A viral metagenomic study was performed to investigate potential viral pathogens associated with a mortality event of three captive California sea lions (Zalophus californianus). This study identified a novel California sea lion anellovirus (ZcAV), with 35 % amino acid identity in the ORF1 region to feline anelloviruses. The double-stranded replicative form of ZcAV was detected in lung tissue, suggesting that ZcAV replicates in sea lion lungs. Specific PCR revealed the presence of ZcAV in the lung tissue of all three sea lions involved in the mortality event, but not in three other sea lions from the same zoo. In addition, ZcAV was detected at low frequency (11 %) in the lungs of wild sea lions. The higher prevalence of ZcAV and presence of the double-stranded replicative form in the lungs of sea lions from the mortality event suggest that ZcAV was associated with the death of these animals.New diseases in marine animals are emerging at an increasing rate and the causative agents of the diseases are mostly unknown (Harvell et al., 1999;Van Bressem et al., 1999). As animals are considered to be the source of .70 % of emerging human infections (Cleaveland et al., 2001), surveillance of pathogens in animals, especially in non-human vertebrates, is important for protecting the health of both humans and wild animal populations (Kruse et al., 2004;Kuiken, 2005).Investigating new viral infections has been difficult due to limitations of current detection methods (reviewed by Delwart, 2007). For example, degenerate PCR and panviral microarrays can detect viruses with close sequence similarity to known viruses, but are limited for discovering novel viruses that share limited similarity with previously sequenced virus genomes. Recent studies have demonstrated the effectiveness of virus-particle purification and shotgun sequencing (viral metagenomics) for describing novel viruses, including those that are unrelated to previously described virus families (Delwart, 2007;Edwards & Rohwer, 2005). The techniques of viral metagenomics were first applied to characterize virus communities present in the environment (Breitbart et al., 2002) One potential application of viral metagenomics is for examination of viral pathogens involved in unusual mortality events of animals. In 2005 and 2006, three California sea lions at the Kansas City Zoo, MO, USA, died in a mortality event of unknown aetiology. Necropsy and histopathology revealed granulomatous, non-suppurative mediastinitis and pleuritis in two of the sea lions, whilst the third sea lion was found to have an accumulation of lymphocytes and macrophages in the upper respiratory submucosa (W. K. Suedmeyer, unpublished results). Chemical toxicity assays using gas chromatography and mass spectroscopy did not detect pesticides, organic toxic compounds or abnormal mineral levels in pool water or gastric contents. All sea lions were negative for pathogenic bacteria, fungi and mycoplasma in culture-based assays and histopathology. The sea lions also tested negative for West Nile virus, ...