The complete genome sequence and genetic analysis of ΦCA82 a novel uncultured microphage from the turkey gastrointestinal system
© Zsak et al; licensee BioMed Central Ltd. 2011
Received: 25 March 2011
Accepted: 29 June 2011
Published: 29 June 2011
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.
Metagenomics analyses have lead to the discovery of a variety of microbial nucleotide sequences from environmental samples . These techniques have also allowed for the discovery of uncultured viral nucleotide sequences that are commonly from bacteriophages [2–4] that has also resulted in the discovery of useful enzymes for molecular biology . There has been a resurgent interest in bacteriophage biology and their use or use of phage gene products as antibacterial agents [6–8]. Bacteriophages are thought to be the most abundant life form as a group  and the importance of phage to bacterial evolution [10, 11], the role of phage or prophage encoded virulence factors that contribute to bacterial infectious diseases [12–14] and their contribution to horizontal gene transfer  cannot be over stated. Additionally, the contribution to microbial ecology  and to agricultural production [17, 18] is also extremely important.
Enteric diseases are an important economic production problem for the poultry industry worldwide. One of the major economically important enteric diseases for the poultry industry are the poult enteritis complex (PEC) and poult enteritis mortality syndrome (PEMS) in turkeys and a runting-stunting syndrome (RSS) in broiler chickens . Consequently, studies have been ongoing to identify novel enteric viruses among poultry species at our laboratory. In a recent study, 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.
Additionally, we have successfully applied a random PCR-based method for detection of unknown microorganisms from enteric samples of turkeys that resulted in identification of genomic sequences and subsequent determination of the full-length genome from a previously uncultured parvovirus . During these ongoing investigations to further characterize the turkey gut microbiome and identify novel viral pathogens of poultry, bacteriophage genomic sequences have also been identified. Herein we report the complete genomic sequence of a putative novel member of the Microviridae obtained from turkey gastrointestinal DNA samples utilizing metagenomics approaches. The protein sequences of ΦCA82 were most similar to those of Chlamydia phages.
Materials and Methods
Assembly of ΦCA82, a novel member of the Microviridae family
Forty-two complete intestinal tracts (from duodenum/pancreas to cloaca, including cecal tonsils) from a turkey farm in California, U.S.A. with histories of enteric disease problems were received at the Southeast Poultry Research Laboratory (SEPRL). The intestines were processed and pooled into a single sample, as previously described . A sequence-independent polymerase chain reaction (PCR) protocol was employed to amplify particle-associated nucleic acid (PAN) present in turkey intestinal homogenates, and has been described elsewhere in detail . Using this approach, a total of 576 clones were identified and sequenced with the M13 forward and reverse primers on an AB-3730 automated DNA sequencer. The sequenced clones were used as query sequences to search the GenBank non-redundant nucleotide and protein databases using the blastn and blastx algorithms . In total, the majority of clones with inserts had no hit in the databases using tblastx . However, 46% of the cloned DNA had homology to cellular DNA, bacterial DNA, bacteriophage DNA, and several eukaryotic viral DNA genomes. Twelve DNA clones had sequence similarity to single-stranded DNA microphages, which have also been identified predominantly in microbialites . A contig, CA82 with an average of eightfold coverage and length of 1962 nt was assembled from eight of those clones. This contig had no significant nucleotide similarity to database sequences, but the deduced amino acid sequence revealed significant similarity to the members of the family Microviridae. This initial contig was used to design PCR primers in the opposite orientation of the circular ssDNA to assemble into a contiguous ΦCA82 genome. The PCR amplification resulted in a 3.4 kb product that closed the gap between the CA82 contig and the rest of the circular genome. The final sequence was confirmed by sub-cloning and primer walking with primers resulting ~1 kb fragments containing 250 bp overlapping sequences across the genome. The circular DNA genome was assembled from contigs exhibiting 100% nucleotide identity within the overlapping regions.
The ΦCA82 genome and ORFs were aligned with selected microvirus sequences using ClustalW . Putative ORFs within the ΦCA82 genome were predicted using the FGENESV Trained Pattern/Markov chain-based viral gene prediction method from the Softberry website . Searches for conserved domains within the ΦCA82 genome were performed with the Conserved Domain Database (CDD) Search Service v2.17 at the National Center for Biotechnology Information (NCBI) website .
Comparative genomics of the Microviridae
Microviridae sequences used for comparative genomic analyses
NCBI Taxon ID
Genome Size (bp)
Chlamydia phage 1
Chlamydia phage 2
Chlamydia phage 3
Chlamydia phage 4
Chlamydia pneumoniae phage CPAR39
Enterobacteria phage G4
Enterobacteria phage St-1
Enterobacteria phage alpha3
Enterobacteria phage phiX174
Guinea pig Chlamydia phage
Spiroplasma phage 4
Genomic functional comparisons were based on pfam categories for each predicted gene as classified by the IMG annotation pipeline . A data table of pfam categories and gene counts for each genome was used to construct a similarity matrix and dendrogram in R. To determine which predicted genes were unique to ΦCA82 and those which were shared with other Microviridae members, the Microviridae pangenome was constructed as the union of all predicted genes from the 14 Microviridae genomes and compared to predicted genes for ΦCA82 using both CD-HIT and our data analysis pipeline as described above and blastp run with default parameters except for an e-value cutoff of 0.01.
Nucleotide accession number
The nucleotide sequence of ΦCA82 genome was deposited in GenBank under accession number HQ264138.
Results and Discussion
The ΦCA82 genome
Capsid proteins of ΦCA82
Putative ΦCA82 ORFs and amino acid (aa) homologies with members of Microviridae
No. of aa
Homologous protein (GenBank accession #)
% amino acid identity (homology)
major capsid protein
minor capsid protein
Recent studies using a comparative metagenomic analysis of viral communities associated with marine and freshwater microbialites indicated that identifiable sequences in these were dominated by single-stranded DNA microphages . Partial sequence analysis of the VP1 gene from these microphages showed that the similarity between metagenomic clones and cultured microphage capsid sequences ranged from 47.5 to 61.2% at the nucleic-acid level and from 37.2 to 69.3% at the protein level, respectively. Interestingly, the VP1 gene of ΦCA82 has a similarly high level of sequence similarity (69.1% at the amino acid level) with the seawater metagenomic phages within the same VP1 region (data not shown). This observation is consistent with an environmental origin of modern poultry phages that have since undergone significant host-specific evolutionary divergence in agricultural settings.
Chipman et al  predicted that the IN5 trimer structure in VP1 may function as a substitute for spike proteins of the ΦX174-like viruses, which are not found in SpV4 or the Chlamydia phages, and as such may be responsible for receptor recognition. It has also been suggested that the diverse sequence in this region is associated with host range of phages [36, 41, 43, 44]. The presence of a large insertion in ΦCA82 further supports that it is closer to the intracellular phage subfamily and the sequence dissimilarity within this region between the ΦCA82 and various other phages strongly indicates that this domain indeed may function as a host range determinant.
Rep protein of ΦCA82
Full genome comparisons of ΦCA82 with other members of the Microviridae
These investigations were supported by ARS-USDA CRIS Project No. 6612-32000-054-00 "Epidemiology, Pathogenesis and Countermeasures to Prevent and Control Enteric Viruses of Poultry" at SEPRL and Project No. 6612-32000-055-00 "Molecular Characterization and Gastrointestinal Tract Ecology of Commensal Human Food-Borne Bacterial Pathogens in the Chicken" at PMSRU. The authors thank to Fenglan Li for excellent technical assistance and to the SEPRL sequencing facility for outstanding support.
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