Molecular evidence for horizontal transmission of chelonid alphaherpesvirus 5 at green turtle (Chelonia mydas) foraging grounds in Queensland, Australia

Abstract

Fibropapillomatosis (FP) is a marine turtle disease recognised by benign tumours on the skin, eyes, shell, oral cavity and/or viscera. Despite being a globally distributed disease that affects an endangered species, research on FP and its likely causative agent chelonid alphaherpesvirus 5 (ChHV5) in Australia is limited. Here we present improved molecular assays developed for detection of ChHV5, in combination with a robust molecular and phylogenetic analysis of ChHV5 variants. This approach utilised a multi-gene assay to detect ChHV5 in all FP tumors sampled from 62 marine turtles found at six foraging grounds along the Great Barrier Reef. Six distinct variants of ChHV5 were identified and the distribution of these variants was associated with host foraging ground. Conversely, no association between host genetic origin and ChHV5 viral variant was found. Together this evidence supports the hypothesis that marine turtles undergo horizontal transmission of ChHV5 at foraging grounds and are unlikely to be contracting the disease at rookeries, either during mating or vertically from parent to offspring.

Fig 1. Samples (n = 62) were collected from six locations along the Queensland coast of Australia; Brisbane (n = 7), Gladstone (n = 4), Airlie Beach (n = 1), Bowen (n = 27), Townsville (n = 22), and Cairns (n = 1).

Five of these sites are located within the Great Barrier Reef (GBR) Marine Park, whilst Brisbane is located just south of the GBR boundary (indicated by hatched area).

Fig 2. Phylogenetic tree using the Maximum Likelihood method generated from the aligned 2565bp ChHV5 glycoprotein B (gB) gene.

The analysis involved 79 nucleotide sequences. Bootstrap values are indicated as a number on each branch and were calculated from 1000 replicates. Individual samples are identified with source location, haplotype, scientific name, tag number, and sample collection year. Sequences retrieved from the GenBank database originating from the Pacific and Altantic are named in the same way, with the accession number in the place of the tag number, and no haplotype information included as it was unknown.

Fig 3. Condensed phylogenetic tree showing the positions of the distinct Australian Variants relative to published sequences.

This tree was constructed using the Maximum Likelihood method generated from the aligned 2565bp ChHV5 glycoprotein B (gB) gene. The analysis involved 29 nucleotide sequences. Bootstrap values are indicated as a number on each branch and were calculated from 1000 replicates. Sequences retrieved from the GenBank database are indicated with source location, scientific name, accession number, and sample collection year. Host haplotype was used to determine the origin composition of each Australian variant in this study, expressed here as a proportion with colour reflecting host origin region; Orange = Coral Sea (CS)/southern Great Barrier Reef (sGBR)/New Caledonia (NC), Pink = NC, Red = Unknown, Purple = north-east Borneo/Sulu Sea, Green = northern Great Barrier Reef (nGBR)/NC and Yellow = nGBR.

Fig 4. Phylogenetic tree using the Maximum Likelihood method generated from the aligned 963bp ChHV5 Sialyltransferase (F-sial) gene.

The analysis involved 68 nucleotide sequences. Bootstrap values are indicated as a number on each branch and were calculated from 1000 replicates. Sequences retrieved from the GenBank database are indicated with the accession number, the source turtle’s scientific name, and sample collection location and year.