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Discovery of a novel single-stranded DNA virus from a sea turtle fibropapilloma by using viral metagenomics.

ABSTRACT: Viral metagenomics, consisting of viral particle purification and shotgun sequencing, is a powerful technique for discovering viruses associated with diseases with no definitive etiology, viruses that share limited homology with known viruses, or viruses that are not culturable. Here we used viral metagenomics to examine viruses associated with sea turtle fibropapillomatosis (FP), a debilitating neoplastic disease affecting sea turtles worldwide. By means of purifying and shotgun sequencing the viral community directly from the fibropapilloma of a Florida green sea turtle, a novel single-stranded DNA virus, sea turtle tornovirus 1 (STTV1), was discovered. The single-stranded, circular genome of STTV1 was approximately 1,800 nucleotides in length. STTV1 has only weak amino acid level identities (25%) to chicken anemia virus in short regions of its genome; hence, STTV1 may represent the first member of a novel virus family. A total of 35 healthy turtles and 27 turtles with FP were tested for STTV1 using PCR, and only 2 turtles severely afflicted with FP were positive. The affected turtles were systemically infected with STTV1, since STTV1 was found in blood and all major organs. STTV1 exists as a quasispecies, with several genome variants identified in the fibropapilloma of each positive turtle, suggesting rapid evolution of this virus. The STTV1 variants were identical over the majority of their genomes but contained a hypervariable region with extensive divergence. This study demonstrates the potential of viral metagenomics for discovering novel viruses directly from animal tissue, which can enhance our understanding of viral evolution and diversity.

SUBMITTER: Ng TF

PROVIDER: S-EPMC2648252 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Discovery of a novel single-stranded DNA virus from a sea turtle fibropapilloma by using viral metagenomics.

Ng Terry Fei Fan TF Manire Charles C Borrowman Kelly K Langer Tammy T Ehrhart Llewellyn L Breitbart Mya M

Journal of virology 20081230 6

Viral metagenomics, consisting of viral particle purification and shotgun sequencing, is a powerful technique for discovering viruses associated with diseases with no definitive etiology, viruses that share limited homology with known viruses, or viruses that are not culturable. Here we used viral metagenomics to examine viruses associated with sea turtle fibropapillomatosis (FP), a debilitating neoplastic disease affecting sea turtles worldwide. By means of purifying and shotgun sequencing the ...[more]

PMID: 19116258

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Project description:Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ∼1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1-2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ∼4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates 'living' protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.

Dataset Information

Discovery of a novel single-stranded DNA virus from a sea turtle fibropapilloma by using viral metagenomics.

Publications

Discovery of a novel single-stranded DNA virus from a sea turtle fibropapilloma by using viral metagenomics.

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