Abstract

Adenovirus has been associated with gastrointestinal signs including abdominal splinting and intermittent blood-tinged diarrhea in sea lions.^1,2 Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated with fatal hepatitis in sea lions. However, in cetaceans, adenoviruses were isolated from gastrointestinal samples from wild cetaceans, including a sei whale (Balaenoptera borealis),³ two bowhead whales (Balaena mysticetus),⁴ a beluga whale (Delphinapterus leucas),⁵ bottlenose dolphin (Tursiops truncatus) (KR024710) without clear association with disease. The role of adenovirus in causing disease in cetaceans remains inconclusive.

In this study, we report an outbreak of gastrointestinal disease in captive bottlenose dolphins (Tursiops truncatus) from Oceanografic of Valencia, Spain. We identified a novel adenovirus, the first well-characterized cetacean adenovirus. The animals were sampled (faecal and urine samples). Viral detection and identification were achieved in part by PCR-amplification and sequencing, of partial adenovirus polymerase (pol)⁶ and hexon genes from faecal samples⁷. The virus was also cultured in HeLa cells, the virions within the cells were observed by EM and immunofluorescence, and the presence of the same gene fragments as from the faecal samples was confirmed by PCR and sequencing. The gene fragments that we obtained were the same for all animals and they were different (< 80% identity) to any sequence in GenBank. We genetically characterized this novel adenovirus by using a high-throughput sequencing approach for whole-genome sequencing using the methodology previously described.⁹

The causative role of this adenovirus for the gastroenteritis was supported by I) our failure to find other potential etiological agents, II) by the exclusive detection in the sick dolphins but not in healthy individuals, III) by the detection of this novel adenovirus in faeces, IV) by the seropositivity for canine adenoviruses 1 and 2, V) and by the disappearance of the virus from faeces soon after the clinical signs disappeared. The high-throughput sequencing allowed the recovery of the complete adenovirus genome and regions of the whole genome were verified by PCR and Sanger sequencing.

The complete genome of the BdAdV-1 is 34 040bp and has an ITR of about 220bp. A total of 26 coding sequences were identified, out of which 3 were assigned as hypothetical and 23 were functionally annotated. The homology analysis indicates that the most similar complete genome is the Bottlenose dolphin adenovirus 1 strain Tt11018 (KR024710) (71% identity), followed by the California sea lion adenovirus 1 strain Zc11-030 (KJ563221),¹⁰ and then by the bovine adenovirus type 2 complete genome (AF252854).

We here describe the complete genome of a recently identified adenovirus most genetically close to adenovirus found previously in a bottlenose dolphin. However, this virus is clearly different from previously published adenoviruses, demonstrating less than 72% sequence identity when compared at the whole genome level. The virus has been associated with gastroenteritis in dolphins, and a more in-depth analysis of the obtained sequence data and predicted proteins should allow predictions to be made regarding its tropism. The study also serves to demonstrate the usefulness of high-throughput sequencing to obtain full-length genome of genetically divergent viruses.

Acknowledgments

This work was carried out under the auspices of a collaborative agreement on virology studies in sea mammals between The Oceanogràfic park of the Ciudad de las Artes y las Ciencias of Valencia, the VISAVET Center of Complutense University of Madrid and the Swedish University of Agricultural Sciences. We thank: Belén Rivera and Rocío Sánchez for technical assistance; Francisco Javier García Peña (Laboratorio Central de Sanidad Animal de Algete) for bacteriological assays; Narcisa Martinez Quiles for providing HeLa cells; and Mario Soriano (Electron Microscopy service of the Centro de Investigación Principe Felipe de Valencia) for assistance with EM sample preparation and analysis. C. R-G. was the recipient of a predoctoral fellowship from the FPU programme of the Spanish Ministry of Education. M. M. is the recipient of a predoctoral fellowship from the PhD student grant programme of Complutense University of Madrid. At the Swedish University of Agricultural Sciences, this work was supported by Epi-SEQ: a research project supported under the 2nd joint call for transnational research projects by EMIDA ERA-NET (FP7 project nr 219235) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, Formas (221-2012-586).

* Presenting author

Literature Cited

1.  Dunn JL, Buck JD, Robeck TR. Bacterial diseases of cetaceans and pinnipeds. In: Dierauf LA, Gulland FMD, eds. CRC Handbook of Marine Mammal Medicine. Boca Raton, FL: CRC Press; 2001:327.

2.  Inoshima Y, Murakami T, Ishiguro N, Hasegawa K, Kasamatsu M. An outbreak of lethal adenovirus infection among different otariid species. Vet Microbiol. 2013;165:455–459.

3.  Goldstein T, Colegrove KM, Hanson M, Gulland FM. Isolation of a novel adenovirus from California sea lions Zalophus californianus. Dis Aquat Organ. 2011;94:243–248.

4.  Smith AW, Skilling DE. Viruses and virus diseases of marine mammals. J Am Vet Med Assoc. 1979;175:918–920.

5.  Smith AW, Skilling DE, Benirschke K, Albert TF, Barlough JE. Serology and virology of the bowhead whale (Balaena mysticetus L.). J Wildl Dis. 1987;23:92–98.

6.  De Guise S, Lagace A, Beland P, Girard C, Higgins R. Non-neoplastic lesions in beluga whales (Delphinapterus leucas) and other marine mammals from the St Lawrence Estuary. J Comp Pathol. 1995;112:257–271.

7.  Wellehan JF, Johnson AJ, Harrach B, Benko M, Pessier AP, Johnson CM, Garner MM, Childress A, Jacobson ER. Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses. J Virol. 2004;78:13366–13369.

8.  Thomson D, Meers J, Harrach B. Molecular confirmation of an adenovirus in brushtail possums (Trichosurus vulpecula). Virus Res. 2002;83:189–195.

9.  Granberg F, et al. Metagenomic detection of viral pathogens in Spanish honeybees: co-infection by aphid lethal paralysis, Israel acute paralysis and Lake Sinai viruses. PLoS One. 2013;8(2):e57459. doi:10.1371/journal.pone.0057459.

10. Cortes-Hinojosa G, et al. Phylogenomic characterization of California sea lion adenovirus-1. Infect Genet Evol. 2015;31:270–276. doi:10.1016/j.meegid.2015.01.024.