Sample collection from sea turtles
In total, 45 serum samples from 33 juvenile green turtles (C. mydas), including 6 sea turtles with tumors, 5 juvenile hawksbill turtles (Eretmochelys imbricate), and 7 olive ridley turtles (Lepidochelys olivacea) (juvenile = 5; sub-adult = 2). All turtles were sourced from: eastern Taiwan (n = 24), southern Taiwan (n = 14), central Taiwan (n = 6), and northern Taiwan (n = 1). Among the 45 sea turtle samples, 6 green turtles developed FP (n = 1 with tumor score 1; n = 1 with tumor score 2; n = 4 with tumor score 3)32, while 39 did not have FP. FP tumor tissues were collected from 6 green turtles (from shoulder/flippers/inguinal regions) with FP during surgical procedures. Regarding the collection of normal skin tissues, one normal skin tissue (from shoulder) was collected from one necropsied dead green turtles (stranding and discovered from southern Taiwan) confirmed without FP. All tissue samples were fixed in 10% neutral buffered formalin prior to further analysis. In this study, all sea turtles were discovered and rescued through the official reporting system of the Marine Animal Rescue Network (established by the Ocean Conservation Administration) and admitted to the National Museum of Marine Biology and Aquarium (NMMBA), between 2017 and 2020.
Detection of ChHV5 DNA by polymerase chain reaction (PCR)
Total DNA was extracted from blood of 45 sea turtles by DNeasy blood & tissue kit (Cat. No. 69504, Qiagen, Valencia, CA, USA) following manufacturer’s instructions. Subsequently, the ChHV5 infection status all 45 sea turtles was determined by PCR using primers targeting on UL18 (capsid protein gene), UL22 (glycoprotein H gene), and UL27 (glycoprotein B gene) regions4. The sequence of primer sets are: UL18F: 5′-CACCACGAGGGGGAAAATGA, UL18R:5′-TCAAATCCCCCGTTCACTCG; UL22F: 5′-ACGGCGTTGGCTAGTGAATC, UL22R: 5′-GCAGTTCGGTACACACCTCT; UL27F: 5′-TAACAAGAAAGAACCGCGCG; UL27R: 5′-ATTTTCCCGGTCAGTGCCAA. PCR amplifications were performed in a total volume of 50 μl. The reaction included 1 μl of the template DNA, 1 μl of each primer (10 μM), 22 μl of distilled water (DDW), and 25 μl of the AmpliTaq Gold® 360 Master Mix (Cat. No. 4398876, Life Technologies, Valencia, CA, USA). The thermocycle for amplification was: Initial denaturing at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, and then a final extension at 72 °C for 7 min. Results were visualized by gel electrophoresis (2% agarose) with SYBR Safe DNA Gel Stain (Cat. No. S33102, Invitrogen, Carlsbad, CA, USA).
Sequence optimization of the UL27 gene for expression of the ChHV5 glycoprotein protein using E. coli
To express large quantities of ChHV5 gB, we adopted the prokaryotic Escherichia coli (E. coli) expression system. The construct (namely UL27/pUC57) containing sequences of the full length UL27 fused with FLAG tag sequence (GenBank accession no. AF035003.3) was synthesized by Allbio Science Co., Ltd, Taiwan. The sequence information of the glycoprotein (gB) datasets used and analyzed for protein expression during the current study was obtained and available from the GenBank repository [https://www.ncbi.nlm.nih.gov/nuccore/AF035003.3]. Considering the difference in tRNA-codon usages between prokaryotes and eukaryotes would possibly affect subsequent protein expression, the optimized UL27 gene sequence, without altering the translated amino acid sequences, to fit the E. coli expression system was synthesized. The codon optimized UL27 gene was further used as the template for amplification of different gene fragments by Polymerase Chain Reaction (PCR).
Construction of plasmids expressing partial fragments of ChHV5 gB protein
To determine the relative antigenicity and also to increase the expression yield, plasmids expressing various regions of gB protein were constructed. Briefly, the five regions covering different fragments of the UL27 gene were amplified from plasmid UL27/pUC57 by PCR using specific primer sets with built-in restriction enzyme sequences shown as underlined in Table 1. The thermal cycling conditions were: 98 °C (5 min) followed by 35 cycles of denaturation (98 °C, 30 s), annealing (58 °C, 1 min), and extension (72 °C, 2 min), and finished with a final extension (72 °C, 10 min). PCR amplicons with expected sizes were isolated from gel and trimmed with the restriction enzymes followed by ligation with vectors either pET24a or pET32b (Novagen, Germany) linearized with the same restriction enzymes. The resulting plasmids with expected insert sizes as confirmed by restriction enzymes were sent for automated DNA sequencing (Mission Biotech, Taipei, Taiwan).
Expression of recombinant gB fragments in E. coli
In the current study, the recombinant gB protein is a key reagent that served as antigen for seroprevalence of ChHV5 as well as for the generation of ChHV5 gB antibody (conducted by Yao-Hong Biotech Inc., Taiwan). The plasmids expressing individual gB fragment were transformed into E. coli host cells, strain BL21 (DE3), Rosetta. Expression of all the recombinant gB fragments was induced by 0.8 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG) at 28 °C for 16 h. As all the gB fragments cloned into the pET series vectors were expressed as a fusion protein with a 6-histidine tag at C-terminus end, they could be further purified by Ni–NTA column chromatography using the chelating Sepharose Fast Flow (GE Healthcare) following the method described in one previous study33. The yield and purity of recombinant gB proteins were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, 6 M urea and 0.4 M imidazole contained in the purified protein were depleted by step-wise dialysis against 1 × PBS buffer (0.02 M phosphate, 0.15 M NaCl) with gradually decreased concentrations of urea at 4 °C. The concentration of recombinant proteins were then estimated by National Institutes of Health ImageJ software (https://imagej.nih.gov/ij/, 1997–2018.) using the standard curve established by bovine serum albumin (BSA) with known concentrations42.
Western blot analysis
Recombinant gB fragments were separated by 12.5% or 15% SDS-PAGE and electrotransferred to PVDF membrane by using Mini Proten III apparatus (Cat. No. 165-3301, BioRad). The filters were blocked in PBS-T buffer (0.02 M phosphate, 0.15 M NaCl, 0.05% Tween-20) containing 5% skimmed milk and reacted with mouse anti-his tag antibody (1:5,000, Cat. No. GTX40628, GeneTex) at 4 °C for overnight. After six-time wash with PBS-T buffer, the PVDF filter was then incubated with the secondary antibody, 1:5000 diluted goat anti-mouse IgG conjugated with horseradish peroxidase (HRP), or 1:500 diluted Protein A/G-HRP (Cat. No. 32400, Thermo fisher scientific™, United States) for sea turtle antibody detection, at room temperature for 1 h followed by PBS-T wash to remove the unbound antibodies. Ultimately, the signal was detected by ECL reagents (Thermo Fisher Scientific, United States) and the image was acquired by ImageQuant LAS 4000 Mini (GE Healthcare).
Immunohistochemical (IHC) analysis
To establish IHC protocol, normal skin tissue from PCR-negative sea turtles served as the negative control. In total, the FP on skin tissue from six individual sea turtles that were detected positive for ChHV5 DNA (positive tissue samples), and one normal tissue detected negative (the negative tissue) were included in the IHC analysis.
IHC procedure was conducted as reported in our previous study34. In brief, sections of formalin-fixed and wax-embedded skin tissues of sea turtles were made using a rotary microtome (Leica RM2245, Leica Biosystems, Germany) and were further deparaffinized and rehydrated. Antigen retrieval was carried out by heat-induced epitope retrieval method: slides immersed into boiled sodium citrate buffer (0.01 M, pH 6.0), which was preheated up to 100 °C, for 20 min and cooled at room temperature for 20 min. Subsequently, the slides were incubated with peroxidase-blocking reagent (Cat. No. S200389, Dako, Denmark) for 30 min, and then treated with or without primary antibodies (the anti-gB serum prepared from this study). In each interval of the following procedures, sections were rinsed with a mixture of TBST buffer. Tissue sections were then reacted with secondary antibody (HRP anti-rabbit/mouse, DAKO, Denmark), followed by incubation of DAB and chromogen (dilution 1 μL in 100 μL) from a commercial ChemMate EnVision detection kit (Cat. No. K5007, Dako, Denmark). Ultimately, tissue sections were counterstained with Mayer’s hematoxylin reagents (Code S3309, Dako, Denmark) for 2 min followed by wash with DDW, and reacted with 37 mM ammonia water for 5 s and rinsed with DDW.
Immunofluorescent assay (IFA)
Human 293 T cells were transfected with plasmids expressing full-length ChHV5 gB protein fused with FLAG tag at its C-terminus. At 24 h post transfection, 293 T cells (CRL-3216, ATCC, USA) were fixed with 2% formaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 for another 10 min. Subsequently, cells were incubated with anti-FLAG antibody (1:500) (F7425; Sigma-Aldrich), or antisera (F1, F2, F3, F2–3) at the dilution of 1:500 for 1 h at room temperature. After six times of washes with PBS containing1% bovine serum, goat anti-mouse IgG (1:2,000 fold diluted) (Cat. No. A28175, Alexa Fluor® 488, Invitrogen) was used as secondary antibody. After one-hour incubation, nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Cat. No. D9542, Sigma-Aldrich) for 10 min, followed by confocal microscopy (FV1000, Olympus, Tokyo, Japan) with Olympus FV10-ASW 1.3 viewer software.
Statistical analysis
To evaluate the association between seropositivity and FP or viremia tested by PCR of UL27 gene, Fisher’s exact test was applied due to very limited number of sea turtles with FP. The statistical significance was determined by p < 0.05. The software R (version 4.0.1) was used for statistical computing.
Ethics statement
No sea turtles were specifically captured for the purpose of this study. All experiments were performed in accordance with relevant guidelines and regulations, and reported in accordance with the ARRIVE guidelines. The scientific permit numbers for research were obtained from the Institutional Animal Care and Use Committee (IACUC) of the NMMBA (Permit no. 2017005, 2018007, and 2020001), the Forestry Bureau (Permit no. 1071655703), and the Ocean Conservation Administration (Permit no. 1070003700 and 1080007820).
Source: Ecology - nature.com
