Metagenomic analysis of the turkey gut RNA virus community

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.

Figure 1

MEGAN tree with taxonomic assignments. The blastx output of the total contigs was assembled using the gsAssembler software. Circles located next to taxa are proportional to the total number of contigs identified in the pyrosequencing run and subsequent assembly (see Materials and Methods).

Figure 2

Picobirnavirus neighbor-joining tree. The evolutionary history for a 201 bp portion of the PBV RdRp gene was inferred using the Neighbor-Joining method, the optimal tree with the sum of branch length = 4.85654838 is shown. The nucleotide tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. The turkey-specific sequence used in the analysis is indicated with a black diamond; its accession number is HM803965. Hu = Human.

Figure 3

Calicivirus neighbor-joining tree. The evolutionary history of a ~300aa portion of the calicivirus ORF1 polyprotein was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 5.99935819 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The turkey-specific sequence used in the analysis is indicated with a black diamond; its accession number is HM803966.

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).