Metagenomic analysis of the turkey gut RNA virus community

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

The complete genome sequence and genetic analysis of ΦCA82 a novel uncultured microphage from the turkey gastrointestinal system

The genomic DNA sequence of a novel enteric uncultured microphage, ΦCA82 from a turkey gastrointestinal system was determined utilizing metagenomics techniques. The entire circular, single-stranded nucleotide sequence of the genome was 5,514 nucleotides. The ΦCA82 genome is quite different from other microviruses as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Only three genes showed significant similarity to microviral proteins as determined by local alignments using BLAST analysis. ORF1 encoded a predicted phage F capsid protein that was phylogenetically most similar to the Microviridae ΦMH2K member's major coat protein. The ΦCA82 genome also encoded a predicted minor capsid protein (ORF2) and putative replication initiation protein (ORF3) most similar to the microviral bacteriophage SpV4. The distant evolutionary relationship of ΦCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system

The pathogenicity of avian metapneumovirus subtype C wild bird isolates in domestic turkeys

<p>Abstract</p> <p>Background</p> <p>Avian metapneumovirus subtype C (aMPV/C) causes severe upper respiratory disease in turkeys. Previous report revealed the presence of aMPV/C in wild birds in the southeast regions of the U.S.</p> <p>Methods</p> <p>In this study, aMPV/C positive oral swabs from American coots (AC) and Canada geese (CG) were passaged three times in the respiratory tract of specific pathogen free (SPF) turkeys and used as aMPV/C P3 virus isolates in subsequent studies.</p> <p>Results</p> <p>Wild bird P3 isolates showed similar growth characteristics when compared to virulent aMPV/C in chicken embryo fibroblast ( CEF) cell cultures and their glycoprotein G gene sequence was closely related to the G gene of aMPV/C Colorado reference virus. Three-day-old commercial or SPF turkeys were inoculated oculonasally with wild bird aMPV/C P3 isolates. At 5 and 7 days post-inoculation (DPI), severe clinical signs were observed in both of the AC and CG virus-exposed groups. Viral RNA was detected in tracheal swabs by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, immunohistochemistry showed virus replication in the nasal turbinate and trachea. All virus-exposed turkeys developed positive antibody response by 14 DPI.</p> <p>Conclusions</p> <p>Our data demonstrate that aMPV/C wild bird isolates induced typical aMPV/C disease in the domestic turkeys.</p

Comparative Analysis of the Intestinal Bacterial and RNA Viral Communities from Sentinel Birds Placed on Selected Broiler Chicken Farms

<div><p>There is a great deal of interest in characterizing the complex microbial communities in the poultry gut, and in understanding the effects of these dynamic communities on poultry performance, disease status, animal welfare, and microbes with human health significance. Investigations characterizing the poultry enteric virome have identified novel poultry viruses, but the roles these viruses play in disease and performance problems have yet to be fully characterized. The complex bacterial community present in the poultry gut influences gut development, immune status, and animal health, each of which can be an indicator of overall performance. The present metagenomic investigation was undertaken to provide insight into the colonization of specific pathogen free chickens by enteric microorganisms under field conditions and to compare the pre-contact intestinal microbiome with the altered microbiome following contact with poultry raised in the field. Analysis of the intestinal virome from contact birds (“sentinels”) placed on farms revealed colonization by members of the <i>Picornaviridae</i>, <i>Picobirnaviridae, Reoviridae</i>, and <i>Astroviridae</i> that were not present in pre-contact birds or present in proportionally lower numbers. Analysis of the sentinel gut bacterial community revealed an altered community in the post-contact birds, notably by members of the <i>Lachnospiracea/Clostridium</i> and <i>Lactobacillus</i> families and genera. Members of the avian enteric <i>Reoviridae</i> and <i>Astroviridae</i> have been well-characterized and have historically been implicated in poultry enteric disease; members of the <i>Picobirnaviridae</i> and <i>Picornaviridae</i> have only relatively recently been described in the poultry and avian gut, and their roles in the recognized disease syndromes and in poultry performance in general have not been determined. This metagenomic analysis has provided insight into the colonization of the poultry gut by enteric microbes circulating in commercial broiler flocks, and has identified enteric viruses and virus communities that warrant further study in order to understand their role(s) in avian gut health and disease.</p></div

Correlation Matrix.

<p>Correlation matrices comparing viral taxa to bacteria at the Family level. The color and size of the cross-referenced circles corresponds to the level of correlation between groups. The legend indicates correlations (r values) ranging from-1 (strong negative correlation) to +1 (strong positive correlation).</p

Phage Taxon Tree.

<p>Taxon tree representing the bacteriophage-associated viral reads present in the gut viral metagenome for the pre-contact MSPF birds and the sentinel birds placed with each flock in the field. Search parameters as described in the text.</p

Clustering and Taxonomic Composition of Flocks.

<p>Clustering (A) and taxonomic composition (B) of the eight flocks according to 16S rRNA sequences of the intestinal microbial communities. Clustering of flocks was performed by canonical correspondence analysis and taxonomic classifications are based on the RDP naïve Bayesian classifier. Taxa with relative abundance <2.5% summed across all samples are excluded for clarity.</p

Virus Family Taxon Tree.

<p>Taxon tree representing the viral Families present in the gut viral metagenome for the pre-contact MSPF birds and the sentinel birds placed with each flock in the field. Search parameters as described in the text.</p

Taxon tree representing the <i>Picornaviridae</i> genera present in the gut viral metagenome for the pre-contact MSPF birds and the sentinel birds placed with each flock in the field.

<p>Taxon tree representing the <i>Picornaviridae</i> genera present in the gut viral metagenome for the pre-contact MSPF birds and the sentinel birds placed with each flock in the field.</p