Development of a Quantitative PCR Assay for Measurement of Trichechid Herpes Virus 1 (TrHV1) Load in the Florida Manatee (Trichechus manatus latirostris)
Abstract
Trichechid herpesvirus 1 (TrHV1), is found in healthy and diseased manatees.4 Stress is well documented as a cause of herpesvirus reactivation in humans.2 For some stress factors, herpesvirus recrudescence can be detected in experimental situations even when cortisol elevation cannot, making it a highly useful biomarker.1 Our hypothesis is that viral counts above threshold values established for healthy manatees may be predictive of immunosuppressive factors. Thus, our objectives are to quantify TrHV1 in manatee peripheral blood and to establish baselines for viral loads in healthy manatees and their relationship to immunosuppression associated with cold stress syndrome.
This phase of the study is to validate and optimize a qPCR assay targeting TrHV1 in manatee buffy coats. Primers and probe were designed for the TrHV1 DNA-dependent DNA polymerase gene. The standard for the qPCR made use of a PCR amplicon of TrHV1 from a known positive manatee sample.3 A series dilution of the product was run using the qPCR protocol and resulted in a suitable standard curve (slope = -3.488; R2 = 0.998; efficiency% = 93.493). Quantification of the polymerase gene abundance of TrHV1 in manatee buffy coat DNA samples was performed. Nine of thirteen buffy coat samples of apparently healthy manatees were positive for TrHV1 with viral concentrations ranging from 22–338 copies. This assay, when optimized and validated, will provide the means for measuring viral loads in manatees and will be assessed for utility as a biomarker for stress and immune function.
Acknowledgements
The authors wish to thank Dr. Bob Bonde and Dr. Maggie Hunter of the USGS Sirenia Project as well as Dr. Mike Walsh, Patrick Thomson and Michelle Davis of the University of Florida, College of Veterinary Medicine for their assistance with sample collection and processing.
*Presenting author
+Student presenter
Literature Cited
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