Complete Genome Sequencing of Two Novel Papillomavirus Types in Indian River Lagoon Bottlenose Dolphins (Tursiops truncatus)
Abstract
A study aimed at discovering novel enteric viruses of bottlenose dolphins (BDs) was conducted using fecal samples collected from 12 free-ranging BDs during 2012 health assessments in the Indian River Lagoon (IRL), FL. Following the creation of two six-sample pools, total nucleic acid was extracted using a QIAamp Viral RNA Mini Kit as previously described.1 Combined DNA and cDNA libraries were created using a sequence-independent, single-primer amplification protocol and a Nextera XT DNA Library Prep Kit.2,3 Normalized samples were sequenced using a V2 chemistry 500-cycle kit on an Illumina MiSeq sequencer. Quality trimming followed by de novo assembly of the paired-end reads were conducted in CLC Genomic Workbench V7 using default settings. The assembled contigs were subjected to BLASTN and BLASTX searches using the National Center for Biotechnology Information (NCBI) non-redundant nucleotide and protein databases. A 693 bp contig was found to be identical to a 437 bp sequence encoding the partial L1 gene sequence previously recovered from a papillomatous penile lesion in a male killer whale (Orcinus orca) that stranded in the UK.4 Primers based on the L1 contig sequence were used to generate the full genome sequence by nested inverse PCR and Sanger sequencing. The full genome was 7,299 bp and found to be most similar to Dyopipapillomavirus 1, a species previously sequenced from a penile papilloma in a harbor porpoise (Phocoena phocoena PV4; PphPV4).4 A second contig was recovered containing the complete genome of a PV most similar to Omikronpapillomavirus 1 (OmikronPV1), a species previously sequenced from a BD penile papilloma (Tursiops truncatus PV; TtPV5).5 Both BD papillomavirus (BDPV) genomes displayed typical PV genome organization, including four early genes, two late genes and a long control region. Phenetic and phylogenetic analyses supported these two BDPV as novel types of DyopiPV1 and OmikronPV1, to be referred to as DyopiPV1 (Tursiops truncatus PV8; TtPV8) and OmikronPV1 (Tursiops truncatus PV9; TtPV9), respectively. Separate specific endpoint PCR assays targeting the L1 gene sequences were used to determine the prevalence of the novel PV types in fecal samples of the 12 BDs sampled in 2012. An adult female BD displaying a genital papilloma was positive by PCR for DyopiPV1 (TtPV8) and was confirmed by Sanger sequencing of the amplified product. Three adult male BDs, one of which displayed a genital papilloma, were positive by PCR for OmikronPV1 (TtPV9) and were confirmed by Sanger sequencing of the amplified products. The genome sequencing of TtPV8 and TtPV9 expands the genomic diversity of PVs infecting BDs. In addition, our report of the identical PV sequences found in a BD and a killer whale5 suggests the ability of certain cetacean PVs to infect multiple delphinid species. These findings may have management implications for facilities that manage mixed cetacean populations. Further investigation of the prevalence and associated health impacts of PVs on BD populations in the IRL, FL are warranted.6,7
Acknowledgments
This study was partially funded through grants provided by the Florida Atlantic University, the Georgia Aquarium, and the Florida Fish and Wildlife Conservation Commission.
* Presenting author
+ Student presenter
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