose gel. 1% agarose gel with RT-PCR amplicons from the turkey calicivirus ORF1 polyprotein region (1369 bp, lane 2) and the turkey picobirnavirus RdRp gene (1135 bp, lane 3). RNA was isolated from the intestinal contents collected from commercial North Carolina turkeys in 2009 (calicivirus positive) and 2010 (picornavirus positive).

Mentions: The amplified cDNA was utilized in high-throughput nucleic acid sequencing using Genome Sequencer FLX Titanium pyrosequencing technology and reagents (Roche). Contigs were assembled using the gsAssembler software (454 Life Sciences) using stringent parameters (50 bp overlap with 95% identity). Using the assembled contigs as query sequences, the blast non-redundant (nr) protein database (GenBank) was searched using the blastx and tblastx programs. The blastx and tblastx output was compiled and contigs were assigned to taxa with MEGAN using the default LCA algorithm parameters [43]. Nucleotide and amino acid sequences were aligned using ClustalW and phylogenetic trees were prepared using MEGA4; the relationship of the sequences was inferred using the Neighbor-Joining method, which was determined to be a computationally efficient method to deal with datasets of this size and produced single trees to illustrate an initial placement of these novel viruses among available sequences [44]. In order to confirm directly the presence of picobirnavirus and calicivirus sequences in gut samples, the metagenomic contigs were utilized to design RT-PCR primers to amplify portions of the picobirnavirus RNA-dependent RNA polymerase (RdRp) and calicivirus ORF1 polyprotein. These primers were subsequently used to amplify viral sequences in the original RNA prep used to create the metagenome and in archived turkey intestinal samples from North Carolina, U.S.A (Figure 4).


Metagenomic analysis of the turkey gut RNA virus community.

Day JM, Ballard LL, Duke MV, Scheffler BE, Zsak L - Virol. J. (2010)

Turkey picobirnavirus and calicivirus diagnostic agarose gel. 1% agarose gel with RT-PCR amplicons from the turkey calicivirus ORF1 polyprotein region (1369 bp, lane 2) and the turkey picobirnavirus RdRp gene (1135 bp, lane 3). RNA was isolated from the intestinal contents collected from commercial North Carolina turkeys in 2009 (calicivirus positive) and 2010 (picornavirus positive).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2991317&req=5

Figure 4: Turkey picobirnavirus and calicivirus diagnostic agarose gel. 1% agarose gel with RT-PCR amplicons from the turkey calicivirus ORF1 polyprotein region (1369 bp, lane 2) and the turkey picobirnavirus RdRp gene (1135 bp, lane 3). RNA was isolated from the intestinal contents collected from commercial North Carolina turkeys in 2009 (calicivirus positive) and 2010 (picornavirus positive).
Mentions: The amplified cDNA was utilized in high-throughput nucleic acid sequencing using Genome Sequencer FLX Titanium pyrosequencing technology and reagents (Roche). Contigs were assembled using the gsAssembler software (454 Life Sciences) using stringent parameters (50 bp overlap with 95% identity). Using the assembled contigs as query sequences, the blast non-redundant (nr) protein database (GenBank) was searched using the blastx and tblastx programs. The blastx and tblastx output was compiled and contigs were assigned to taxa with MEGAN using the default LCA algorithm parameters [43]. Nucleotide and amino acid sequences were aligned using ClustalW and phylogenetic trees were prepared using MEGA4; the relationship of the sequences was inferred using the Neighbor-Joining method, which was determined to be a computationally efficient method to deal with datasets of this size and produced single trees to illustrate an initial placement of these novel viruses among available sequences [44]. In order to confirm directly the presence of picobirnavirus and calicivirus sequences in gut samples, the metagenomic contigs were utilized to design RT-PCR primers to amplify portions of the picobirnavirus RNA-dependent RNA polymerase (RdRp) and calicivirus ORF1 polyprotein. These primers were subsequently used to amplify viral sequences in the original RNA prep used to create the metagenome and in archived turkey intestinal samples from North Carolina, U.S.A (Figure 4).

Bottom Line: High-throughput nucleic acid pyrosequencing is a powerful diagnostic technology capable of determining the full genomic repertoire present in a complex environmental sample.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Southeast Poultry Research Laboratory Agricultural Research Service United States Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA. michael.day@ars.usda.gov

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 disease. 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.

Show MeSH
Related in: MedlinePlus