A capsid gene-based real-time reverse transcription polymerase chain reaction assay for the detection of marine vesiviruses in the Caliciviridae

Abstract

A real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was developed for the identification of marine vesiviruses. The primers were designed to target a 176-nucleotide fragment within a highly conserved region of the San Miguel sea lion viruses (SMSVs) capsid gene. The assay detected viral RNA from nine marine vesivirus serotypes described previously, including two serotypes (SMSV-8 and SMSV-12) not identified with presently available molecular assays, a highly-related bovine vesivirus strain (Bos-1), a mink vesivirus strain (MCV), and two novel genotypes isolated recently from Steller sea lions (SSL V810 and V1415). The real-time assay did not amplify sequences from the corresponding genomic regions of feline calicivirus (also in the genus Vesivirus) and representative members of the genus Norovirus. The rtRT-PCR assay described below may prove useful as a diagnostic tool for the detection of currently circulating, emerging and previously described marine vesiviruses in clinical samples, especially when large numbers are screened in surveillance studies of these restricted viruses.

Pinnipediae

“…Marine caliciviruses are within the vesivirus genus and include over 40 serotypes including San Miguel San Lion viruses (SMSV-1-17) and Walrus calicivirus (WCV) (McClenahan et al, 2009). Isolates and serologic evidence of exposure is documented in multiple species and in both clinically affected and healthy individuals.…”

“…). Because of the importance in differentiating these viruses from foreign animal diseases, a real-time reverse transcription PCR has recently been developed that can identify most serotypes and differentiate them from reportable vesicular diseases(McClenahan et al, 2009).Miscellaneous viral diseases are reported onSupplemental Table e3.…”

Emerging viruses in marine mammals.

“…The pinniped vesiviruses SMSV-8 and SMSV-12 are genetically different from VESV and likely represent other vesivirus species [179]. Over 20 marine mammal vesivirus serotypes have been detected in Pacific marine mammals including California sea lions, northern fur seals, Hawaiian monk seals, Steller sea lions, Pacific walrus (Odobenus rosmarus divergens), bottlenose dolphins, grey whales (Eschrichtius robustus), fin whales, sei whales (B. borealis) and sperm whales (P. macrocephalus) [46,180,181]. Some serotypes can infect terrestrial mammals [46,182].…”

Multiplex reverse transcription quantitative PCR detection of a single-stranded RNA virus HcRNAV infecting the bloom-forming dinoflagellate Heterocapsa circularisquama

“…Real-time PCR, which allows rapid and accurate detection and quantification of target DNA sequences, is widely used for diagnosing viral infections in both research and clinical settings (Mackay et al 2002;Mackay 2004). It has also been employed for rapidly detecting aquatic environments of harmful dinoflagellates (Bowers et al 2000;Handy et al 2006;Kamikawa et al 2006;Perini et al 2011), bacteria (Rueckert and Cary 2009;Blazejak and Schippers 2010;Halliday et al 2010), cyanophages (Takashima et al 2007;Yoshida et al 2010), and pathogenic viruses (Sano et al 2006;McClenahan et al 2009).…”