Quantifying chelonid herpesvirus 5 in symptomatic and asymptomatic rehabilitating green sea turtles

A Page-Karjian,TM Norton,M Jackwood,C Mancia,C Brown,NL Gottdenker,B Ritchie

doi:10.3354/esr00687

Abstract

Fibropapillomatosis (FP), an infectious disease of sea turtles, is characterized by tumors of the skin, eye(s) and/or internal organ(s), and is associated with chelonid herpesvirus 5 (ChHV5). Despite extensive research on FP, the pathogenesis of ChHV5 remains poorly under- stood, particularly regarding asymptomatic infections. Here, we provide evidence for detectable ChHV5 DNA in biological samples from symptomatic and asymptomatic green turtles Chelonia mydas. Using a probe-based quantitative PCR (qPCR) assay for ChHV5, we evaluated the rela- tionship between ChHV5 DNA loads and FP disease status, and investigated potential routes of ChHV5 shedding. Samples of tissue, blood, urine, and feces were collected from 67 green turtles at 3 rehabilitation facilities in the southeastern USA. Turtles were divided into 3 study groups: clinical signs of FP (n = 23), history of FP but no clinical signs (n = 13), and no known history of FP (n = 31). Via qPCR, ChHV5 DNA was reliably detected in FP tumors, non-tumored skin, blood, urine, cloacal swabs, and plasma from green turtles in all 3 groups. Our results provide novel evi- dence for ChHV5 DNA in blood cells, which may represent a critical phase of the ChHV5 life cycle and provide a mechanism for viral transport, and documents that viral DNA can be detected in the urine of symptomatic and asymptomatic turtles. As molecular diagnostics become more afford- able, sea turtle health experts can use qPCR to monitor ChHV5 gene copies and thereby detect early signs of viral presence in blood, urine, and tissue samples.

Highlights

Fibropapillomatosis (FP) is an infectious disease of sea turtles with both individual and population level effects (Aguirre & Lutz 2004)
The primers and probe developed for quantitative polymerase chain reaction (PCR) (qPCR) were specific to the chelonid herpesvirus 5 (ChHV5) DN A polymerase (UL30) gene target: qPCR primers were unable to amplify DNA from the related herpesviruses (BHV3, phocine herpesvirus 1 (PhHV-1), phocine herpesvirus 2 (PhHV-2)) assayed, and multiple sequence alignment (BLAST; Altschul et al 1990) revealed that the nucleotide sequence within the span of the qPCR product does not align at all with lung-eye-tracheaassociated herpesvirus (LETV), loggerhead orocutaneous herpesvirus (LOCV), or LRGV at the nucleotide level
Via qPCR, ChHV5 DNA was detected in FP tumors, non-tumored skin, blood, urine, cloacal swabs, and plasma sampled from green turtles in all 3 study groups

Summary

Introduction

Fibropapillomatosis (FP) is an infectious disease of sea turtles with both individual and population level effects (Aguirre & Lutz 2004). Endang Species Res 28: 135–146, 2015 tified in FP tumors via molecular tests such as polymerase chain reaction (PCR; Lackovich et al 1999, Quackenbush et al 2001, Ene et al 2005), Koch’s postulates have not been fulfilled due to an inability to culture the virus to date (Moore et al 1997, Lu et al 1999, Work et al 2009). In addition to identification of ChHV5 in tumors, ChHV5 DNA has been identified in tissues of apparently healthy turtles via PCR, suggesting the detection of early, subclinical or latent viral infection (Quackenbush et al 2001, Page-Karjian et al 2012, Alfaro-N úñez & Gilbert 2014). Viremia is usually indicative of viral replication, and can be detectable during either the primary infection event or during viral reactivation following a period of latency (Paillot et al 2008)

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Conclusion