ASSESSING IMMUNE GENE ASSOCIATION WITH FIBROPAPILLOMATOSIS IN GREEN SEA TURTLES (CHELONIA MYDAS) IN KENYA.

Abstract

Background: Sea turtles are reptiles that play indispensable roles in marine ecosystems, especially coral reefs and sea grass. However, their populations are in danger of extinction due to fibropapillomatosis (FP), a viral tumor-causing disease whose prevalence is linked to human impacts on turtle foraging areas. Of the turtle species, green sea turtles (Chelonia mydas) are mostly affected by the disease. In Kenya, for instance, it was reported that nearly all C. mydas unintentionally caught by fishers had tumors. The reasons for tumor development were unknown, which raised the need to investigate functional genetic factors influencing FP in C. mydas. The likely causative agent of Fp is Chelonid alpha herpes virus 5 (ChHV5). ChHV5 has a gB gene that codes for glycoproteins which are present on the surface of the virus, allowing it to come into contact with the host immune system. Sea turtles possess Major Histocompatibility Complex (MHC) molecules that recognize peptides and present them to T cells. Materials and methods: The study sampling technique was scrapping the skin ( for sea turtles without tumors) and cutting (for sea turtles with tumors). A total of 207 samples were collected from the North Coast (Watamu and Marereni) and South Coast (Diani) in 2021. The prevalence of Chelonid alpha herpes virus 5 (ChHV5) was determined by PCR amplification of DNA-pol gene that is used to determine the presence or absence of ChHV5 virus. The α1 peptide binding region of MHC I exon 2 (~220 bp) was sequenced using Sanger sequencing technique. The sequences were used to determine genetic diversity using nuc.div() function from Pegas v.0.12 and seg.sites() function from Ape v.5.3 packages in R. Phylogenetic trees were also generated to determine evolutionary relationship in C. mydas. Haplotype network was used to check for clustering among the haplotypes of C. mydas without tumors and with tumors. Variant calling was done using Genious Prime, SNPs were generated to determine association of MHC I SNPs and FP. Results: The ChHV5 was detected in all 50 tumors of C. mydas with Fp. In all the 157 C. mydas without tumors ChHV5 was not detected. The C. mydas showed a high level of MHC I polymorphism based on the number of polymorphic sites (191) and nucleotide diversity. The C. mydas phylogenetic tree had no distinct clustering pattern between C. mydas with tumors and those without tumors. The phylogenetic tree of Kenya, Florida, and Cape Verde, C. mydas had three clades that did not cluster by species. The haplotypes network, had no distinct clustering pattern between C. mydas with tumors and those without tumors, only 5 haplotypes were shared. Lastly, SNP 1(RR=5.77, 95% CI=0.77- 43.25) and SNP 4.3 (RR=2.00, 95% CI=0.54-7.47) had elevated risk ratio values greater than one indicating potential risk factors. Conclusion: Detection of ChHV5 in all tumors indicates that ChHV5 could be the possible causative agent of FP infection. The study revealed high MHC I polymorphism in C. mydas indicating high genetic diversity and no association between the SNPS of MHC I and FP.