A review of fibropapillomatosis in Green turtles (Chelonia mydas)

Abstract

Despite being identified in 1938, many aspects of the pathogenesis and epidemiology of fibropapillomatosis (FP) in marine turtles are yet to be fully uncovered. Current knowledge suggests that FP is an emerging infectious disease, with the prevalence varying both spatially and temporally, even between localities in close proximity to each other. A high prevalence of FP in marine turtles has been correlated with residency in areas of reduced water quality, indicating that there is an environmental influence on disease presentation. Chelonid herpesvirus 5 (ChHV5) has been identified as the likely aetiological agent of FR The current taxonomic position of ChHV5 is in the family Herpesviridae, subfamily Alphahmesvirinae, genus Scutavirus. Molecular differentiation of strains has revealed that a viral variant is typically present at specific locations, even within sympatric species of marine turtles, indicating that the disease FP originates regionally. There is uncertainty surrounding the exact path of transmission and the conditions that facilitate lesion development, although recent research has identified atypical genes within the genome of ChHV5 that may play a role in pathogenesis. This review discusses emerging areas where researchers might focus and theories behind the emergence of FP globally since the 1980s, which appear to be a multi-factorial interplay between the virus, the host and environmental factors influencing disease expression.

Full text

Accepted Manuscript
Title: A review of fibropapillomatosis in green turtles (Chelonia mydas)
Author: K. Jones, E. Ariel, G. Burgess, M. Read
PII: S1090-0233(15)00452-9
DOI: http://dx.doi.org/doi:10.1016/j.tvjl.2015.10.041
Reference: YTVJL 4679
To appear in: The Veterinary Journal
Accepted date: 16-10-2015
Please cite this article as: K. Jones, E. Ariel, G. Burgess, M. Read, A review of
fibropapillomatosis in green turtles (Chelonia mydas), The Veterinary Journal (2015),
http://dx.doi.org/doi:10.1016/j.tvjl.2015.10.041.
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Review 1
2
3
A review of fibropapillomatosis in Green turtles (Chelonia mydas) 4
5
6
K. Jones
a,
*, E. Ariel
a
, G. Burgess
a
, M. Read
b
7
8
a
College of Public Health, Medical and Veterinary Sciences, James Cook University, 4811 9
Townsville, Queensland, Australia 10
b
Reef Recovery Group, Great Barrier Reef Marine Park Authority, 2-68 Flinders Street, 11
Townsville 4810, Queensland, Australia 12
13
14
15
16
* Corresponding author. Tel.: +61 7 47816915. 17
E-mail address: karina.jones@my.jcu.edu.au (K. Jones).18
Page 1 of 40

Highlights 19
 Fibropapillomatosis (FP), an emerging disease in green turtles, is reviewed 20
 Chelonid herpesvirus 5 is the likely aetiological agent of FP 21
 The route of transmission and conditions facilitating lesion development are uncertain 22
 High prevalence of FP is observed in areas of reduced water quality 23
 A multi-factorial interplay between a range of factors is likely to be occurring 24
25
Abstract 26
Despite being identified in 1938, many aspects of the pathogenesis and epidemiology of 27
fibropapillomatosis (FP) in marine turtles are yet to be fully uncovered. Current knowledge 28
suggests that FP is an emerging infectious disease, with the prevalence varying both spatially and 29
temporally, even between localities in close proximity to each other. A high prevalence of FP in 30
marine turtles has been correlated with residency in areas of reduced water quality, indicating 31
that there is an environmental influence on disease presentation. 32
33
Chelonid herpesvirus 5 (ChHV5) has been identified as the likely aetiological agent of 34
FP. The current taxonomic position of ChHV5 is in the family Herpesviridae, subfamily 35
Alphaherpesvirinae, genus Scutavirus. Molecular differentiation of strains has revealed that a 36
viral variant is typically present at specific locations, even within sympatric species of marine 37
turtles, indicating that the disease FP originates regionally. There is uncertainty surrounding the 38
exact path of transmission and the conditions that facilitate lesion development, although recent 39
research has identified atypical genes within the genome of ChHV5 that may play a role in 40
pathogenesis. This review discusses emerging areas where researchers might focus and theories 41
behind the emergence of FP globally since the 1980s, which appear to be a multi-factorial 42
interplay between the virus, the host and environmental factors influencing disease expression. 43
44
Keywords: Fibropapillomatosis; Marine turtle; Herpesvirus; Chelonid herpesvirus 5; Green turtle45
Page 2 of 40

Introduction 46
The Green turtle (Chelonia mydas) is one of seven species of marine turtle and is 47
internationally recognised as endangered by the International Union for the Conservation of 48
Nature (Seminoff, 2004). Eleven discrete population segments of Green turtles have been 49
identified, each of which is considered biologically and ecologically significant (NMFS and 50
USFWS, 2014). Green turtles also hold great cultural significance for many indigenous peoples 51
and are of economic interest, playing a significant role in ecotourism (Dobbs, 2001; Gulko, 52
2004). The species has a global distribution and a complex life history, occupying a range of 53
habitats. Hatchling turtles have a pelagic existence and recruit into benthic inshore waters at the 54
age of 3-5 years (Reich et al., 2007). With the exception of migration for breeding, turtles 55
typically remain in these inshore environments, which are commonly associated with seagrass 56
meadows or coral reefs, for the remainder of their life (Musick and Limpus, 1997) (Fig. 1). 57
58
Green turtles are exposed to a number of threats including ingestion of marine debris, 59
degradation, urbanisation and pollution of nesting habitats and foraging areas, nest and hatchling 60
depredation by wild, feral and domestic animals, boat strike, traditional hunting and egg harvest, 61
the impacts of climate change on the marine and terrestrial environment, and entanglement in 62
fishing nets and lines (Bjorndal, 1995; Herbst and Klein, 1995a; Lutz, 2002; Van Houtan et al., 63
2010). Conservation efforts which aim to abate many of these threats have assisted in the 64
recovery of some of the major Green turtle populations (Chaloupka et al., 2008a). However, 65
outbreaks of disease are also contributing to morbidity and mortality in this already vulnerable 66
species (Foley et al., 2005; Chaloupka et al., 2008; Flint et al., 2010c). 67
68
Fibropapillomatosis (FP) is a disease that has now been reported in every species of 69
marine turtle; Green (Smith and Coates, 1938), Loggerhead (Caretta caretta) (Harshbarger, 70
Page 3 of 40

1991), Kemp’s Ridley (Lepidochelys kempii) (Barragan and Sarti, 1994), Hawksbill 71
(Eretmochelys imbricata) (D'Amato and Moraes-Neto, 2000), Olive Ridley (Lepidochelys 72
olivacea) (Aguirre et al., 1999), Flatback (Natator depressus) (Limpus et al., 1993), and 73
Leatherback (Dermochelys coriacea) (Huerta et al., 2002) turtles. FP is of greatest concern in 74
Green turtles as it has only reached a panzootic status in this species (Williams et al., 1994). 75
76
FP is a neoplastic condition which may lead to the growth of lesions on the skin, oral 77
cavity, shell, eyes and internal organs of the affected turtle, which in severe cases reduces the 78
probability of survival (Flint et al., 2010a; Herbst, 1995; Work et al., 2004). The disease was first 79
identified in a Green turtle with multiple wart-like lesions on display at the New York Aquarium, 80
although originally from Key West, Florida (Smith and Coates, 1938). Despite being described 81
in 1938 (Lucke, 1938; Smith and Coates, 1938), FP did not reach epizootic proportions until the 82
1980s (Herbst et al., 1994, 2004) and has now been reported from every major ocean basin that 83
Green turtles inhabit (Herbst, 1994). 84
85
This review covers the epidemiology and proposed aetiology of FP in Green turtles, with 86
considerable emphasis on the primary candidate for the aetiological agent, chelonid herpesvirus 87
5 (ChHV5). 88
89
Disease presentation 90
FP can be identified in marine turtles by the presence of single or multiple benign 91
fibroepithelial lesions. The characteristic lesions are easily noticed and are pathognomonic for 92
FP, often limiting or obstructing the vision, feeding and locomotive ability of the affected turtle 93
(Herbst, 1994, 1995; Work et al., 2004; Flint et al., 2010a). Cutaneous lesions are typically 94
present on the external soft tissue of the turtle, but may grow on the carapace, plastron (Smith 95
and Coates, 1938; Jacobson et al., 1989; Balazs and Pooley, 1991; Brooks et al., 1994; Herbst, 96
Page 4 of 40

1994) and cornea of affected turtles (Brooks et al., 1994; Flint et al., 2010a). The lesions can be 97
observed on all visceral organs (Herbst 1994; Work et al., 2004; Foley et al. 2005) and are 98
thought to develop during later stages of the disease (Herbst et al. 1999; Wyneken et al. 2006). 99
However, as most visceral lesions are observed during post mortem investigations, the data 100
available on the prevalence of this type of lesion are skewed. Individual lesions can range from 101
0.1 to 30 cm in diameter and can be sessile or pedunculated. The appearance of these lesions can 102
vary from smooth to verrucous and the colour is dependent on the pigment at the site of origin 103
(Herbst, 1994) (Fig. 2). 104
105
Myxofibromas, fibrosarcomas, papillomas, fibromas and fibropapillomas have all been 106
found to be associated with FP (Norton et al., 1990; Work et al., 2004). Three of these lesions are 107
thought to be linked with different stages of lesion development (Herbst, 1994; Kang et al., 108
2008). The early development phase is associated with papilloma lesions, proliferation of 109
epidermal cells, with little or no involvement of the dermal layer. The chronic phase of lesion 110
development is marked by the presence of fibromas, with proliferation of the dermal layer, while 111
the epidermal layer remains normal. Fibropapillomas represent the intermediate phase of lesion 112
development and consist of characteristics of both the papillomas and fibromas (Herbst, 1994; 113
Kang et al., 2008). 114
115
Histological studies on FP lesions have observed orthokeratotic hyperkeratosis and 116
varying degrees of epidermal hyperplasia. Key features observed in FP lesions include 117
cytoplasmic vacuolation and ballooning degeneration of superficial epidermal cells (Jacobson et 118
al., 1989, 1991; Herbst, 1994; Adnyana et al., 1997). 119
120
Page 5 of 40

Haematological and biochemical signs of immunosuppression, chronic stress, and 121
chronic inflammation such as anaemia, lymphocytopenia, neutrophilia, monocytosis, 122
hypoproteinaemia and hyperglobulinaemia have been observed in turtles with clinical signs of 123
FP (Aguirre et al., 1995; Work et al., 2001; dos Santos et al., 2010; Page-Karjian et al. 2014). 124
Although it is still unclear whether the immunosuppression occurs as a result of or as a precursor 125
to FP development, it has been suggested that immunosuppression occurs as a result of FP 126
(Work et al., 2001). While further study is essential to confirm the relationship between 127
immunosuppression and FP infection, it is clear that immunosuppression leaves turtles with FP 128
lesions susceptible to secondary infections and opportunistic pathogens (Work et al., 2001, 2003; 129
Stacey et al., 2008; dos Santos et al., 2010). Impacts of such secondary infections, combined 130
with FP in marine turtles, are a major cause for concern in an already vulnerable species. 131
132
Epidemiology of fibropapillomatosis in marine turtles 133
FP typically occurs in marine turtles inhabiting neritic tropical and sub-tropical areas 134
(Herbst, 1994; Adnyana et al., 1997; Work et al., 2004; Ene et al., 2005). The disease is most 135
frequently observed in juvenile turtles; FP has also been reported in sub-adults and less 136
commonly in adults (Herbst, 1994; Herbst and Klein, 1995b; Adnyana et al., 1997; Work et al., 137
2004; Ene et al., 2005; Patrício et al., 2012; Page-Karjian et al., 2014). This apparent age 138
differentiation in certain locations may indicate that affected juveniles perish from the population 139
altogether or recover with acquired immunity that protects them as adults (Van Houtan et al., 140
2010). Alternatively, it is possible that these adults were never exposed to this disease. 141
142
There are no reports of FP in pelagic post hatchlings or new recruits that have recently 143
taken up residence in inshore foraging habitats (Herbst, 1994). Sex is not thought to be a 144
Page 6 of 40

contributing factor, as no significant difference has been observed in prevalence between males 145
and females (Work et al., 2004). 146
147
Disease prevalence and impact 148
Smith and Coates (1938) reported a prevalence of 1.5% in the Florida Keys region. The 149
disease was not documented in the area again until the 1980s, where the prevalence was then 150
reported to range between 20-60% throughout the subsequent decade. The early to mid-1990s 151
saw FP emerge in the Eastern Pacific, Hawaiian Islands, Indonesia and Australia. As the disease 152
reached epizootic status in several locations globally, it is now considered a panzootic (Williams 153
et al., 1994). Due to the conspicuous presentation of FP, any prior presence would have been 154
noticed in a region where it currently occurs. The incidence of turtles with FP lesions as a 155
percentage of total turtles captured is reported in the Appendix (Supplementary Table 1). 156
Although age class is a risk factor, not all reports of FP prevalence have been corrected by 157
demographic proportions and future reports would benefit from making this distinction. 158
159
The prevalence of FP varies both spatially and temporally - see Appendix 160
(Supplementary Table 1). The sporadic reports of the disease over time, in combination with a 161
lack of oral history prior to the 1980s, indicate that FP is globally emerging (Greenblatt et al., 162
2005b; Duarte et al., 2012). In several cases, a significantly different prevalence of the disease in 163
nearby regions has been observed. In Florida, a prevalence of approximately 50% was observed 164
in Green turtle aggregations in the Indian River region. However, less than 1 km away at the 165
Sabellariid worm reef, FP was not observed at all (Herbst, 1994). At Pala’au, Molokai, FP was 166
not observed at all until 1985, with the prevalence increasing from 1% in 1987 to 60.7% in 1995 167
- see Appendix (Supplementary Table 1). 168
169
Page 7 of 40

A shift in FP prevalence at two closely monitored sites in Puerto Rico has been observed 170
in recent years; FP prevalence began decreasing Puerto Manglar and increasing at Tortuga Bay 171
in 2009 (Patrício et al., 2011). In Australia, FP has been reported in a number of locations since 172
it was first observed in Queensland in the early 1970s (C. Limpus, personal communication). 173
174
The contribution of this disease to morbidity and mortality in affected turtles has also 175
been widely discussed (Herbst, 1994; Ene et al., 2005; Foley et al., 2005; Chaloupka et al., 2008, 176
2009; Flint et al., 2010c). A study on Green turtles at Palaau, Hawaii found that this population 177
was already recovering from previous overharvesting at the time of the FP outbreak in this 178
region. The FP prevalence in this region has also been in decline since the mid-1990s 179
(Chaloupka et al., 2009). 180
181
Studies on regions in Australia (Flint et al., 2010c), Puerto Rico (Patrício et al., 2011) and 182
Florida (Hirama and Ehrhart, 2007) have all concluded that FP is not a significant factor in 183
mortality of turtles. Conversely, a study conducted on data accumulated over 21 years from 184
Hawaii implicated FP as the primary cause of strandings (Chaloupka et al., 2008). 185
186
Despite some conflicting conclusions, the overwhelming consensus is that FP does not 187
significantly impact the survival of turtle populations. However, Hamann et al. (2010) highlights 188
that understanding and managing this disease is a priority research area for sea turtle 189
conservation. Without a more complete understanding of the fundamental elements of this 190
disease, FP cannot be discounted as a threat to the survival of this species. 191
192
Aetiology of fibropapillomatosis in marine turtles 193
Page 8 of 40

Research to date suggests that FP is associated with a herpesvirus infection (Herbst et al., 194
1995; Quackenbush et al., 1998, 2001; Lackovich et al., 1999). Despite ongoing research, this 195
virus cannot be cultured in vitro and therefore Koch's postulates have not been fulfilled (Herbst, 196
1994, 1995; Moore et al., 1997; Lu et al., 1999; Work et al., 2009). Molecular techniques 197
(Quackenbush et al. 1998, 2001; Lackovich et al. 1999) have proven a strong association 198
between FP and a herpesvirus and, according to the criteria established by Hill (1965), the 199
relationship seems to be that of cause and effect. Chelonid herpesvirus 5 (ChHV5) is now the 200
primary focus of research in this area and belongs to the subfamily Alphaherpesvirinae, genus 201
Scutavirus (Davison and McGeoch, 2010). However, there are still some uncertainties 202
surrounding the transmission of the virus, the circumstances that lead to lesion development and 203
the role of environmental factors in the development of this disease. 204
205
Infectious nature of fibropapillomatosis 206
The epizootic nature of FP and the significant variation in the prevalence of FP between 207
different populations of marine turtles, even between nearby localities, led to speculation that FP 208
was primarily caused by an infectious agent. 209
210
Herbst et al. (1995) successfully transferred FP between animals by using cell-free lesion 211
extracts from turtles with lesions to inoculate young captive-reared turtles that were theoretically 212
naive to FP. All turtles in 3/4 experimental groups developed FP lesions. Control animals, which 213
were housed in the same facility and conditions as the experimental turtles, did not develop FP 214
during the same study period. The lesion extracts used in this experiment were filtered through a 215
0.45 µm syringe tip filter to prevent most pathogens, other than viruses, from being transferred. 216
These findings support the case for the role of a viral agent in FP transmission in marine turtles. 217
218
Page 9 of 40

Although in their initial description of FP, Smith and Coates (1938) did not identify any 219
viral elements in histological examination of FP lesions, modern theories have focused on 220
viruses as the primary aetiological agent of FP. A range of viruses are capable of producing 221
neoplasms such as those seen in Green turtle FP. As a result, papillomavirus (Herbst, 1994), 222
papova-like virus (Lu et al., 2000a), retrovirus (Casey et al., 1997) and herpesviruses (Jacobson 223
et al., 1991; Quackenbush et al., 1998; Herbst et al., 1994, 2004) have all been proposed as 224
potential candidates for the aetiological agents of FP in marine turtles. 225
226
Current research suggests that FP is associated with ChHV5 infection. Early molecular 227
studies tested a range of tissues from turtles both with and without FP lesions and all concluded 228
that while ChHV5 could be detected in lesion biopsies from turtles with FP, the virus was rarely 229
detected in normal skin samples from the same turtles (Quackenbush et al., 1998; Lackovich et 230
al., 1999). Samples from turtles without FP lesions did not react in any of the PCR assays 231
conducted in these early studies (Quackenbush et al., 1998; Lackovich et al., 1999; Lu et al., 232
2000b). These results support a strong association between the presence of ChHV5 and the 233
presence of FP lesions. 234
235
Quackenbush et al. (2001) first successfully amplified ChHV5 from skin samples 236
collected from turtles without FP lesions. Although only a subset of samples from turtles without 237
FP lesions reacted in the assay, the results showed that the virus may be present in turtles despite 238
a lack of clinical signs of disease. More recently, ChHV5 sequences have been amplified from 239
skin samples of turtles without FP lesions with greater success (Page-Karjian et al., 2012; 240
Alfaro-Núñez et al., 2014). These results indicate that early or latent infection with ChHV5 is 241
more common than previously thought. The prevalence of turtles with FP lesions may be small 242
relative to the number of turtles infected with ChHV5. Therefore, an absence of FP lesions does 243
Page 10 of 40

not imply absence of ChHV5 infection. As latency is a typical feature of herpesviruses (Fields et 244
al., 2013), such results are to be expected. The improved sensitivity and specificity of the assays 245
used in these studies have revealed a feature of the disease that was undetectable using earlier 246
assays. 247
248
If disease presentation is not dependent on viral infection alone, other factors contributing 249
to lesion development must be considered. An interaction between host, pathogen and the 250
environment (García-Sastre and Sansonetti, 2010) which tips the balance in favour of lesion 251
development may be at play. Differences in host immunity may be preventing certain turtles 252
from mounting a response to the virus (Griffin et al., 2010). Studies on other viral infections 253
have shown that variants of a virus can have different levels of virulence and as such, disease 254
presentation and severity may differ with each variant (Laegreid et al., 1993; Kaashoek et al., 255
1996; Berumen et al., 2001; Zhang et al., 2001; Yunis et al., 2004). 256
257
It is possible that the development of FP lesions is dependent on which viral variant a 258
turtle is infected with. It is also possible that turtles infected with the virus only develop lesions 259
when the viral load surpasses a certain threshold. While the relationship between viral titre and 260
lesion development has not been resolved for ChHV5, this relationship has been described in 261
other viral infections (Brodie et al., 1992; Liu et al., 2000; Zhang et al., 2000; Rosell et al., 2000; 262
Quintana et al., 2001; Ladekjær-Mikkelsen et al., 2002; Rovira et al., 2002; Olvera et al., 2004; 263
Islam et al., 2006; Ravazzolo et al., 2006; Nsubuga et al., 2008; Haralambus et al., 2010). The 264
consistent association of high viral load and lesion development provides support for the theory 265
that this may be the case for ChHV5. 266
267
Chelonid herpesvirus 5 268
Page 11 of 40

Nomenclature and taxonomy 269
There are currently six herpesviruses documented in chelonids, named chelonid 270
herpesvirus 1 to 6 (ChHV1-6). Chelonid herpesvirus 1, 5 and 6 are described in marine turtles 271
whilst the others have been reported in freshwater turtles (Tidona and Darai, 2011). In the 272
absence of sequence data, ChHV1, ChHV2, ChHV3 and ChHV4 remain unrecognised by the 273
International Committee on Taxonomy of Viruses (ICTV) and their taxonomic place is unclear 274
(Davison and McGeoch, 2010). With respect to the marine turtle herpesviruses, ChHV1 is 275
described in association with grey patch disease (Haines et al., 1974; Rebell et al., 1975), 276
ChHV5 is associated with FP and ChHV6 is known to be associated with lung-eye-trachea 277
disease (Jacobson et al., 1986; Curry et al., 2000; Coberley et al., 2001a, 2002). 278
279
Chelonid fibropapilloma-associated herpesvirus (CFPHV) or ChHV5 (Davison and 280
McGeoch, 2010) is now the more commonly used name for this virus. However, it should be 281
noted that previous studies have used a range of names for this virus – see Appendix 282
(Supplementary Table 2). This review refers to the virus as ChHV5. 283
284
Histological investigations of FP lesions showed indications of herpesvirus infection and 285
subsequent studies using electron microscopy concluded that the virus-like particles that were 286
observed were likely to belong to the family Herpesviridae based on location, size and 287
morphology (Jacobson et al., 1989, 1991; Herbst et al., 1995). 288
289
More recent studies using a range of molecular techniques have confirmed herpesviral 290
elements are present in FP lesions (Quackenbush et al., 1998, 2001; Lackovich et al., 1999; Lu et 291
al., 2000a, b, 2003; Yu et al., 2000, 2001; Nigro et al., 2004a, b). Phylogenetic analysis of the 292
ChHV5 genes DNA polymerase and DNA binding protein sequences revealed that ChHV5 293
Page 12 of 40

clusters closely with, but separate to, other members of the Alphaherpesvirinae subfamily 294
(Greenblatt et al., 2005b; McGeoch and Gatherer, 2005). Davison and McGeoch (2010) targeted 295
the single-stranded DNA-binding protein, glycoprotein B, the major capsid protein, DNA 296
polymerase and two subunits of the DNA packaging terminase (genes UL29, UL27, UL19, 297
UL30, UL15 and UL28, respectively). The resulting Bayesian phylogenetic tree shows that 298
ChHV5 exists as an out-group, clearly separate from the current genera. A Minimum Evolution 299
phylogenetic tree of Alphaherpesvirinae based on full length DNA polymerase sequence further 300
supports this result (Fig. 3). Consequently, it has been proposed that ChHV5 be placed in its own 301
genus. The proposed genus, Scutavirus, sits within the Alphaherpesvirinae subfamily of 302
Herpesviridae. 303
304
Variants of chelonid herpesvirus 5 305
Based on nucleotide sequence diversity, four viral variants of ChHV5 have been recorded 306
in waters around Florida. At present, they are known as A, B, C and D (Herbst et al., 2004; Ene 307
et al., 2005). Variant A is the most prevalent in the region, yet there is variation in relative 308
prevalence of variants at each site. Co-infection with variants A and B was also found in one 309
Green turtle (Ene et al., 2005). Perhaps even more significantly, different species of marine turtle 310
shared the same variant if they were present in the same locality (Herbst et al., 2004; Ene et al., 311
2005). This indicates a strong geographic role in the transmission of the virus. 312
313
In a recent study, ChHV5 was examined using samples from a variety of locations in 314
order to create a global phylogeography of the virus. Four phylogeographical groups of ChHV5 315
were identified: eastern Pacific, western Atlantic/eastern Caribbean, mid-west Pacific and 316
Atlantic (Patrício et al., 2012). The results of the study showed that the viral variant is similar 317
between nearby foraging grounds while distant regions are considerably divergent. The study by 318
Page 13 of 40

Patrício et al. (2012) also found that sympatric species of marine turtle were infected with the 319
same viral variant, further supporting the results of Herbst et al. (2004) and Ene et al. (2005). 320
These findings indicate that individual turtles are likely to be infected with the virus through 321
horizontal transmission in neritic bays (Patrício et al., 2012). 322
323
Co-evolution of virus and host 324
Herbst et al. (2004) suggested that the virus diverged prior to the separation of avian and 325
mammalian alphaherpesviruses. This would mean that ChHV5 became specific to marine turtles 326
approximately 300 million years ago (mya). In addition, it was estimated that the two most 327
divergent clades were separated approximately 1.6-4.0 mya. These results led to speculation that 328
the rise of the Isthmus of Panama (3.1-3.5 mya) was responsible for the divergence as it 329
prevented genetic exchange between these clades. Patrício et al. (2012) found that the most 330
recent common ancestor of the currently known variants of this virus existed 193-430 years ago. 331
This estimate is considerably more recent than the work of Herbst et al. (2004) but both studies 332
demonstrate that ChHV5 has evolved with marine turtles and, in either case, it is likely ChHV5 333
has undergone region specific co-evolution with its host. 334
335
While further research is needed to resolve the time of divergence, there is one clear 336
conclusion; it is not a new virus, or even recent mutations in an old virus, that is causing lesions 337
to develop. This evidence further supports the theory that the recent emergence of FP is linked to 338
modern day extrinsic environmental factors promoting lesion development. 339
340
Genome organisation 341
The herpesvirus genome is divided into two unique regions, one composed of a unique 342
long (UL) sequence and the other region is composed of a unique short (US) sequence. These 343
Page 14 of 40

unique sequences are flanked by repeat sequences. The number, position and direction of these 344
sequences can vary and as a result, there are multiple types of herpesvirus genome structures. 345
Current literature lists between four and six known herpesvirus genome types. Fauquet et al. 346
(2005) recognised four herpesvirus genome types (denoted Type 1-4) while Pellet and Roizmann 347
(2007) described six different genome types (denoted Type A-F). 348
349
A recent study has described the entire genome of ChHV5 (Ackermann et al., 2012). The 350
extensive sequence data generated from this study showed a clear division of the genome into 351
UL and US regions. Inverted repeat sequences (IRS) were also found to flank the US sequence. 352
This configuration is consistent with ChHV5 having a type D genome (Ackermann et al., 2012). 353
354
Ackermann et al. (2012) also described four genes that are atypical for an 355
alphaherpesvirus genome. Two members of the C-type lectin-like domain superfamily (F-lec1, 356
F-lec2), an orthologue to the mouse cytomegalovirus M04 (F-M04) and a viral sialyltransferase 357
(F-sial) were all found to be present in the ChHV5 genome (Ackermann et al., 2012). While the 358
products of these genes may not be critical for viral replication, each one has a potential role in 359
pathogenesis or immune deviation (Ackermann et al., 2012). Orthologues to these genes have 360
been described in other viral families and host cells (Neilan et al., 1999; Wilcock et al., 1999; 361
Voigt et al., 2001; Markine-Goriaynoff et al., 2004). However, until now, none of these genes 362
has ever been reported in the genome of an alphaherpesvirus. Two of these atypical genes (F-sial 363
and F-M04) were found to be expressed in the FP lesions and it has been suggested that these 364
genes may play a role in FP pathogenesis (Ackermann et al., 2012). 365
366
Transmission of chelonid herpesvirus 5 367
Page 15 of 40

As this disease has not been observed in pelagic juveniles, it is thought that turtles are 368
exposed to ChHV5 upon recruitment to neritic zones, indicating horizontal transmission (Herbst, 369
1994; Ene et al., 2005; Patrício et al., 2012). These new recruits may be exposed to several 370
stressors associated with migration, adaptation to a new environment, and changes in population 371
density, diet and pathogen exposure, which may all combine to reduce the efficacy of the 372
immune system and make these juveniles more susceptible to infection (Ritchie, 2006) with 373
ChHV5 and development of FP. It is also possible that these stressors combine to enhance 374
transmission or elicit herpesviral recrudescence in latently infected turtles (Ritchie, 2006) 375
leading to the development of FP lesions. Alternatively, direct transmission may be occurring 376
between co-habiting turtles via interactions such as mating and aggression. 377
378
Researchers have speculated on means of transmission of FP as an infectious disease and 379
possible vectors. Marine turtles host a range of parasites and correlations have been made 380
between parasite load and individual health. Spirorchid trematodes (Jacobson et al., 1989, 1991; 381
Norton et al., 1990; Aguirre et al., 1994, 1998b; Williams et al., 1994), coral reef cleaner fish 382
(Booth and Peters, 1972; Losey et al., 1994; Lu et al., 2000c), saddleback wrasse (Thalassoma 383
duperrey) (Lu et al., 2000c) and marine leeches (Ozobranchus spp.) (Greenblatt et al., 2004) 384
have all been proposed as potential vectors of ChHV5. Significantly higher viral loads were 385
detected in marine leeches when compared with the other parasites examined (Greenblatt et al., 386
2004) and they are currently the leading candidate for a mechanical vector. Although 387
Ozobranchus leeches are the most likely candidates for transmission vectors of ChHV5, their 388
exact role has not yet been confirmed. This is partly due to the possible latent state of the virus 389
and involvement of other co-factors in disease expression of FP (Greenblatt et al., 2004). 390
391
Page 16 of 40

Other marine turtle epibiota, including bladder parasites (Pyelosomum longicaecum), 392
barnacles (Platylepas spp.), amphipods of the skin and oral cavity (order Talitroidea) and blood 393
flukes of the genera Carretacola, Hapalotrema and Laeredius have been ruled out as potential 394
vectors (Greenblatt et al., 2004). 395
396
Environmental factors 397
Marine turtles are particularly susceptible to changes in their environment as they are 398
long-lived animals with a complex life history (Aguirre and Lutz, 2004). A marine turtle will 399
access a range of habitat types during its lifetime, but exhibits a high degree of site fidelity once 400
recruited into a near shore foraging area. Mature female turtles are known to return to the natal 401
area from which they originated as hatchlings in order to lay their eggs (Limpus, 2008). Due to 402
this site fidelity, marine turtles are likely to persist in, or return to, their chosen localities despite 403
unfavourable changes to the environment. As a result, any damage to or destruction of these sites 404
could have extremely detrimental effects on populations that inhabit them (Hawkes et al., 2009; 405
Poloczanska et al., 2010; GBRMPA, 2014). 406
407
It has been suggested that environmental factors may play a role in the development of 408
FP (Herbst, 1994; Herbst and Klein, 1995a; Adnyana et al., 1997; Aguirre and Lutz, 2004; 409
Chaloupka et al., 2009; dos Santos et al., 2010; Van Houtan et al., 2014). Moreover, the presence 410
of chemical contaminants may be part of a multifactorial problem that leads to FP (Herbst, 411
1994). Early proponents of a possible relationship between degraded water quality and the 412
presence of FP proposed that chemical contaminants present in the water acted as immunotoxins 413
or were causing damage at the cellular or genetic level (Herbst, 1994). 414
415
Page 17 of 40

Indirect disturbances to the immune system may occur if the chemical contaminants 416
create a disruption of neuroendocrine function (Zeeman and Brindley, 1981; Anderson et al., 417
1984; Dean et al., 1990; Colborn et al., 1993; Arkoosh et al., 1994; Dunier, 1994). Herbst (1994) 418
demonstrated that a positive correlation exists between the prevalence of FP in Green turtle 419
populations adjacent to regions associated with agriculture, industry and urban development. 420
Subsequent studies have observed the same correlation (Adnyana et al., 1997; Foley et al., 2005; 421
dos Santos et al., 2010; Van Houtan et al., 2010). Although initial reports in Puerto Rico 422
observed the same relationship, this trend was reversed after several years; the prevalence of FP 423
at the more pristine site is now considerably higher than at the site which is subjected to high 424
levels of human activity (Patrício et al., 2011; Page-Karjian et al., 2012). Researchers attempted 425
to quantify this relationship in Hawaii by developing an information-rich index of eutrophication 426
from the analysis of 82 different watersheds. The results showed a strong association between FP 427
rates, nitrogen-footprints and macroalgae consumed by turtles (Van Houtan et al. 2010). 428
Different quantification studies were also undertaken in waters around Brazil and found that 429
Green turtles residing in areas with degraded water quality had a higher prevalence of FP. 430
However, this study based the assessment of water quality on the presence of benthic 431
macrophytes and nutrient levels; pollution and the presence of chemical contaminants were not 432
considered (dos Santos et al., 2010). 433
434
Only very low concentrations of persistent organic pollutants (Keller et al., 2014) and 435
selected trace metals and organic pollutants (Aguirre et al., 1994) have been detected in turtles 436
with FP lesions. Although these results suggest that the pollutants examined do not significantly 437
contribute to FP development, it is possible that further investigations will uncover a relationship 438
between this disease and other environmental contaminants (Keller et al., 2014). 439
440
Page 18 of 40

Water temperature may also be a factor in lesion development and growth rate. It is 441
possible that warmer water temperatures during summer promote lesion growth, resulting in 442
lesions of a debilitating size by autumn (Herbst, 1994; Herbst et al., 1995). This seasonal trend 443
has been observed in Florida, where a higher rate of FP is observed in turtles that strand in winter 444
(Herbst, 1994). However, no seasonal trends have been observed in Hawaii (Murakawa et al., 445
2000), which may be because there is less seasonal fluctuation in water temperature in this 446
region (Foley et al., 2005). 447
448
Natural biotoxins have also been implicated as a co-factor involved in FP development. 449
Landsberg et al. (1999) identified a correlation between high-risk FP areas in the Hawaiian 450
Islands and prevalence of Prorocentrum, a species that produces okadaic acid, a known tumour 451
promoter (Suganuma et al., 1988; Haystead et al., 1989; Cohen et al., 1990; Huynh et al., 1997). 452
Similarly, tissue concentrations of lyngbyatoxin A, produced by Lyngbya majuscula, have been 453
correlated with the presence of FP lesions in dead Green turtles (Arthur et al., 2006, 2008). 454
However, this species constituted less than 2% of total dietary intake and subsequently, any 455
biotoxins would be at a low concentration in the turtles (Arthur et al., 2008). If the dietary items 456
containing these biotoxins form a natural component of the diet of Green turtles and the amount 457
being consumed was not altered, these toxins should have no influence on the development of 458
FP. 459
460
An increased concentration of arginine in the diet of Green turtles as a result of invasive 461
macroalgae blooms has also been linked to an increasing prevalence of FP (van Houtan et al., 462
2010). Arginine is a regulator of immune activity (Peranzoni et al., 2008) and is known to 463
promote herpesviruses and contribute to tumour formation (Mannick et al., 1994). This amino 464
Page 19 of 40

acid is also a major component of glycoproteins on the viral envelope of herpesviruses (van 465
Houtan et al. 2010; van Houtan et al. 2014). 466
467
The results of a subsequent study found an association between eutrophication and 468
arginine content of macroalgae, with the intake of arginine in turtles at eutrophied sites being up 469
to 14 times the background level. This increased arginine content may metabolically promote 470
ChHV5, leading to FP lesion development (Van Houtan et al., 2014). Although the conclusions 471
from this study were subsequently challenged (Work et al., 2014), the epidemiological link 472
between the prevalence of disease and feeding ecology found in Van Houtan et al. (2014) 473
provides strong support that environmental factors play a role in the development of this disease. 474
However, the environmental factors leading to the bloom of macroalgae may be causing the 475
development of FP lesions directly, and the algal blooms may not be involved in lesion 476
development at all. If this is the case, it is difficult to link cause and effect. 477
478
Despite there being a strong positive correlation between the prevalence of FP in Green 479
turtle populations and areas with degraded water quality, it is difficult to identify one specific 480
causal contaminant or a combination of such working synergistically to the detriment of the 481
turtles. Studies on toxicity usually focus on chemicals that are persistent in the environment or 482
can bio-accumulate. Genetic damage as a result of a toxin may occur as a consequence of 483
transient exposure and as such, future studies would need to be expanded to include transient 484
chemicals that could have this effect on Green turtles. The practicality of such investigations is 485
daunting considering the vast marine environment and the known and unknown possible causes 486
of FP (Herbst, 1994; Herbst and Klein, 1995a). 487
488
Page 20 of 40

One way that potential links between FP and anthropogenic contaminants might be 489
identified is to develop a monitoring program that records and compares contaminant residue 490
levels, genetic changes and viral load in blood and/or tissue samples collected from turtles with 491
and without FP lesions over a wide geographic area and across several seasons. Such a program 492
could be integrated into existing turtle monitoring activities. Controlled laboratory studies in a 493
closed experimental system may be needed to conclusively evaluate the roles of various 494
environmental factors in FP development (Herbst and Klein 1995a). Alternatively, results from 495
both field and laboratory based studies may work synergistically to fully resolve this 496
relationship. 497
498
Direction of future research 499
The longevity of marine turtles, coupled with their close association with inshore habitats 500
and seagrass meadows and coral reefs in these habitats, has led to the proposal that they may act 501
as sentinel indicators of marine ecosystem health (Aguirre and Lutz, 2004). Gaining a better 502
understanding of the health and prevalence of diseases in marine turtle populations provides a 503
critical link between ecosystem health and turtle health. Effective management of both the 504
habitat and the species that rely on it is critical for effective species conservation. As FP has been 505
found to be associated with turtles resident in areas exposed to poor water quality (Herbst, 1994; 506
dos Santos et al., 2010; Van Houtan et al., 2010, 2014), FP prevalence may be a vital tool in 507
monitoring inshore marine habitats. Many of these marine environments are also utilised by 508
humans and consequently, research into the epidemiology of this disease could be mutually 509
beneficial for Green turtles, other species in these ecosystems and humans alike (Aguirre and 510
Lutz, 2004; Flint et al., 2010c). Long term monitoring of populations will allow researchers to 511
more accurately establish disease prevalence, corrected by demographic proportions. 512
513
Page 21 of 40

Whether the development of FP lesions is a result of a single agent or the interaction 514
between multiple factors is yet to be determined. It is clear that it is an infectious disease with a 515
strong link to ChHV5. In addition, the strong influence of different geographic regions on the 516
prevalence of FP and each of the viral variants indicate that FP is geographically specific (Herbst 517
et al., 2004; Ene et al., 2005; Patrício et al., 2012). The results from molecular studies targeting 518
ChHV5 in samples from turtles show that the virus is present in turtles with and without FP 519
lesions (Quackenbush et al., 2001; Page-Karjian et al., 2012; Alfaro-Núñez et al., 2014). Future 520
molecular studies targeting ChHV5 should consider these results and screen all samples for 521
ChHV5, not only those from turtles with FP lesions. Biosecurity and potential zoonosis should 522
always be considered by those handling marine turtles in both field and captive situations. 523
However, future research should prioritise understanding the triggers for lesion development. 524
525
Conclusions 526
There are many aspects of FP in marine turtles that are yet to be resolved and future 527
research needs to target those gaps which will ultimately aid in managing the disease. 528
Understanding how ChHV5 is transmitted between turtles and between regions is a key priority. 529
Molecular epidemiology is a useful tool for revealing genetic differences in this virus between 530
regions; possible relationships between host lineage and viral strain and the genes responsible for 531
pathogenesis and viral replication. Molecular investigations on ChHV5 from different regions 532
are essential to improve our understanding of the epidemiology and pathogenesis of this virus 533
which will in turn inform the management and conservation of a vulnerable species, the Green 534
turtle. 535
536
Acknowledgements 537
Page 22 of 40

We gratefully acknowledge the anonymous reviewers of this paper for providing 538
constructive comments which helped to improve this manuscript. We also thank Dr Colette 539
Thomas for assistance with water quality information. 540
541
Appendix: Supplementary material 542
Supplementary data associated with this article can be found in the online version 543
at…..setters please insert doi number 544
545
Conflict of interest statement 546
None of the authors of this paper has a financial or personal relationship with other 547
people or organisations that could inappropriately influence or bias the content of the paper. 548
549
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1348
Figure legends 1349
1350
Fig. 1. The complex life history of Green turtles. Adapted from Lanyon et al. (1989). 1351
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Fig. 2. The plastron and hind flippers of a Green turtle severely affected by fibropapillomatosis 1353
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