ABSTRACT
The genome sequence of a papillomavirus was determined from fecal samples collected from bottlenose dolphins in the Indian River Lagoon, FL. The genome was 7,772 bp and displayed a typical papillomavirus genome organization. Phylogenetic analysis supported the bottlenose dolphin papillomavirus as being a novel type of Omikronpapillomavirus 1.
GENOME ANNOUNCEMENT
The family Papillomaviridae includes 48 genera of double-stranded DNA viruses (1, 2). Papillomaviruses (PVs) display a predilection for squamous epithelium and can cause benign or, less commonly, malignant neoplasia (3). In bottlenose dolphins (BDs), eight PVs have been characterized within the genera Omikronpapillomavirus (OmikronPV), Upsilonpapillomavirus (UpsilonPV), and Dyopipapillomavirus (4–9). Herein, we describe a novel type of OmikronPV1 sequenced from BD fecal samples.
Fecal swabs from 12 BDs were collected during a 2012 health assessment conducted under U.S. NMFS permit no. 14352 in the Indian River Lagoon (IRL), FL. Swabs were suspended in 1 ml phosphate-buffered saline and stored at −80°C. After thawing, the swabs were vortexed and centrifuged at 5,000 × g for 10 min. The clarified supernatant was filtered through a sterile 0.22-μm-pore-size filter and treated with a mixture of nucleases (10). Following the creation of two 6-sample pools, total nucleic acid was extracted using a commercial kit (10). For both pools, combined DNA and cDNA libraries were prepared as previously described (11, 12). Samples were sequenced using a V2 chemistry 500-cycle kit on an Illumina MiSeq sequencer.
De novo assembly of paired-end reads was performed in CLC Genomics Workbench. BLASTN searches of the resulting contigs, using the nonredundant nucleotide database maintained by the NCBI, recovered a 7,772-bp contig containing the full genome of a PV with highest sequence identity to previously reported cetacean OmikronPVs (4, 7, 8). Open reading frames (ORFs) were predicted using CLC Genomics Workbench, and the gene functions were determined using BLASTP searches. Six ORFs were predicted, with four early proteins (E1, E2, E4, and E6), two late proteins (L1 and L2), and a noncoding control region.
Seventy-six PV L1 nucleotide sequences were retrieved from the Papillomavirus Episteme resource (http://pave.niaid.nih.gov/) and aligned in TranslatorX (13) guided by amino acid translations with the MUSCLE alignment option. Maximum Likelihood phylogenetic analysis performed in IQ-TREE (14) supported the bottlenose dolphin papillomavirus (BDPV) within the OmikronPV1 clade. OmikronPV1 types have previously been determined from papillomatous lesions in BDs (7), harbor porpoise (Phocoena phocoena) (4), and Burmeister’s porpoise (Phocoena spinipinnis) (8). The L1 gene sequence of the BDPV was compared to those of all cetacean PVs and the type species of the other 46 PV genera using the Sequence Demarcation Tool (15). The BDPV displayed greatest nucleotide identity (75.8%) to OmikronPV1 (Tursiops truncatus papillomavirus 6 [TtPV6]) (7). Given that this is the ninth PV type described from a BD, it is referred to here as OmikronPV1 (Tursiops truncatus PV9 [TtPV9]).
An endpoint PCR assay targeting the OmikronPV1 (TtPV9) L1 gene sequence was designed to screen the 12 BD fecal samples. Three adult males, one displaying a genital papilloma noted during sampling, produced the expected 491-bp amplicons. Sequencing of the purified PCR products revealed that they were identical to OmikronPV1 (TtPV9). The reported OmikronPV1 (TtPV9) expands the genomic diversity of BDPVs. Further investigation into its prevalence and potential health impacts on free-ranging BDs is warranted.
Accession number(s).
The sequence for OmikronPV1 (TtPV9) is available in GenBank under accession no. MG905161.
ACKNOWLEDGMENTS
This study was partially funded by Florida Atlantic University (award no. 00086229), the Georgia Aquarium (award no. 03184), and the Florida Fish and Wildlife Conservation Commission (award no. 00126905). The views expressed herein are those of the authors and not necessarily those of the Commission.
Footnotes
Citation Rodrigues TCS, Subramaniam K, Cortés-Hinojosa G, Wellehan JFX, Jr, Ng TFF, Delwart E, McCulloch SD, Goldstein JD, Schaefer AM, Fair PA, Reif JS, Bossart GD, Waltzek TB. 2018. Complete genome sequencing of a novel type of Omikronpapillomavirus 1 in Indian River Lagoon bottlenose dolphins (Tursiops truncatus). Genome Announc 6:e00240-18. https://doi.org/10.1128/genomeA.00240-18.
REFERENCES
- 1.Burk R, Chen Z, van Doorslaer K, Bernard H-U, Chan PKS, Desalle R, Dillner J, Forslund O, Haga T, McBride A, Villa LL. 2015. ICTV taxonomic proposal 2015014a-atDAv2Papillomaviridae_10gen. Creation of 21 new species and 10 new genera in the family Papillomaviridae. https://talk.ictvonline.org/files/ictv_official_taxonomy_updates_since_the_8th_report/m/animal-dna-viruses-and-retroviruses/5818.
- 2.Bernard HU, Burk RD, DeVilliers EM, zur Hausen H. 2012. Papillomaviridae, p 235–248. In King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (ed), Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, New York, NY. [Google Scholar]
- 3.Maclachlan NJ, Dubovi EJ, Barthold SW, Swayne DF, Winton JR. 2017. Fenner's veterinary virology, 5th ed Elsevier Academic Press, New York, NY. [Google Scholar]
- 4.Gottschling M, Bravo IG, Schulz E, Bracho MA, Deaville R, Jepson PD, Van Bressem MF, Stockfleth E, Nindl I. 2011. Modular organizations of novel cetacean papillomaviruses. Mol Phylogenet Evol 59:34–42. doi: 10.1016/j.ympev.2010.12.013. [DOI] [PubMed] [Google Scholar]
- 5.Rector A, Stevens H, Lacave G, Lemey P, Mostmans S, Salbany A, Vos M, Van Doorslaer K, Ghim SJ, Rehtanz M, Bossart GD, Jenson AB, Van Ranst M. 2008. Genomic characterization of novel dolphin papillomaviruses provides indications for recombination within the Papillomaviridae. Virology 378:151–161. doi: 10.1016/j.virol.2008.05.020. [DOI] [PubMed] [Google Scholar]
- 6.Rehtanz M, Ghim SJ, Rector A, Van Ranst M, Fair PA, Bossart GD, Jenson AB. 2006. Isolation and characterization of the first American bottlenose dolphin papillomavirus: Tursiops truncatus papillomavirus type 2. J Gen Virol 87:3559–3565. doi: 10.1099/vir.0.82388-0. [DOI] [PubMed] [Google Scholar]
- 7.Robles-Sikisaka R, Rivera R, Nollens HH, St Leger J, Durden WN, Stolen M, Burchell J, Wellehan JF Jr. 2012. Evidence of recombination and positive selection in cetacean papillomaviruses. Virology 427:189–197. doi: 10.1016/j.virol.2012.01.039. [DOI] [PubMed] [Google Scholar]
- 8.Van Bressem MF, Cassonnet P, Rector A, Desaintes C, Van Waerebeek K, Alfaro-Shigueto J, Van Ranst M, Orth G. 2007. Genital warts in Burmeister's porpoises: characterization of Phocoena spinipinnis papillomavirus type 1 (PsPV-1) and evidence for a second, distantly related PsPV. J Gen Virol 88:1928–1933. doi: 10.1099/vir.0.82694-0. [DOI] [PubMed] [Google Scholar]
- 9.Cortés-Hinojosa G, Subramaniam K, Wellehan JF Jr, Ng TF, Delwart E, McCulloch SD, Goldstein JD, Schaefer AM, Fair PA, Reif JS, Bossart GD, Waltzek TB. 2018. Genomic sequencing of a novel type of Dyopipapillomavirus 1 in an Indian River Lagoon bottlenose dolphin provides evidence of host jumping in a cetacean papillomavirus. Arch Virol, in press. [DOI] [PubMed] [Google Scholar]
- 10.Ng TF, Chen LF, Zhou Y, Shapiro B, Stiller M, Heintzman PD, Varsani A, Kondov NO, Wong W, Deng X, Andrews TD, Moorman BJ, Meulendyk T, MacKay G, Gilbertson RL, Delwart E. 2014. Preservation of viral genomes in 700-y-old caribou feces from a subarctic ice patch. Proc Natl Acad Sci U S A 111:16842–16847. doi: 10.1073/pnas.1410429111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ng TF, Marine R, Wang C, Simmonds P, Kapusinszky B, Bodhidatta L, Oderinde BS, Wommack KE, Delwart E. 2012. High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage. J Virol 86:12161–12175. doi: 10.1128/JVI.00869-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ng TF, Kondov NO, Deng X, Van Eenennaam A, Neibergs HL, Delwart E. 2015. A metagenomics and case-control study to identify viruses associated with bovine respiratory disease. J Virol 89:5340–5349. doi: 10.1128/JVI.00064-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Abascal F, Zardoya R, Telford MJ. 2010. TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations. Nucleic Acids Res 38:W7–W13. doi: 10.1093/nar/gkq291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Mol Biol Evol 32:268–274. doi: 10.1093/molbev/msu300. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Muhire BM, Varsani A, Martin DP. 2014. SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One 9:e108277. doi: 10.1371/journal.pone.0108277. [DOI] [PMC free article] [PubMed] [Google Scholar]