Abstract

Diseases amongst organisms are emerging at an increasing rate, but many of the infections have unknown causative agents. Current methods for diagnosing viral infections in animals are limited to previously identified viruses. The methods are PCR, immunologic assays, and replication in cell culture, all of which require prior knowledge about the type of virus expected. Diagnosing novel viral infections is difficult due to our inability to culture many viruses on cell lines in the laboratory, and the lack of a conserved genetic element that is found in all viral genomes that can be used for PCR-based analyses. Therefore, the current methods are only applicable when trying to identify a previously described virus, but cannot be used to discover novel viruses.

Therefore, our study attempts to identify novel viral pathogens in marine animals using the shotgun sequencing method, which is capable of detecting unidentified viruses. The shotgun sequencing technique involves selection for viral particles (based on size, density, and nuclease resistance), nucleic acid extraction, sequence-independent amplification, fragmenting the DNA, and subsequent sequencing to retrieve genetic information from the unknown virus. The complete sequence is obtained by reassembling sequences from overlapping fragments of nucleic acid. Since shotgun sequencing can retrieve any nucleic acids sequence in the sample, it is applicable for detection of both known and unknown viruses. This approach can be used to sequence single- and double-stranded DNA and RNA viruses. It can also retrieve the whole genome of the unidentified viruses. Here we demonstrate the use of this technique to investigate the herpesvirus associated with sea turtle fibropapillomatosis in green turtles, and unknown viral infections in captive marine animals.