Abstract

The polymerase chain reaction (PCR) followed by immediate sequencing of the generated uncloned DNA fragments have been performed on total DNA extracted of tissues of marine animals showing lesions suspicious of viral activity. DNA was extracted from tissues such as brain, lung and genitals and from fibropapillomas of stranded marine animals from the Clearwater Marine Aquarium, Dolphins Plus and Mote Marine Laboratory. Oligonucleotide primers designed from conserved sequences of herpesviruses allowed the demonstration in the brain of a striped dolphin (Stenella coeruleoalba) and the lung and genital lesion of a bottlenose dolphin (Tursiops truncatus) of a 346-bp fragment whose sequences seemed to correspond to that of the large subunit of the ribonucleotide reductase (RR) gene of herpesviruses. These fragments showed 99% nucleotide homology and 98% amino acid homology to each other. The fragment amplified from the brain of the striped dolphin showed 52%, 50%, 49%, 47%, 44%, 43% amino acid homology, respectively, with the RR sequence of EHV-4, PRV, EHV-1, HSV-2, BHV-2, and HSV-1. The DNA fragments amplified from the bottlenose dolphin genital lesion and lung had 52%, 50%, 49%, 49%, 47%, 45%, 43% amino acid homology, respectively, with EHV-4, PRV, EHV-1, FeHV-1, HSV-2, BHV-2, and HSV-1. PCR amplification of total DNA from the genital lesion using primers based on genomic sequences of serotype 3 herpes virus of turkeys amplified a fragment of approximately 1.4 Kb. This fragment showed 48.5% homology with the IE2 gene of human herpesvirus 6. Consensus primers designed from the L1 gene of papillomaviruses amplified a 154-bp DNA fragment from a fibropapilloma excised from a Kemp's ridley sea turtle (Lepidochelys kempi) that showed 96% nucleotide homology with a 178-bp amplicon obtained from fibropapillomatous tissue from a green sea turtle (Chelonia mydas).¹ This sequence showed nucleotide homologies ranging from 36%-42.8% with the L1 gene of mammalian papillomaviruses and 46% with the E2 gene of bovine papillomavirus type 1. The utilization of consensus primers in PCR and RT-PCR amplification of viral sequences from nucleic acids extracted from tissues of aquatic animals provides a rapid and sensitive alternative to virus isolation in the diagnosis of viral infections.

References

1.  Lu Y, AA Aguirre, TM Work, GH Balazs, VR Nerurkar, R Yanagihara. 2000. Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas. Journal of Virological Methods 86:25-33.