Genetic Characterization of Alpha and Gamma Herpesviruses from Cutaneous and Mucosal Lesions of Cetaceans
Extracted DNA from lesions of various captive and stranded cetaceans were analyzed by direct polymerase chain reaction (PCR) and nested PCR using consensus primers.2,3 The targeted sequence corresponded to a region of the DNA polymerase gene containing multiple conserved amino acid motifs. Herpesvirus genomic DNA fragments were amplified from lesions of seven individual animals using nested PCR. Sequence analysis of the small DNA fragments indicated that alpha or gamma herpesviruses were present in the lesions. Alphaherpesvirus DNA fragments of 244-bp in length were detected in skin lesions of two Atlantic bottlenose dolphins (Tursiops truncatus). Gammaherpesvirus DNA fragments of 221-bp were obtained from genital lesions from three Atlantic bottlenose dolphins, one dwarf sperm whale (Kogia sima) and one Blainville's beaked whale (Mesoplodon densirostris). In order to increase the amount of sequence derived from these lesions, direct PCR was performed using the primary primers of the nested PCR. This assay resulted in the amplification of fragments 731-bp in length in the case of four of the five gammaherpesvirus contained in the mucosal lesions. These longer fragments could not be amplified in the case of the two alphaherpesviruses identified by nested PCR. Alphaherpesviruses have previously been found to be associated with disseminated lethal infections in two Atlantic bottlenose dolphins.1 Interestingly, the amino acid sequence deduced from the short DNA polymerase fragment of one of the two alphaherpesviruses from our study was 96.8% identical to one of the viruses associated with generalized lethal infection.1 However, both alphaherpesviruses identified by us originated from skin lesions and did not induce systemic lethal infection, as the skin lesions of both dolphins eventually resolved. The amino acid sequences of the alphaherpesviruses small DNA polymerase fragments from the skin lesions shared 77.2% identity. All five cases diagnosed as gammaherpesvirus infection were associated with genital lesions, either penile or vaginal, strongly indicating that cetacean gammaherpesviruses are sexually transmitted. The amino acid sequences deduced from the four long DNA polymerase fragments of these viruses were between 88.1 and 96.3% similar and between 84.8 and 93.8% identical. Overall, the nucleotide and amino acid identities between the alpha- and gammaherpesvirus sequences ranged between 40.6 to 65.5% and 45.1 to 55.6%, respectively. In summary, alpha and gamma herpesviruses have been found, respectively, in lesions localized in the skin and genital mucosa of captive and free-ranging dolphins and whales.
These viruses were rapidly characterized after sequencing of DNA polymerase gene fragments, multiple sequence alignments and phylogeny.
This work was supported by a grant from Harbor Branch Oceanographic Institution and Florida Fish and Wildlife Commission through the Marine Mammal Animal Health Program of the University of Florida.
1. Blanchard TW, NT Santiago, TP Lipscomb, RL Garber, WE McFee, S Knowles. 2001. Two novel alphaherpesviruses associated with fatal disseminated infections in Atlantic bottlenose dolphins. Journal of Wildlife Diseases, 37:297-305.
2. Ehlers B, K Borchers, C Grund, K Frölich, H Ludwig, H-J Buhk. 1999. Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers. Virus Genes, 18: 211-220.
3. VanDevanter DR, P Warrener, L Bennett, ER Schultz, S Coulter, RL Garber, TM Rose. 1996. Detection and analysis of diverse herpesviral species by consensus primer PCR. Journal of Clinical Microbiology, 34: 1666-1671.