Differences in Antibody Responses against Chelonid Alphaherpesvirus 5 (ChHV5) Suggest Differences in Virus Biology in ChHV5-Seropositive Green Turtles from Hawaii and ChHV5-Seropositive Green Turtles from Florida

Abstract

Fibropapillomatosis (FP) is a tumor disease associated with a herpesvirus (chelonid herpesvirus 5 [ChHV5]) that affects mainly green turtles globally. Understanding the epidemiology of FP has been hampered by a lack of robust serological assays to monitor exposure to ChHV5. This is due in part to an inability to efficiently culture the virus in vitro for neutralization assays. Here, we expressed two glycoproteins (FUS4 and FUS8) from ChHV5 using baculovirus. These proteins were immobilized on enzyme-linked immunosorbent assay plates in their native form and assayed for reactivity to two types of antibodies, full-length 7S IgY and 5.7S IgY, which has a truncated Fc region. Turtles from Florida were uniformly seropositive to ChHV5 regardless of tumor status. In contrast, in turtles from Hawaii, we detected strong antibody reactivity mainly in tumored animals, with a lower antibody response being seen in nontumored animals, including those from areas where FP is enzootic. Turtles from Hawaii actively shedding ChHV5 were more seropositive than nonshedders. In trying to account for differences in the serological responses to ChHV5 between green turtles from Hawaii and green turtles from Florida, we rejected the cross-reactivity of antibodies to other herpesviruses, differences in viral epitopes, or differences in procedure as likely explanations. Rather, behavioral or other differences between green turtles from Hawaii and green turtles from Florida might have led to the emergence of biologically different viral strains. While the strains from turtles in Florida apparently spread independently of tumors, the transmission of the Hawaiian subtype relies heavily on tumor formation.IMPORTANCE Fibropapillomatosis (FP) is a tumor disease associated with chelonid herpesvirus 5 (ChHV5) that is an important cause of mortality in threatened green turtles globally. FP is expanding in Florida and the Caribbean but declining in Hawaii. We show that Hawaiian turtles mount antibodies to ChHV5 mainly in response to tumors, which are the only sites of viral replication, whereas tumored and nontumored Floridian turtles are uniformly seropositive. Tumor viruses that depend on tumors for replication and spread are rare, with the only example being the retrovirus causing walleye dermal sarcoma in fish. The Hawaiian strain of ChHV5 may be the first DNA virus with such an unusual life history. Our findings, along with the fundamental differences in the life histories between Floridian turtles and Hawaiian turtles, may partly explain the differential dynamics of FP between the two regions.

Keywords: Chelonia mydas; IgY; chelonid herpesvirus 5; fibropapilloma; fibropapillomatosis; green turtle; herpesvirus; serology.

FIG 1

Primary characterization of baculovirus-derived antigens by Western blotting. (A) Cell culture supernatants from baculovirus-inoculated Sf9 cells expressing Signal-FUS4-c-myc-GST (lane 1), Signal-FUS8-c-myc-GST (lane 2), and Signal-c-myc-GST (lane 3) were resolved on a 10% polyacrylamide gel, transferred to nitrocellulose, and probed with a monoclonal antibody against c-myc. (B) Western blot of a nonreducing gel analyzed with a monoclonal antibody against GST to further characterize the Signal-FUS4-c-myc-GST protein (lane 4). The black arrow points to the band of the expected size; red arrows point to bands of unexpected sizes. Note that the gel shown in panel B had to be overloaded to make the bands clearly visible. The positions of relevant molecular size markers are indicated (lanes M), and the numbers on the left are in kilodaltons.

FIG 2

Hawaiian turtles were uniformly larger than Floridian turtles. Box plots of the straight carapace length (SCL) for green turtles from Florida and Hawaii partitioned by tumor score (TS) categories, ranging from nontumored (TS0) to severely tumored (TS3). The boxes represent the 25th and 75th percentiles, whiskers are 1.5 times the interquartile range, and the black dots are the medians.

FIG 3

Negative sera have uniformly low absorbances. Box plot of delta OD (OD for FUS4 or FUS8 minus the OD for the c-myc backbone) from 1 positive-control (Pos) and 10 negative-control (Neg) plasma samples tested at a 1:25 dilution for 7S or 5.7S IgY antibody reactivity against FUS4 and FUS8 on seven ELISA plates over 3 days (four plates, day 1; three plates, day 2; two plates, day 3). The boxes represent the 25th and 75th percentiles, whiskers are 1.5 times the interquartile range, black dots are medians, and open circles are outliers.

FIG 4

Delta OD and the percentage of the value for the positive control correlate. Scatter plot ELISA results are expressed as delta OD₄₅₀ values versus the delta OD₄₅₀ values expressed as a percentage of the value for positive-control plasma for tumored and nontumored Floridian (n = 74, left) and Hawaiian (n = 110, right) green turtles assayed for the reactivity of 7S or 5.7S IgY against FUS8 or FUS4.

FIG 5

Plasma dilutions correlate with absorbance. The plots show the delta OD₄₅₀ values versus the log of serial 10-fold dilutions of plasma partitioned by antigen and antibody reactivity for 18 Hawaiian green turtles (gray), with the mean OD (solid black line) ± 1 standard deviation (dotted lines) being shown.

FIG 6

Increasing ELISA reactions with increasing tumor score. Delta OD values for different groups of animals according to tumor score categories are shown. SLP, Sea Life Park negative controls; Kona, nontumored turtles from Kona, HI (an FP-free area); TS0 to TS3, tumor scores of 0 to 3, respectively, for animals from areas in Hawaii and Florida where FP is enzootic. Thick dark lines are the mean OD values for a particular tumor score category, dotted lines are the high-specificity cutoff, and solid lines are the high-sensitivity cutoff. Sample sizes were as follows: SLP, 10 plasma samples; Kona, 20 plasma samples; TS0, 40 plasma samples; TS1, 44 plasma samples; TS2, 44 plasma samples; TS3, 26 plasma samples.

FIG 7

Contrasting seroresponses of Hawaiian and Floridian turtles. The percentages of green turtles from Hawaii and Florida testing ELISA positive (given as the percentage of seropositive turtles) for 7S or 5.7S IgY antibody reactivity to FUS4 or FUS8 according to the high-sensitivity (High Se) or high-specificity (High Sp) cutoffs for the following six groups of turtles are shown: Sea Life Park negative controls (SLP); tumor-free turtles from the Kona coast, west Hawaii (Kona); and turtles from Kaneohe Bay, Oahu, where tumors are endemic, with tumor scores (TS) ranging from 0 (TS0; no tumors) to 3 (TS3; severely tumored).

FIG 8

Some but not all virus shedders showed strong ELISA reactions, which inferred chronic antigen exposure. Delta OD values are for 10 shedders and 8 nonshedders of ChHV5. Thick dark lines are the mean OD values for a particular shedding category, dotted lines are the high-specificity cutoff, and solid lines are the high-sensitivity cutoff.

FIG 9

Cloning strategy. The amino acid sequences of extracellular domains (exodomains) of the FUS4 and FUS8 glycoproteins (top), depicted here with their signal peptides (red), transmembrane region, and intracellular regions, were selected for gene synthesis. The synthetic constructs (blue arrow, middle) were bracketed by BamHI restriction enzyme sites. A third synthetic construct (red arrows, bottom), comprising a baculovirus signal sequence, an internal BamHI restriction enzyme site, a c-myc tag, and a GST tail, flanked by attB recombination sites, was cloned into pDONR221 before two variants were constructed. One variant had the FUS4 exodomain inserted into the BamHI site, and the other had the FUS8 exodomain inserted into the BamHI site.