A Real-Time RT-PCR Assay for the Detection of Marine Caliciviruses
IAAAM Archive
Shasta D. McClenahan1; John D. Neill2; Kathy A. Burek3; Kimberlee B. Beckmen4; Carlos H. Romero1
1University of Florida, College of Veterinary Medicine, Gainesville, FL, USA; 2National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, USA; 3Alaska Veterinary Pathology Services, Eagle River, AK, USA; 4Alaska Department of Fish and Game, Division of Wildlife Conservation, Fairbanks, AK, USA


Marine caliciviruses, RNA viruses belonging to the family Caliciviridae, genus Vesivirus, were first isolated from pinnipeds in 1972. Since the initial isolation, more than 40 different serotypes of marine caliciviruses have been isolated from several species of marine mammals in the Pacific Ocean, and from wild and domestic animals within the United States. Marine calicivirus have been reported to cause many different clinical signs including, vesicle formation on the skin and oral mucosa, abortions, reproductive failures, diarrhea, myocarditis, and encephalitis. At least one isolate has also been shown to be zoonotic, causing blisters on the hands and feet of humans.

The current methods for identification of caliciviruses are serological assays, including ELISA, and molecular assays such as RT-PCR. These assays may not identify all marine caliciviruses in a single test, and may fail to identify emerging isolates. This is due to the high mutation rate of RNA viruses, and the large number of resulting serotypes and genotypes. We previously reported the isolation and genomic characterization of two novel genotypes of marine caliciviruses from Steller sea lions (SSL) from Alaska. We used these isolates, and other existing marine caliciviruses to develop a rapid and sensitive diagnostic real-time RT-PCR assay to identify any marine calicivirus in a single test.

Multiple alignments of genomic sequences from our SSL isolates, along with sequences from other marine caliciviruses available in the GenBank database, allowed us to design primers to amplify a conserved 176-nucleotide fragment within the capsid gene. A fluorescently labeled oligonucleotide probe was designed to hybridize within this amplicon for the real-time detection. The probe includes locked nucleic acids (LNA) to increase its melting temperature and stability during the assay.

This new assay successfully identified eight known serotypes of marine caliciviruses including San Miguel sea lion virus (SMSV) type 1, SMSV-2, SMSV-4, SMSV-5, SMSV-6, SMSV-13, SMSV-14, bovine calicivirus (Bos-1), and our two novel SSL isolates. The newly developed real-time RT-PCR assay was shown to be specific for marine caliciviruses, as it did not crossreact with three different isolates of feline calicivirus, another Vesivirus, not considered to be of marine origin. The assay described here can be used as a diagnostic tool for the rapid and sensitive detection marine caliciviruses in clinical samples.

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Shasta D. McClenahan

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