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.

Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes

“…Hepadnaviruses, bornaviruses and circoviruses, however, have not been reported in this group, with the exception of one transcript of a truncated bornavirus nucleoprotein gene (94 aa) found in a Gaboon viper transcriptome [44] and a TBLASTN hit corresponding to our python EBLN3 reported in a supplementary table of Horie et al [28]. In fact, bornaviruses and hepadnaviruses have never been observed in non-avian reptiles, and only one member of the Circoviridae is known from a nonavian reptile (green sea turtle [61]). The multiple endogenization events we report across taxa, however, show that snakes have been repeatedly exposed to divergent members of these three viral families during the last 50 Myr (table 1).…”

Metagenomic identification, seasonal dynamics, and potential transmission mechanisms of a Daphnia‐associated single‐stranded DNA virus in two temperate lakes

“…The filtrate was subjected to density gradient ultracentrifugation, with density steps at 1.3, 1.5, and 1.7 g mL 21 , and performed in a swing-bucket rotor at 60,000 3 g for 2 h. Viruses at the 1.3-1.5 g mL 21 interface (, 1 mL) were removed using a sterile syringe and refiltered through a 0.2 mm polyethylsulfone filter. The concentrate was treated with nucleases (2.5 U of deoxyribonuclease and 0.25 U ribonuclease) for 2 h at 37uC to eliminate nucleic acids not incorporated into viral capsids (Ng et al 2009a). DNA was extracted from 500 mL of each using the cetyltrimethylammonium bromide and ethylenediaminetetraacetic acid extraction procedure outlined in Thurber et al (2009).…”

“…The viral metagenomic approach has elucidated a wide diversity of free-living viruses in marine (Breitbart et al 2002(Breitbart et al , 2004Angly et al 2006) and freshwater (Breitbart et al 2009;Djikeng et al 2009;Rodriguez-Brito et al 2010) habitats, and viral metagenomes prepared directly from animal and plant tissues have identified the presence of novel viral lineages and potential pathogens (Kapoor et al 2008;Ng et al 2009a;Li et al 2010). Viral metagenomics also provides targets for molecular quantification of viruses in individuals (Thurber et al 2008), their prevalence in populations (Ng et al 2009b), and their distribution in the environment (Rosario et al 2009a;Hewson et al 2012).…”

Viral metagenomics as an emerging and powerful tool in veterinary medicine

“…For short linear DNA viruses and cDNA, amplification is less efficient, and additional steps, such as ligation, may have to be included for amplification (Berthet et al 2008). This strategy has also been successful in studying different viral metagenomes, and several novel viruses have been discovered by this method, such as a fibropapilloma virus in sea turtles (Ng et al 2009), Anellovirus in harbor seals (Ng et al 2011), Bocavirus in pigs (Blomstro¨m et al 2009), and Papillomaviruses in humans (Rector et al 2004). …”