Evidence of Chelonid Herpesvirus 5 (ChHV5) in Green Turtles (Chelonia mydas) from Sabah, Borneo

Fibropapillomatosis (FP) is characterized by cutaneous tumours and is associated with Chelonid herpesvirus 5 (ChHV5), an alphaherpesvirus from the family Herpesviridae. Here, we provide the first evidence of ChHV5-associated FP in endangered Green turtles (Chelonia mydas) from Sabah, which is located at the northern region of Malaysian Borneo. The aims of this study were firstly, to determine the presence of ChHV5 in both tumour exhibiting and tumour-free turtles using molecular techniques and secondly, to determine the phylogeography of ChHV5 in Sabah. We also aim to provide evidence of ChHV5 infection through histopathological examinations. A total of 115 Green turtles were sampled from Mabul Island, Sabah. We observed three Green turtles that exhibited FP tumours and were positive for ChHV5.In addition, six clinically healthy turtles were also positive for the virus based on Polymerase Chain Reaction of three viral genes (Capsid protein gene UL18, Glycoprotein H gene UL22 and Glycoprotein B gene UL27). The prevalence of the ChHV5 was 5.22% in asymptomatic Green turtles. Epidermal intranuclear inclusions were identified in tumour lesions upon histopathological examination. Thus, the emergence of ChHV5 in Green turtle in the waters of Sabah could indicate a possible threat to sea turtle populations in the future and requires further monitoring of the populations along the Bornean coast.


INTRODUCTION
Fibropapillomatosis (FP) is a debilitating neoplastic disease of sea turtles (Alfaro-Núñez et al., 2014; Ene et al., 2015). The first reported case of FP was in Florida in 1938 where a Green turtle was captured with tumour-like characteristics (Smith & Coates 1938). However, it has been reported that disease outbreaks in the wild have increased since the 1980s (Patrício et al., 2016). In Asia, it was reported that FP tumour was first observed in nesting Green turtles (Chelonia mydas) from Sarawak Turtle Islands in 1958, but there have been no further reports of possible etiological agents in the waters of Malaysia (Li et al., 2017).
However, a high prevalence of FP was observed in Indonesia, a neighbouring country to Malaysia (Adnyana et al., 1997). In addition, FP has been be classified as a pandemic disease which appears to be increasing in some parts of the world (Work et al., 2009). This further serves a need to determine the presence of FP and to better understand how it might affect turtle population in Borneo, a region where the health of sea turtles is poorly understood. Even though, FP is widely distributed, reports on FP in Asia are few and limited (Hargrove et al. 2015;Li et al., 2017). The identification of ChHV5 in the pristine waters of Sabah's east coast is important as the site is ecologically rich and home to many marine species. Anecdotal reports indicate that several Green turtles have been spotted with FP tumours. Through this study, we have verified the claims with the aim to document the presence of FP in Green turtles in this region using microscopy and molecular approach. To the best of our knowledge, we present here the first record of ChHV5 in Green turtles from Borneo.

Field sampling
The field sampling was carried out at Mabul Island (4°14′45″N 118°37′52″E) located at the east coast of Sabah, Malaysia, in May and November 2015, and again in November 2016 ( Figure 1). Each field trip was conducted for four days, with a total of 13 dives per trip. The study site is a feeding ground for both the Green and Hawksbill (Eretmochelys imbricata) turtles. The turtles were caught by hand while SCUBA diving during the day at the seven established dive sites ( Figure 1) at depths not exceeding 20 m. The turtles were photographed, measured and tagged on board the research vessel. Two individually numbered Inconel tags from Sabah Parks, Malaysia were applied to the axial scale of each front flipper (Limpus, 1992a(Limpus, , 1992b. The turtles were measured using standard procedures (Limpus & Reed, 1985;Bolten, 1999) and all curved carapace length measurements were taken using a flexible tape to the nearest mm. Tissue samples (n=131) from 115 Green turtles and 16 Hawksbills were obtained from the fore flipper using a 6mm skin biopsy punch after disinfection with Betadine, following the method described by (Dutton, 1996) and stored in lysis buffer. The turtles were also evaluated physically immediately after capture. All 131 samples were subjected for molecular analysis while three of the turtles with tumours were proceeded for pathological analysis. The turtles were then released into the sea after all measurements and tissues were collected. This study was conducted with the permission of the Sabah Wildlife Department.

Histopathological Examinations
Tissue samples from the tumours (n=3) were fixed in a 10% buffered formaldehyde solution prior to fixation and processed for routine histopathology (Bancroft et al., 2012) and TEM examination (Hunter 1993). The tissues were then embedded in paraffin wax. The paraffinembedded samples were then sectioned to 4 μm and finally stained by hematoxylin and eosin

RESULTS AND DISCUSSION
We              Brazilian strain. This could be due to interoceanic connection and ad-mixture of population.
Although further analysis is needed to support this theory. As for the DNA polymerase gene Atlantic. As such, we suggest that a comprehensive study on the migratory routes of the sea turtle population and oceanic current is required to further understand the missing link between geographical connection and ChHV5.

CONCLUSION
The results of this study facilitate the understanding of the origin and extent of FP in Mabul Island as the disease is considered to be one of the major threats to Chelonia mydas. Turtles infected with FP often do not survive as the tumour obstructs vision and impedes daily activities such as feeding and locomotion. This hampers organ functions and eventually causes death. The 5.22% prevalence in asymptomatic green turtles in this study implies that despite living in different geographical regions, Green turtles may be exposed to a common pathogen or other environmental factors that aggravates FP. Even though ChHV5 has evolved for millions of years with its turtle host, for the conservation of these endangered sea turtles, ChHV5 needs to be considered as a re-emerging virus, which threatens Green turtles in marine waters surrounding Borneo and worldwide. In particular, the need to gather more information on sea turtle disease risk analysis and disease hazards from various regions is vital. Identification of the cause of FP will be the first step towards developing effective measures for management and control programs.