Presence of chelonid fibropapilloma-associated herpesvirus in tumored and non-tumored green turtles, as detected by polymerase chain reaction, in endemic and non-endemic aggregations, Puerto Rico

Abstract

Fibropapillomatosis (FP), a transmissible neoplastic disease of marine turtles characterized by a likely herpesviral primary etiology, has emerged as an important disease in green sea turtles (Chelonia mydas) over the past three decades. The objectives of this study were to determine the suitability of three different chelonid fibropapilloma-associated herpesvirus (CFPHV) gene targets in polymerase chain reaction (PCR) assays of affected tissues; to explore the presence of CFPHV in non-affected skin from turtles with and without tumors; and to better understand tissue localization of the CFPHV genome in a tumor-free turtle by evaluating CFPHV presence in microanatomic tissue sites. Two aggregations of green sea turtles (Chelonia mydas) in Puerto Rico were evaluated, with six sampling intervals over the three-year period 2004–2007. Primary and nested PCR for three different herpesviral gene targets- DNA polymerase, capsid maturation protease, and membrane glycoprotein B- were performed on 201 skin biopsies taken from 126 turtles with and without external tumors. Laser capture microdissection and nested PCR were used to identify tissue localizations of CFPHV in skin from a normal turtle. Of the turtles sampled in Manglar Bay, 30.5% had tumors; at the relatively more pristine Culebrita, 5.3% of turtles sampled had tumors. All three PCR primer combinations successfully amplified CFPHV from tumors, and from normal skin of both tumored and tumor-free turtles. Via nested PCR, the polymerase gene target proved superior to the other two gene targets in the positive detection of CFPHV DNA. CFPHV infection may be common relative to disease incidence, supporting the idea that extrinsic and/or host factors could play a transforming role in tumor expression. Laser capture microdissection revealed CFPHV in skin from a tumor-free turtle, harbored in both epidermal and dermal tissues. Identification of CFPHV harbored in a non-epidermal site (dermis) of a tumor-free turtle indicates that virus is latent in a non-tumored host.

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

“…Our finding that 83.3% of non-tumored skin samples from FP+ turtles (turtles with FP) tested positive for ChHV5 DNA is similar to a previous study, in which 88% of FP+ green turtles had non-tumored skin samples that tested positive for ChHV5 DN A by qPCR (Alfaro-N úñez et al 2014). Our findings that skin samples from 7/28 (25%) of tumor-free turtles were positive for ChHV5 DN A via qPCR is also similar to the results of another study, in which 32.4% of skin samples from tumor-free turtles were positive for ChHV5 DNA via nested PCR (Page-Karjian et al 2012).…”

“…Although several previous studies have identified ChHV5 DN A in FP tumors and non-tumored skin (Lackovich et al 1999, Lu et al 2000, Quackenbush et al 2001, Page-Karjian et al 2012, AlfaroNúñez et al 2014), here we provide novel evidence for the presence of ChHV5 DNA in blood, urine, cloacal swabs, and plasma from turtles with and without FP tumors. This study provides critical information for a deeper understanding of the complex ChHV5 pathogenesis in symptomatic and asymptomatic turtles.…”

“…Samples were tested in replicates of 8, and an intra-assay coefficient of variation (CV) of 0 to 20% was considered acceptable (Pfaffl 2004). …”

“…In sea turtle rehabilitation centers, FP is a key concern because it requires extensive quarantine measures and complicates prognoses, and, intified in FP tumors via molecular tests such as polymerase chain reaction (PCR; Lackovich et al 1999, Quackenbush et al 2001, Ene et al 2005, although 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). In addition to identification of ChHV5 in tumors, ChHV5 DNA has also 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.…”

“…Determining how ChHV5 is transmitted in apparently healthy FP-free populations could have major implications in designing effective prevention and disease control strategies (Herbst et al 2008). Recently, a nested PCR assay identified ChHV5 DNA in 32.4% of skin samples taken from tumor-free, wild-caught green turtles in Puerto Rico, suggesting that these asymptomatic turtles may be capable of shedding viral DN A from normal skin (Page-Karjian et al 2012). Such asymptomatic turtles may remain disease-free or may experience a transient viral incubation phase followed later by clinical signs (FP tumors).…”