A Quantitative PCR Assay for Measurement of Trichechid Herpesvirus 1 (TRHV1) Load in the Florida Manatee (Trichechus manatus latirostris)
Trichechid herpesvirus 1 (TrHV1) is found in both healthy and diseased manatees.1 Stress can reactivate herpesviruses. For some stress factors, herpesvirus reactivation can be detected in experimental situations even when cortisol elevation cannot, making the virus a potential biomarker of stress.2 Viral DNA is likely to be more stable than host cytokine mRNA postmortem.
Quantitative PCR (qPCR) primers and probe were designed for the TrHV1 polymerase gene, and the assay was optimized and validated according to MIQE standards.3 To assess baseline healthy manatee TrHV1 loads, we performed qPCR using DNA extracted from buffy coats. Blood was collected during routine health assessments from 26 wild animals. Manatees were all sub-adult to adult size manatees (≥ 236 cm), had good or excellent body condition scores, and were of both sexes. The range of TrHV1 copy numbers was between < 10 (limit of detection) and 59.4 counts/100 ng DNA, with an average of 41.3 counts/100 ng (± 19.7 StdDev).
This assay will next be used to measure TrHV1 loads in immunosuppressed manatees and we will look for correlations with host cytokine mRNA levels as well as test the hypothesis that stressed FL manatees have viral loads significantly higher than expected in a healthy manatee. The potential diagnostic benefit of these assays is the ability to monitor manatee immunological processes more closely and to help evaluate treatment regimen/care.
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
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2. Buske-Kirschbaum A, Geiben A, et al. Preliminary evidence for Herpes labialis recurrence following experimentally induced disgust. Psychother Psychosom. 2001;70(2):86–91.
3. Bustin SA, Benes V, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–622.