Fibropapillomatosis and the Chelonid Alphaherpesvirus 5 in Green Turtles from West Africa.

Fibropapillomatosis (FP) is a tumorigenic panzootic disease of sea turtles, most common in green turtles (Chelonia mydas). FP is linked to the chelonid alphaherpesvirus 5 (ChAHV5) and to degraded habitats and, though benign, large tumours can hinder vital functions, causing death. We analyse 108 green turtles, captured in 2018 and 2019, at key foraging grounds in Guinea-Bissau and Mauritania, West Africa, for the presence of FP, and use real-time PCR to detect ChAHV5 DNA, in 76 individuals. The prevalence of FP was moderate; 33% in Guinea-Bissau (n = 36) and 28% in Mauritania (n = 72), and most turtles were mildly affected, possibly due to low human impact at study locations. Juveniles had higher FP prevalence (35%, n = 82) compared to subadults (5%, n = 21), probably because individuals acquire resistance over time. ChAHV5 DNA was detected in 83% (n = 24) of the tumour biopsies, consistent with its role as aetiological agent of FP and in 26% (n = 27) of the 'normal' skin (not showing lesions) from FP turtles. Notably, 45% of the asymptomatic turtles were positive for ChAHV5, supporting multifactorial disease expression. We report the first baselines of FP and ChAHV5 prevalence for West Africa green turtles, essential to assess evolution of disease and future impacts of anthropogenic activities.

The Concurrent Detection of Chelonid Alphaherpesvirus 5 and Chelonia mydas Papillomavirus 1 in Tumoured and Non-Tumoured Green Turtles.

Characterised by benign tumours, fibropapillomatosis (FP) is a debilitating disease that predominantly afflicts the endangered green turtle (Chelonia mydas). A growing body of histological and molecular evidence has associated FP tumours with Chelonid alphaherpesvirus 5 (ChHV5). However, a recent study which detected both ChHV5 and Chelonia mydas papillomavirus 1 (CmPV1) DNA in FP tumour tissues has challenged this hypothesis. The present study aimed to establish a probe-based qPCR to assess the wider prevalence of CmPV1 and co-occurrence with ChHV5 in 275 marine turtles foraging in waters adjacent to the east coast of Queensland, Australia: three categories: Group A (FP tumours), Group B (non-tumoured skin from FP turtles) and Group C (non-tumoured skin from turtles without FP). Concurrent detection of ChHV5 and CmPV1 DNA is reported for all three categories, where Group A had the highest rate (43.5%). ChHV5 viral loads in Group A were significantly higher than loads seen in Group B and C. This was not the case for CmPV1 where the loads in Group B were highest, followed by Group A. However, the mean CmPV1 load for Group A samples was not significantly different to the mean load reported from Group B or C samples. Collectively, these results pivot the way we think about FP; as an infectious disease where two separate viruses may be at play.

Transcriptomic Profiling of Fibropapillomatosis in Green Sea Turtles (Chelonia mydas) From South Texas.

Sea turtle fibropapillomatosis (FP) is a tumor promoting disease that is one of several threats globally to endangered sea turtle populations. The prevalence of FP is highest in green sea turtle (Chelonia mydas) populations, and historically has shown considerable temporal growth. FP tumors can significantly affect the ability of turtles to forage for food and avoid predation and can grow to debilitating sizes. In the current study, based in South Texas, we have applied transcriptome sequencing to FP tumors and healthy control tissue to study the gene expression profiles of FP. By identifying differentially expressed turtle genes in FP, and matching these genes to their closest human ortholog we draw on the wealth of human based knowledge, specifically human cancer, to identify new insights into the biology of sea turtle FP. We show that several genes aberrantly expressed in FP tumors have known tumor promoting biology in humans, including CTHRC1 and NLRC5, and provide support that disruption of the Wnt signaling pathway is a feature of FP. Further, we profiled the expression of current targets of immune checkpoint inhibitors from human oncology in FP tumors and identified potential candidates for future studies.

Molecular Characterization of Chelonid Alphaherpesvirus 5 in a Black Turtle (Chelonia mydas) Fibropapilloma from Baja California Sur, Mexico.

During routine monitoring in Ojo de Liebre Lagoon, Mexico, a juvenile black turtle (Chelonia mydas) was captured, physically examined, measured, weighed, sampled, and tagged. The turtle showed no clinical signs suggestive of disease. Eleven months later, this turtle was recaptured in the same area, during which one lesion suggestive of fibropapilloma on the neck was identified and sampled for histopathology and molecular analysis. Histopathology revealed hyperkeratosis, epidermal hyperplasia, acanthosis, papillary differentiation and ballooning degeneration of epidermal cells, increased fibroblasts in the dermis, and angiogenesis, among other things. Hematological values were similar to those reported for clinically healthy black turtles and did not show notable changes between the first capture and the recapture; likewise, clinicopathological evaluation did not show structural or functional damage in the turtle's systems. The chelonid alphaherpesvirus 5 (ChHV5) UL30 gene was amplified and sequenced for phylogeny; Bayesian reconstruction showed a high alignment with the genus Scutavirus of the Eastern Pacific group. This is one of the first reports of ChHV5 in a cutaneous fibropapilloma of a black turtle in the Baja California peninsula.

Primary Multicentric Pulmonary Low-grade Fibromyxoid Sarcoma and Chelonid Alphaherpesvirus 5 Detection in a Leatherback Sea Turtle (Dermochelys coriacea).

We describe the gross, microscopical, histochemical and immunohistochemical features of a sclerosing pneumopathic disease process resembling primary multicentric pulmonary low-grade fibromyxoid sarcoma in a juvenile female leatherback sea turtle (Dermochelys coriacea). The animal was fresh, presented in good body condition and stranded dead in Aracaju, Sergipe state, Brazil, in September, 2017. Grossly, the lungs were enlarged bilaterally and the parenchyma was replaced by large, coalescing, white, firm masses that extended into the bronchi and bronchioles and to the pleura. Microscopically, these masses consisted of paucicellular populations of well-differentiated, spindle-shaped fibroblasts with low pleomorphism and low mitotic count, but tissue invasion. Abundant collagen in compact areas merged with peripheral fibromyxoid foci and inflamed stroma. Antibodies specific for cytokeratins AE1/AE3 and smooth muscle actin (SMA) labelled pneumocytes lining the remaining distorted alveoli and the hypertrophied and hyperplastic bronchial muscles, respectively. Tumour cells were negative for SMA; neither neoplastic nor normal tissues cross-reacted with antibodies specific for vimentin or Ki67. Chelonid alphaherpesvirus 5 (ChHV5) polymerase chain reaction analysis from formalin-fixed, paraffin wax-embedded lung tissue sections amplified a 450 base pair fragment of DNA-polymerase (UL30 region) that had 100% homology to sequences previously detected in green sea turtles (Chelonia mydas) on the Brazilian coast. Enterocolitis was a concomitant condition that likely caused morbidity in this case. These findings contribute to the body of knowledge on sea turtle health and expand the known geographical range for ChHV5 in the southern hemisphere.

Fibropapillomatosis in a Green Sea Turtle ( Chelonia mydas) from the Southeastern Pacific.

Fibropapillomatosis is a neoplastic disease that afflicts sea turtles. Although it is disseminated worldwide, cases of the disease have not been reported in the southeastern Pacific region. We describe a case of fibropapillomatosis in a green sea turtle ( Chelonia mydas) during its rehabilitation at the Machalilla National Park Rehabilitation Center, Ecuador. Viral presence was confirmed by PCR, targeting fragments of the chelonid alphaherpesvirus 5 (ChHV5) unique long (UL) genes, UL27, UL28, and UL30. The amplicons were sequenced and included in a global phylogenetic analysis of the virus with other reported sequences from GenBank. Results showed that the available viral sequences segregated into five phylogeographic groups: western Atlantic and eastern Caribbean, central Pacific, western Pacific, Atlantic, and eastern Pacific groups. The concatenated ChHV5 sequences from Ecuador clustered with the eastern Pacific sequences.

Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis.

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

Examining the Role of Transmission of Chelonid Alphaherpesvirus 5.

Marine turtle fibropapillomatosis (FP) is a devastating neoplastic disease characterized by single or multiple cutaneous and visceral fibrovascular tumors. Chelonid alphaherpesvirus 5 (ChHV5) has been identified as the most likely etiologic agent. From 2010 to 2013, the presence of ChHV5 DNA was determined in apparently normal skin, tumors and swab samples (ocular, nasal and cloacal) collected from 114 olive ridley (Lepidochelys olivacea) and 101 green (Chelonia mydas) turtles, with and without FP tumors, on the Pacific coasts of Costa Rica and Nicaragua. For nesting olive ridley turtles from Costa Rica without FP, 13.5% were found to be positive for ChHV5 DNA in at least one sample, while in Nicaragua, all olive ridley turtles had FP tumors, and 77.5% tested positive for ChHV5 DNA. For green turtles without FP, 19.8% were found to be positive for ChHV5 DNA in at least one of the samples. In turtles without FP tumors, ChHV5 DNA was detected more readily in skin biopsies than swabs. Juvenile green turtles caught at the foraging site had a higher prevalence of ChHV5 DNA than adults. The presence of ChHV5 DNA in swabs suggests a possible route of viral transmission through viral secretion and excretion via corporal fluids.