JPC SYSTEMIC PATHOLOGY
SPECIAL SENSES 
April 2021
S-V04

Signalment (JPC #4141684): Female juvenile green sea turtle

HISTORY: This turtle presented with a mass on the cornea and multiple additional masses on the skin. Initial surgery was performed to remove the masses. However, the corneal mass regrew, and the eye was enucleated and submitted for histology.

HISTOPATHOLOGIC DESCRIPTION: Eye, parasaggital section: Arising from the cornea, expanding the corneal epithelium and stroma, and forming multiple, variably sized, pedunculated, exophytic outgrowths is a well-demarcated, non-encapsulated, moderately cellular neoplasm composed of polygonal cells progressing from a hypertrophied stratum basale to a thickened stratum spinosum (acanthosis) and forming deep, anastomosing rete ridges overlying a stroma composed of a robust fibroblastic spindle cell proliferation. Neoplastic polygonal cells have distinct cell borders with intercellular bridges, a moderate amount of finely granular eosinophilic cytoplasm, and a round to oval nucleus with either finely stippled chromatin or marginated chromatin with a 3-5 µm diameter, homogenous, brightly eosinophilic intranuclear viral inclusion. Anisocytosis and anisokaryosis are moderate, and there are 9 mitotic figures per 2.37 mm². Supporting these polygonal cells, and confluent with the corneal stroma, are variably sized, moderately cellular proliferations of haphazardly arranged, variably plump to attenuated, fibroblastic to fibrocytic spindle cells on a moderate collagenous matrix.  These spindle cells have indistinct cell borders, a moderate amount of eosinophilic, fibrillar cytoplasm, and an ovoid to elongate nucleus with finely stippled chromatin and up to two prominent, magenta nucleoli. Admixed with this spindle cell proliferation and extant corneal stroma are aggregates of lymphocytes, plasma cells, fewer macrophages (keratitis), numerous melanocytes and melanophages (hyperpigmentation), and small caliber blood vessels (vascularization). There is multifocal necrosis of the superficial corneal stroma, characterized by amorphous eosinophilic material admixed with necrotic heterophils, cellular, and karyorrhectic debris (lytic necrosis). Multifocally, inflammatory cells and scant fibrosis surround anisotropic material - either suture material (suture granuloma), or yellow-golden, 3 µm thick, oval, approximately 20 x 30 µm, trematode eggs. Multifocally, there is both orthokeratotic hyperkeratosis of the cornea, and ulceration and replacement by a serocellular crust. There is bilateral diffuse anterior synechia with the iris adhered to the cornea, with extensive fibrosis and complete loss of Descemet's membrane. The lens is rotated (artifact), with abundant melanin adhered to the anterior capsule, and lens epithelium adhered to a robust fibrovascular membrane (synechia). There are few lymphocytes, plasma cells, and rare heterophils within the stroma of the ciliary body and fibrovascular membranes (uveitis). Ciliary body epithelium is regionally characterized by marked cytoplasmic swelling and vacuolization (degeneration). There are few dilated myelin sheaths within the optic nerve (degeneration). There are few trematode eggs within the choroid, with most containing a central miracidium.

MORPHOLOGIC DIAGNOSIS:

1. Eye, cornea: Viral fibropapilloma, green sea turtle, chelonid.
2. Eye: Trematode eggs, few.

ETIOLOGIC DIAGNOSIS: Herpesviral papilloma

CAUSE: Chelonid herpesvirus 5 (ChHV-5)

CONDITION: Fibropapillomatosis of sea turtles

GENERAL DISCUSSION:

ChHV-5:

• Neoplasia other than fibropapillomatosis is rare in sea turtles (Diaz-Delgado, J Comp Pathol, 2019)
• ChHV-5 causes cutaneous fibropapillomas and internal sarcomas of marine turtles
• Primarily affects green sea turtles, but documented in every species of sea turtle
• Primary cause of green sea turtle strandings in Hawaii (Work, Vet Pathol, 2015)
• Double stranded DNA virus (dsDNA), Alphaherpesvirus (Scutavirus genus) -> Alphaherpesvirinae contains all herpesviruses that infect non-avian reptiles
• ChHV-5 has been documented in healthy animals, suggesting other factors may be important in the development of disease
• Suspected horizontal transmission; superspreaders implicated in propagating the virus in naïve populations
• Factors that may predispose juveniles to infection: recent migration, adapting to new environments, social dynamics when encountering new populations, parasitism, elevated water temperatures, increased local human activity, and toxic algal blooms

Spirorchiid trematodes:

• Spirorchids are blood flukes of turtles, analogous to schistosomes in bird and mammals, and a common co-infection in animals identified with viral induced fibropapillomatosis (Yonkers, J Vet Diag Invest, 2015)

PATHOGENESIS:

• Viral attachment occurs when virion glycoprotein spikes bind host cell receptors, the viral envelope fuses with host cell plasma membrane, the nucleocapsid enters the cytoplasm, and then the DNA-protein complex is released and enters the nucleus and halts host cell macromolecule production; following viral replication, virions bud through host cell plasma membrane to gain envelope (Maclachlan, Fenner's Veterinary Virology, 2017)
• Capsid protein VP26 is common component of many herpesviruses, which remains in the cytoplasm when no viral replication is occurring; VP26 translocates to nucleus during viral replication
• Demonstrated F-VP26 in the nucleus of cells with intranuclear viral inclusions confirms active viral replication takes place in these cells
• Similar to papillomaviruses: requires stratified keratinocytes for lytic replication (Work, J Virol, 2017)
• ChHV-5 production requires close interplay between fibroblasts and keratinocytes (Work, J Virol, 2017)
• Transmission is sporadic, and not necessarily correlated with cells with intranuclear viral inclusions

TYPICAL CLINICAL FINDINGS:

• Vision, breathing, or feeding may be compromised, depending on location of lesions
• May interfere with normal swimming locomotion

TYPICAL GROSS FINDINGS:

• Tumors may be flat and plaque-like lesions, to raised and exophytic, gray to black, verrucous masses, possibly greater than 30 cm in diameter
• Typically arise from the skin around the eyes, mouth, and limbs, but are reported on the cornea, and the surfaces of the carapace and plastron
• Internal lesions may include variably sized fibrous nodules on different organs
  • Parenchyma of organs replaced by multifocal large, poorly demarcated, pale tan to white, firm masses that extend into adjacent areas

TYPICAL LIGHT MICROSCOPIC FINDINGS:

ChHV-5:

• Early bland fibroblast proliferation, followed by epidermal hyperplasia
• Exophytic nodules composed of papilliferous, hyperkeratotic projections
• Acanthosis, basal cell necrosis, clefting at the dermo-epidermal junction
• Possible subepidermal clefts, spongiosis, and epidermal inclusion cysts
• Prominent interdigitating rete ridges extend to deep dermis
• Eosinophilic intranuclear viral inclusions are observed only rarely, and are transient
• Internal tumors (oral cavity, glottis, lungs, kidney, heart, liver, spleen, GI tract) arise late in disease progression
• Internal lesions have highly variable morphologies
  • Linear, interlacing, whorling patterns, or any combination
  • Compact stroma, honeycomb appearance, or myxoid appearance

Spirorchiid trematodes:

• Foreign body granulomas with multinucleated giant cells are common to see when surrounding spirorchiid trematode ova in wild animals

ULTRASTRUCTURAL FINDINGS:

• Single, linear dsDNA packaged in protein capsid, ~125 nm in diameter
• Variable envelope size, leading virions to range from ~200-300 nm in diameter
• Capsids aggregate near the nucleus and associate with keratin filaments

ADDITIONAL DIAGNOSTIC TESTS:

• Myxoid morphology internal lesions have been Alcianophilic
• Epithelial component of fibropapilloma is AE1/AE3 cytokeratin immunopositive
• In situ hybridization has demonstrated ChHV-5 in tumor cells

DIFFERENTIAL DIAGNOSIS:

• Non-viral fibropapilloma

COMPARATIVE PATHOLOGY:

Chelonid herpesvirus-5 in other species:

• ChHV-5 was detected with PCR in a pulmonary low-grade fibromyxoid sarcoma in a leatherback sea turtle (vimentin and SMA immunonegative) (Diaz-Delgado, J Comp Pathol, 2019)

Other Chelonid herpesviruses:

• Chelonid herpesvirus 1 (Gray patch disease): Epidermal necrosis, papules, hyperkeratosis of head, neck, flippers in green sea turtle hatchlings
• Chelonid herpesvirus 6 (Lung, eye, tracheal disease virus [LETV]): Ocular, pharyngeal, and tracheal necrosis, pneumonia in juvenile green sea turtles

Other virally induced neoplasms:

• Eastern box turtles: Terrapene herpesvirus 2 (TerHV-2) is associated with fibropapillomas (Andersson, J Vet Diagn Invest, 2021)
• Cervids: Cutaneous fibromas (I-V02) caused by fibroma viruses (FV), members of Papillomaviridae; intranuclear viral inclusions are inconsistent
• Rabbits: Rabbit (Shope) fibroma virus induces cutaneous fibromas (I-V09); eosinophilic cytoplasmic viral inclusions are common in both epithelial cells and fibroblasts
• Rabbits, hares, jack-rabbits: Shope papilloma virus (cottontail rabbit papilloma virus) induces firm, white warts that may progress to "keratinous carcinomas", and resemble horns on the head, neck, pinnae, and eyelids
• Gray squirrels: Squirrel fibroma virus causes lesions similar to Shope fibromas in rabbits
• Freshwater and marine fish: Lymphocystis disease virus (LDV, I-V15) induces variably sized raised, white to tan masses on the skin, oral mucous membranes, and gills; dermal fibroblasts are extremely cytomegalic and hypertrophied

REFERENCES:

1. Andersson, KE, Adamovicz L, Mumm LE, et al. Detection of a novel herpesvirus associated with squamous cell carcinoma in a free-ranging Blanding's turtle. J Vet Diag Invest. 2021;33(2):348-351.
2. Diaz-Delgado J, Gomes-Borges JC, Silveira AM, et al. Primary multicentric pulmonary low-grade fibromyxoid sarcoma and Chelonid alphaherpesvirus 5 detection in a leatherback sea turtle (Dermochelys coriacea). J Comp Pathol. 2019;168:1-7.
3. MacLachlan NJ, Dubovi EJ, eds. Herpesvirales. In: Fenner's Veterinary Virology, 5^th San Diego, CA:Elsevier. 2017:208.
4. Rodriguez CE, Duque AMH, Steinberg J, Woodburn DB. Chelonia. In: Terio KA, McAloose D, St. Leger J, eds, Pathology of Wildlife and Zoo Animals. San Diego, CA:Elsevier. 2018:837-840.
5. Work TM, Dagenais J, Balazs GH, Schettle N, Ackermann M. Dynamics of virus shedding and in situ confirmation of Chelonid herpesvirus 5 in Hawaiian green turtles with fibropapillomatosis. Vet Pathol. 2015;52(6):1195-201.
6. Work TM, Dagenais J, Weatherby TM, Balazs GH, Ackermann M. In vitro replication of Chelonid herpesvirus 5 in organotypic skin cultures from Hawaiian green turtles (Chelonia mydas). J Virol. 2017;91(17):e00404-17.
7. Yonkers SB, Schneider R, Reavill DR, et al. Coinfection with a novel fibropapilloma-associated herpesvirus and a novel Spirorchis in an eastern box turtle (Terrapene carolina) in Florida. J Vet Diag Invest. 2015;27(4):408-413.

Click the slide to view.

---