Identification of a novel cetacean polyomavirus from a common dolphin (Delphinus delphis) with Tracheobronchitis

Abstract

A female short-beaked common dolphin calf was found stranded in San Diego, California in October 2010, presenting with multifocal ulcerative lesions in the trachea and bronchi. Viral particles suggestive of polyomavirus were detected by EM, and subsequently confirmed by PCR and sequencing. Full genome sequencing (Ion Torrent) revealed a circular dsDNA genome of 5,159 bp that was shown to form a distinct lineage within the genus Polyomavirus based on phylogenetic analysis of the early and late transcriptomes. Viral infection and distribution in laryngeal mucosa was characterised using in-situ hybridisation, and apoptosis observed in the virus-infected region. These results demonstrate that polyomaviruses can be associated with respiratory disease in cetaceans, and expand our knowledge of their diversity and clinical significance in marine mammals.

Figure 1. There is multifocal attenuation and necrosis of the epithelium and filling of the lumen with debris and sloughed epithelial cells.

Multifocal sloughed cells demonstrate karyomegaly with basophilic intranuclear inclusions (arrows). HE stain at 20× magnification.

Figure 2. Infection of dolphin trachea with DPyV-1 and demonstration of polyomavirus ISH staining and apoptosis in virus infected tissue.

A = ISH and DAPI infected trachea at ×40 magnification. Panel Ai = DPyV-1 ISH staining (red) showing intranuclear infection of mucosal tissue. Panel Aii = DAPI staining for cell nuclei. Aiii = Merge of ISH and DAPI. B = TUNEL-TMR staining (red) for apoptosis at ×100 magnification. Panel Bi = TUNEL staining of apoptotic cell nuclei. Bii = DAPI staining. Biii = Merge of TUNEL and DAPI. Tracheal mucosa of an uninfected common dolphin was used as a negative control and no staining for virus or apoptosis was observed (data not shown).

Figure 3. Genome arrangement of DPyV-1 (accession number: KC594077).

Possible intron in VP2 indicated, containing three in-frame stop codons. Truncation of the VP2 by any of these stop codons considered unlikely as the region downstream of the intron (a) shows strong homology to VP2.

Figure 4. Phylogenetic trees based on complete A) early (T Antigen) and B) late (VP1, VP2 and VP3) transcripts.

Alignments were generated using Muscle (executed in Geneious V6.0.4), and Maximum Likelihood trees generated in Mega V.5.