abstract

Four California sea lions died with no known etiology at Kansas City Zoo during 2005-2006. Granulomatous nonsuppurative mediastinitis and pleuritis were observed in two of the sea lions by necropsy and histopathology. Toxicity assays and tests for pathogenic bacteria and fungi were negative. The sea lions tested negative for West Nile Virus and in viral cultures using green monkey and canine cell lines.

To investigate the possible involvement of viruses in the etiology of these sea lion deaths, we used viral metagenomics to examine the viral community present in lung tissue from one of the diseased sea lions. The viral metagenomics technique involves selection for viral particles (based on size, density, and nuclease resistance), nucleic acid extraction, sequence-independent amplification, DNA fragmentation, and sequencing. These methods led to the discovery and complete genome sequencing of a novel virus from the deceased sea lions. The virus has a 2.1 kilobase, single-stranded, circular genome, with amino acid level similarity to a feline Anellovirus.

PCR primers were designed to amplify this newly discovered sea lion Anellovirus, and lung tissue of three sea lions in the mortality event tested positive. Abdominal fluid, blood, oral and nasal swabs of those sea lions were negative for the virus. To determine if this virus was involved in the mortality event, lung biopsies from four other sea lions that died of unrelated causes (cancer or kidney disease) were also tested. All of these sea lions were negative for the sea lion Anellovirus. Although larger sample sizes are needed, this suggests that the sea lion Anellovirus is associated with the mortality event. This is the first Anellovirus discovered in marine animals and it demonstrates our ability to discover novel pathogenic viruses from animal tissues using metagenomic sequencing.

acknowledgements

This project was funded by a research fellowship to Mya Breitbart from the Alfred P. Sloan Foundation. Terry Ng was supported by the Gulf Oceanographic Trust Fellowships. He also wishes to thank IAAAM, USF GPSC Conference Presentation Grant and USF Graduate Student Achievement Award for their support.