Abstract
New diseases in marine animals are emerging at an increasing rate and the causative agents of these diseases are mostly unknown. Currently, diagnosing novel viral infections is difficult due to limitations of virus discovery methods. PCR, immunological assays, and pan-viral microarrays are only useful in identifying viruses with high levels of similarity to known viruses. Viral metagenomics, which involves viral particle purification and shotgun sequencing, has proven to be useful for describing novel viruses, including those with limited homology to previously described viral families. Viral metagenomics is useful for investigating unknown etiological agents in mortality events, characterizing viruses in known disease, and assessing existing viral pathogens (both known and unknown) in endangered species.
Viral metagenomics was used to examine the possible involvement of viruses in a mortality event with no known etiology of four California sea lions at the Kansas City Zoo during 2005-2006. Necropsy and histopathology revealed granulomatous nonsuppurative mediastinitis and pleuritis in two of the sea lions, while the third sea lion was found to have an accumulation of lymphocytes and macrophages in the upper respiratory submucosa. Toxicity assays and tests for known pathogenic bacteria, fungi, and viruses were negative.
Viral metagenomics was performed on a lung tissue samples from one of the mortality event sea lions, revealing a novel California sea lion anellovirus (ZcAV),2 with 35% amino acid identity to feline anelloviruses in the ORF1 region. 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 that died of unrelated causes. In addition, ZcAV was detected at a 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.
Viral metagenomics was also used to characterize viruses associated with known diseases and for assessing viral pathogens in endangered species - it was performed to examine viruses associated with sea turtle fibropapillomatosis (FP), a debilitating neoplastic disease affecting sea turtles worldwide. A novel single-stranded DNA virus, sea turtle tornovirus 1 (STTV1) was discovered.1 The single-stranded, circular genome of STTV1 was approximately 1800 nucleotides long. STTV1 only has weak amino acid level identities (25%) to Chicken Anemia Virus (CAV) in short regions of its genome, hence STTV1 may represent the first member of a novel virus family. A total of 35 healthy turtles and 27 turtles with FP were tested for STTV1 using PCR, and only two turtles severely afflicted with FP were positive. The affected turtles were systemically infected with STTV1, as STTV1 was found in blood and all major organs. STTV1 exists as a quasi species, with several genome variants identified in the fibropapilloma of each turtle, suggesting rapid evolution of this virus.
Acknowledgements
This project was funded by grants to MB from the Florida Sea Turtle Grants Program (08-003R) and the Alfred P. Sloan Foundation (BR-4772). TFFN was supported by the Gulf Oceanographic Charitable Trust Fellowship and the Elsie and William Knight, Jr. Endowed Fellowship.
Contact Information: Terry Fei Fan Ng, College of Marine Science, University of South Florida, 140 Seventh Avenue South, St. Petersburg, FL 33701, USA. Tel: (727) 553-3930. Email: fng@mail.usf.edu website: http://www.marine.usf.edu/genomics/
References
1. Ng T.F.F., C. Manire, K. Borrowman, T. Langer, L. Ehrhart, and M. Breitbart. 2009. Discovery of a novel single-stranded DNA virus from a sea turtle fibropapilloma using viral metagenomics. J Virol 83: 2500-2509.
2. Ng T.F.F., W.K. Suedmeyer, F. Gulland, E. Wheeler, and M. Breitbart. 2009. Discovery of a novel anellovirus from a mortality event of four captive California sea lions using viral metagenomics. J Gen Virol, in press.