Abstract

Marine caliciviruses as exemplified by San Miguel sea lion viruses (SMSV) are a group of single stranded, nonenveloped, icosahedral, positive-sense RNA viruses of ocean origin belonging to the genus Vesivirus within the Caliciviridae. The first isolation of a SMSV was in 1972 from a pinniped. This virus was named SMSV-1 and since then, at least 17 serotypes of SMSV have been identified. Blister fluids and oral and rectal swabs were harvested from two declining populations of Steller sea lions (Eumetopias jubatus) in southeast Alaska. Samples were inoculated onto African green monkey kidney cells (Vero) and Madin-Darby canine kidney cells (MDCK) resulting in the isolation of several caliciviruses identified by RT-PCR and sequencing of an approximately 700-bp fragment within the capsid protein gene. Two of the isolates were randomly selected for further characterization. Isolate V810 was recovered from an oral swab harvested in 2002 from an apparently healthy animal while isolate V1415 originated from a flipper vesicle fluid harvested in 2004 from a young animal. Isolate V810 grew well in Vero and MDCK cultures while isolate V1415 only grew in MDCK cells. Total RNA was extracted from the infected cell monolayers and reversed transcribed to produce cDNA. Primers specific for the capsid gene of marine caliciviruses were used to amplify the full capsids from the cDNA using PCR. Both capsids were approximately 2 kbp in length, consistent with other calicivirus capsids' size. The PCR fragments were cloned into pCR2.1-TOPO T/A and sequenced. The capsid gene from isolate V1415 was 2166-bp in length while that of V810 was 2157-bp. Pair-wise alignments of the full capsid genes showed that they were 79.6% and 88.4% identical at the nucleotide and amino acid levels, respectively. Multiple sequence analyses and phylogeny of the amino acid sequences deduced from both full capsids and their homologues from other marine caliciviruses available in the GenBank database revealed that these isolates constituted new marine calicivirus genotypes. Sera from Steller sea lions older than 14 weeks-of-age generally had antibody titers greater than 128 against the recent local isolate V1415. On the other hand, sera from animals about 2-month-old had antibody titers that varied greatly between <4 and >8192, most likely due to waning of maternal antibodies or early calicivirus infection. The capsid gene of isolate V1415 was subcloned into the baculovirus expression vector pFastBac and transfected into Sf-21 (Spodoptera frugiperda) insect cell cultures to generate a recombinant baculovirus that expresses the full capsid protein. This antigen is being used in the development of an ELISA for the detection of calicivirus antibodies.

Acknowledgments

This work was supported by a grant from Florida Fish and Wildlife Commission through the Marine Mammal Animal Health Program of the University of Florida. The work was also supported by the Alaska Department of Fish and Game under permit number 358-1564-03.