Abstract

Isolation, identification, and characterization of cetacean viruses has been limited in part due to the lack of established, characterized tissue culture lines for any cetacean species (but especially for species commonly held in captivity), and in part by the inherent difficulties associated with conditions typically encountered when collecting clinical samples from cetaceans. One of the focuses of this laboratory has been the development and characterization of cell culture lines from several target species of cetaceans. These cell lines have not yet been "immortalized" and remain as diploid cultures capable (presumably) of a limited number of subcultures. Regardless of whether immortalization of the lines is achieved, it is possible to stockpile large numbers of cultures in liquid nitrogen storage. These frozen stocks provide essential tools for evaluation of clinical samples from cetaceans for viruses and for isolation and characterization of cetacean virus isolates. Reported here is a preliminary analysis of the susceptibility of two developmental cetacean cell lines to a range of mammalian viruses. The two cell lines being characterized were developed from tissue samples from the kidney and lung of two stillborn Tursiops truncatus calves from a facility housing captive dolphins. Aliquots of early subcultures (passages <5) of these lines from 1994 and 1996 were suspended in 10% glycerol, 90% L_15 media and were stored in liquid nitrogen pending need. Pursuant to characterization of viral susceptibility, aliquots of these T. truncatus primary kidney and lung lines were recovered from liquid nitrogen and expanded into monolayers on multiwell plates. Evaluation of susceptibility of the lines to stock cultures of viruses was performed with viruses represented by the following families: Picornaviridae, Reoviridae, Rhabdoviridae, Herpesviridae, Poxviridae, Togaviridae, and Paramyxoviridae (morbillivirus). All viruses used in this evaluation were obtained from the American Type Culture Collection. As a control to confirm viral culture viability, cultures of a monkey kidney cell line known to be susceptible to the viruses being tested were inoculated with each of the viruses. Cell lines were monitored for cytopathic effects (CPE) and the monolayers were stained and photographed at various times post-inoculation. Infectivity titers were determined by performing TCID 50 on cell lines showing CPE.

Acknowledgements

This research was supported by a DEPSCoR grant through the Office of Naval Research.