In 1992, an interdisciplinary research team headquartered at the University of Florida

began studies in key targeted areas of fibropapillomatosis (FP) etiology and

pathogenesis. At that time, little was known about FP outside of field studies

documenting its prevalence in different areas of the world and studies of tumor

histopathology. Our primary objective was to develop a broad-based scientific

understanding of FP by applying principles of tumor biology, immunology, pathology,

virology, molecular biology, and epidemiology to FP in the green turtle, Chelonia

mydas. Long-term goals included the development of assays for FP and study of any

role of environmental co-factors in the disease. This report is a continuation of that

effort and the results reported here bring us closer to understanding the role of a

tumor-associated herpesvirus in marine turtle fibropapillomatosis. 

This research has demonstrated that marine turtle herpesviruses can persist for

extended periods of time as infectious agents in the marine environment and that wild

green turtles in Florida are exposed to the LETD-associated herpesvirus. This is the

first description of LETV infection in free-ranging. marine turtles. In addition, data is

presented that supports the hypothesis that LETV and FPHV infections are

independent. These data reveal new levels of complexity that must be addressed

before reliable serodiagnostic assays for herpesvirus infections of chelonians can be

developed for widespread application. The results reported here also raise new

concerns about the potential impact of infections by new herpesviruses on populations

of wild marine turtles, an area which has previously been unexplored by turtle

biologists. (8 page document

Jacobson, Elliott

Klein, Paul A.

English

Aquatic Commons

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1
Final Report
Research Work Order #194
November 11 t h, 2001
Project Title: Further Strategies for Evaluating the Etiological Role of a
Tumor-associated Herpesvirus in Marine Turtle Fibropapillomatosis
Principal Investigator: Paul A. Klein, Ph.D.
Professor, Pathology, Immunology, and
Laboratory Medicine
University of Florida, Gainesville, FL
Co-principal investigator: Elliott Jacobson, D.V.M., Ph.D.
Professor, Small Animal Clinical Sciences
College of Veterinary Medicine
University of Florida, Gainesville, FL
Collaborators: Larry Herbst, D.V.M, Ph.D
Assistant Professor
Albert Einstein College of Medicine, Bronx, NY
Daniel Brown, Ph.D.
Assistant Scientist
Department of Pathobiology
College of Veterinary Medicine
University of Florida, Gainesville, FL
Llewellyn Ehrhart, Ph.D., Professor,
Department of Biology, University of Central Florida
Orlando, Florida
Ritchie Moretti and Sue Schaf
Hidden Harbor Marine Environmental Project, Inc.
and The Turtle Hospital, Marathon, Florida
Graduate Student:
Project Duration:
Sadie S. Coberley (Ph.D. Program)
Interdisciplinary Program in Biomedical Sciences
College of Medicine
University of Florida, Gainesville, FL
Two years: 9/15/99-9/14/01
2INTRODUCTION
In 1992, an interdisciplinary research team headquartered at the University of Florida
began studies in key targeted areas of fibropapillomatosis (FP) etiology and
pathogenesis. At that time, little was known about FP outside of field studies
documenting its prevalence in different areas of the world and studies of tumor
histopathology' Our primary objective was to develop a broad-based scientific
understanding of Fp by apl3lying principles of tumor biology, immunology, pathology,
virology, molecular biology, and epidemiology to FP in the green turtle, Chelonia
mydas. Long-term goals included the development of assays for FP and study of any
role of environmental co-factors in the disease. This report is a continuation of that
effort and the results reported here bring us closer to understanding the role of a
tumor-associated herpesvirus in marine turtle fibropapillomatosis.
OVERALL SUMMARY
Sea turtle conservation efforts must include the means to monitor the health status of
sea turtle populations for exposure to disease-associated microorganisms. Critically
needed are improvements in diagnostics, inclUding the development of defined
pathogen-specific antigens for use in immunoassays that measure disease exposure.
Herpesviruses are associated with several diseases of marine turtles including lung-
eye-trachea disease (LETD) and fibropapillomatosis. While the LETD-associated
herpesvirus (LETV) can be cultivated in the laboratory, efforts to cultivate the FP-
associated herpesvirus (FPHV) have been unsuccessful, limiting diagnostic assay
development and epidemiological studies. In this study, we have extensively studied
LETV in order to gain critical information about both LETV and FPHV. In doing this we
have developed a better understanding of FP and the potential role of FPHV in its
etiology. This work moves us closer to the development of an assay to detect exposure
of wild sea turtles to FPHV.
This research has demonstrated that marine turtle herpesviruses can persist for
extended periods of time as infectious agents in the marine environment and that wild
green turtles in Florida are exposed to the LETD-associated herpesvirus. This is the
first description of LETV infection in free-ranging. marine turtles. In addition, data is
presented that supports the hypothesis that LETV and FPHV infections are
independent. These data reveal new levels of complexity that must be addressed
before reliable serodiagnostic assays for herpesvirus infections of chelonians can be
developed for widespread application. The results reported here also raise new
concerns about the potential impact of infections by new herpesviruses on populations
of wild marine turtles, an area which has previously been unexplored by turtle
biologists.
3RESULTS SUMMARIES
Note: Copies of 3 papers published or in press are attached to this report.
These contain the full details of results, materials, and methods
1. Persistent Infectivity of a Green Turtle Disease-Associated Herpesvirus
after Exposure to Seawater Note: Certain aspects of this research were funded by
RWO 180 to P.A. Klein as well as this RWO.
Herpesviruses are associated with several diseases of marine turtles including Lung-
Eye-Trachea disease and Gray Patch Disease (GPO) of green turtles (Chelonia
mydas) and fibropapillomatosis of green, loggerhead (Carettacaretta), and olive ridley
turtles (Lepidochelys olivacea). Stability of chelonian herpesviruses in the marine
environment has not been previously studied. In these experiments, LETD-associated
herpesvirus (LETV) was used as a model chelonian herpesvirus to test viral infectivity
after exposure to seawater. The LETV was grown in terrapene heart (TH-1) cells and
then virus preparations were dialyzed for 24 to 120 hr against aerated artificial or
natural seawater or Hank's balanced salt solution (HBBS). TH-1 cell cultures were
inoculated with seawater-exposed LETV, and on day 10 post-infection cells were
scored for cytopathic effect (CPE). Virus samples tested up to 120 hr after seawater
exposure were positive for the herpesvirus DNA polymerase gene by polymerase
chain reaction. Electron microscopy revealed intact LETV nucleocapsids after
exposure of LETV to artificial seawater or HBSS for 24 hr at 23 C. LETV preparations
were then titered for infectivity and were found to remain infectious after 120 hr of
exposure to natural and artificial seawater at 23 C. Similar results were obtained with
a second cultivable chelonian herpesvirus, HV2245. LETV infectivity could not be
detected after 48 hr exposure to artificial seawater at 30 C. Since LETV remains
infectious for extended periods of time in the marine environment, it is possible that
FP-associated and GPO-associated herpesviruses also may be stable. These findings
are significant both for researchers studying the epidemiological association of
herpesviruses with diseases of marine turtles and for individuals who handle turtles in
marine turtle conservation efforts.
2. Detection of Antibodies to a Disease-associated Herpesvirus
of the Green Turtle, Chelonia mydas
Lung-eye-trachea disease-associated herpesvirus-is linked with morbidity and
mortality in mariculture-reared green turtles, but its prevalence among and impact on
wild marine turtle populations is unknown. An enzyme-linked immunosorbent assay
(ELISA) was developed for detection of anti-LETV antibodies and could distinguish
LETV exposed green turtles from those with antibodies to fibropapillomatosis-
associated herpesvirus. Plasma from two captive-reared green turtles immunized with
inactivated LETV served as positive controls. Plasma from 42 healthy captive-reared
green turtles, and plasma from 30 captive-reared green turtles with experimentally
induced fibropapillomatosis (FP) and anti-FPHV antibodies had low ELISA values on
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4
LETVantigen. A survey of wild green turtles with (n:19) and without FP (n:27) (with
and without anti-FPHV antibodies, respectively) identified individuals with antibodies
to LETV regardless of their FP status. The seroprevalence of LETV infection was 13%.
The presence of antibodies to LETV in plasma samples was confirmed by Western blot
and immunohistochemical analyses. These results are the first to suggest that wild
Florida green turtles are exposed to LETV or to antigenically closely related
herpesvirus(es) other than FPHV and that infection with FPHV and LETV are most
likely independent events, This is the first ELISA developed to detect antibodies for a
specific herpesvirus infection of marine turtles. The specificity of this ELISA for LETV
(ability to distinguishLETV from FPHV) makes it valuable for detecting exposure to this
specific herpesvirus and enhances our ability to conduct seroepidemiological studies
of these disease-associated agents in marine turtles.
3. Survey of Florida Green Turtles for Exposure to a Disease-associated
Herpesvirus
A recently developed enzyme-linked immunosorbent assay (ELISA) was used
to assess exposure of Florida wild green turtles, Chelonia mydas, to LETV, the
herpesvirus associated with lung-eye-trachea disease. Plasma samples from 329 wild
juvenile green turtles netted in the Indian River lagoon, along the Sebastian reef, or in
the Trident basin (Indian River and Brevard Counties, Florida, USA) were tested by
ELISA for the presence of antibodies to LETV. Plasma samples from 180 wild juvenile
green turtles were tested from these study sites to compare the prevalence of anti-
LETVantibodies. While some plasma samples from each site contained anti-LETV
antibodies (confirmed by Western blot analysis), plasma samples collected from the
Indian River lagoon had statistically higher optical density values measured in the
ELISA. No statistical differences were observed when these same plasma samples
were analyzed for changes in the level of anti-LETV antibodies over three years (1997,
1998, and 1999). To explore the relationship between anti-LETV antibodies and
fibropapillomatosis (FP), plasma from 133 green turtles scored for fibropapilloma
tumor severity were tested by ELISA. There was no correlation between tumor severity
and the presence of antibodies against LETV. Additional plasma samples collected
from 16 tagged green turtles captured and sampled more than once (recaptures) were
also tested to monitor antibody levels to LETV relative to the FP status of individual
turtles over time. Again there was no clear relationship between FP tumor status and
the presence of antibodies to LETV. Finally, ELISA tests on plasma from 13 nesting
female turtles (9 green and 4 loggerhead) revealed high levels of anti-LETV
antibodies in 11 individuals, including 2 loggerhead turtles. These results provide
strong evidence that wild Florida green turtle populations at these three study sites are
exposed to LETV or a closely related virus and that loggerhead turtles may be
exposed as well. Based on a cutoff optical density value of 0.310, 71 out of the 329
wild Florida green turtles tested were seropositive for LETV antibodies
(seroprevalence : 21.6%). In addition, no relationship between FP tumor severity or
status and the presence of anti-LETV antibodies was found, further supporting the
5hypothesis that LETV and the FP-associated herpesvirus are separate infections of
marine turtles.
4. Development of a LETV genetic library
Terrapene heart cells (1.OX107) were infected with LETV clone 221. After 6-7 days post
infection, cells with. 1QO% cytopathic effect were collected from the supernatant by
centrifugation 2500 g 10 min at 4 ac. Infected pelleted cells were resuspended in L
buffer (0.1 M EDTA pH 8.0, 0.01 M Tris HCI pH 7.6, 0.02M NaC!), mixed with equal
volume 1.2% low temperature agarose, and poured in plug mold apparatus.
Embedded infected cells were lysed in situ with 2 changes of L buffer plus 1% v/v
Sarkosyl plus 0.1 % wlv proteinase K for 48 hrs at 50 C. After 3 washes in L buffer,
plugs were kept in TE pH 8.0 at 4 ac. A 10 well 1.0% 0.5X TBE low temperature
aqarose gel was poured and _ of a plug was loaded into each of 8 wells. Lambda
Ladder PFG marker (New England Biolabs, Beverly MA) and Low Range PFG Marker
(New England Biolabs, Beverly MA) were loaded into the remaining wells. LETV 221
genomic DNA was separated by pulse-field gel electrophoresis (200V, 24 hrs, 50-
90sec) at 4 C. The gel was stained in ethidium bromide bath and bands visualized on
long wavelength UV box. Bands corresponding to the LETV genome (approx 120 kb)
were cut out of the gel and were melted at 70 C for 20 min. Melted gel slices were
digested in ~ agarase (5 units/1 00 mg) overnight at 45 C. The mixture was transferred
to Centricon YM-100 (Millipore, Medford MA) and concentrated by centrifugation at
500 g for 20 min @ room temperature. The centricon was washed with 20%
isopropanol, reconcentrated, and then washed with 2 volumes of TE pH 7.5. The
centricon was inverted and the DNA sample was collected by centrifugation. 20 I-tg of
LETV 221 DNA was sonicated to generate fragments 1-2 kb in size and was size
fractionated on CHROMA SPIN-100 gel filtration columns (Clontech, Palo Alto CAl to
remove DNA molecules smaller than 300 bp. The DNA was eluted, ethanol
precipitated, and blunt ends were formed using T4 DNA polymerase. The DNA was
ligated into Smal linearized pUC18 cloning vector and then the ligated product was
ethanol precipitated. The ligated product was electroporated into E. coli host TOP10
and clones were blue/white selected on LB plates plus X-gal and IPTG. Clones were
grown in LB plus ampicillin for 20 hours in 96 well plate format with agitation. Bacterial
clones were harvested and DNA extracted with Qiagen Biorobot (Qiagen Valencia
CAl. DNA samples were quantified, and then sequenced on MegaBACE sequencer
using ET terminator mix in MJ thermocycler (Amersham Pharmacia, Biotech,
Piscataway NJ). Sequences were compared to other sequences by blastx (basic local
alignment search tool in protein database) searching the NCBI (National Center for
Biotechnology Information) database. Clones matching herpesvirus genes were
arranged according to the gene order of the model alpha-herpesvirus, human simplex
virus 1.
65. Identification of immunodominant LETV antigens
We have initiated studies to identify the specific LETV proteins that green turtles
respond to with antibody formation following infection with this herpesvirus. We used
two-dimensional Western blots to study a 38 kD protein that is recognized by
antibodies in the plasma of green turtles exposed to LETV. Figure 1 shows that this
protein can be resolved and visualized by two-dimensional gel electrophoresis (Figure
1 A) and Western blottinq (Figure 1B). This protein is currently being sequenced in the
Protein Core of the Universityof Florida Biotechnology Program.
DISCUSSION
Seroepidemiology, which uses a variety of laboratory-based serological assays,
can determine whether an individual has been infected with the disease-
associated microorganism and how many individuals in a population are infected
(seroprevalence of the disease). Seroepidemiology can track the spread of an
infection through a population or to new populations that have previously been
disease free. Seroepidemiology can help to determine the routes of disease
spread and identify mechanisms of disease transmission (vectors, high risk
environmental conditions, etc.).
Seroepidemiology can also be used to provide evidence which links infection with
an infectious agent and the development of the full blown clinical disease. It is
especially useful in cases where the infectious agent cannot be isolated and grown
in culture for use in transmission studies as is currently the case for FP. The
seroepidemiological approach is the cornerstone of Hill's Criteria for Disease
Causation. This seroepidemiological approach has been used successfully in
human diseases in which transmission studies are not feasible, such as Kaposi's
sarcoma, a cancer syndrome involving infection with a newly identified
herpesvirus.
The critical factor in using seroepidemiology as a tool to monitor and study
infectious disease in populations is having reliable, sensitive, and specific
serological assays that can detect antibodies against the infectious agent. Such
assays require two main components; antigens from the microorganism and
specific secondary antibodies which can detect the primary anti-infectious agent
antibodies in plasma samples from animals. Previously we have developed
unique monoclonal antibody reagents against marine turtle IgY, IgM, and 5.7s
immunoglobulin subclasses as secondary antibodies. These can detect turtle
antibody responses to infectious agents. The research described here will lead to
the development of pathogen-specific recombinant proteins and antigenic peptides
(antigens) of both the LETV and FPHV for use in seroepidemiological studies of
these two diseases of marine turtles.
7PRESENTATIONS AND PUBLICATIONS FROM THIS PROJECT
Presentations.
Curry, S. S., D. R. Brown, E. R. Jacobson, and P. A. Klein. Persistent infectivity of
Chelonian herpes viruses after exposure to artificial seawater. In Proceedings of the
Nineteenth Annual Symposium on Sea Turtle Biology and Conservation, March 2-5,
1999, South Padre Island, Texas. U.S. Dep.Commer. NOAA Tech. Memo. NMFS-
SEFSC., In Press.
Origgi, F.e., Jacobson, E., Herbst, L.H., Klein, P.A., and Curry, S.S.. Development of
Serological Assays for herpesvirus Infections in Chelonians. 20th Annual Symposium
on Sea Turtle Biology and Conservation, Orlando, FI, February 29th-March 4th, 2000,
In pres~.
Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama, S., Shaf, S.A., Moretti, RH.,
Jacobson, E.R., and P.A. Klein. Serological Detection of Herpesvirus Infections in Green
Turtles. Abstract 267.7, The FASEBJournal, Part I, Abstracts 2.1-537.42, Experimental
Biology 2001, March 31-April 4th, 2001.
Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama, S., Shaf, S.A., Moretti, RH.,
Jacobson, E.R., and P.A. Klein. Serological Detection of Herpesvirus Infections in Green
Turtles. 21st Annual Symposium on Sea Turtle Biology and Conservation, Philadelphia, PA
February 23rd-February 28th,2001, In press.
Coberley, S., Herbst, L.H., Ehrhart, L., Bagley, D., Hirama, S., Schaf, S., Moretti, R.,Jacobson,
E, Condit, R. and P. Klein. Detection of Antibodies to a Disease-associated Herpesvirus of
the Green Turtle, Chelonia mydas. American Society for Virology. Madison, Wisconsin. July
21-25th, 2001.
Publications.
1.Curry, S. S., D. R Brown, E R Jacobson, and P. A. Klein., Ehrhart, L.M.,
Gaskin,J.M., Blahak, S., Herbst, L.H. and P. A. Klein. , Persistent Infectivity of a
Chelonian Herpesvirus after Exposure to Seawater. J. Wildlife Diseases 36,792-797,
2000.
2. Coberley, S.S., Herbst, L.H, Brown, D.R., Ehrhart, L.M., Bagley, D.A., Schaf, S.A.,
Moretti, R.H., Jacobson, ER., and P.A. Klein. Detection of Antibodies to a Disease-
associated Herpesvirus of the Green Turtle, Chelonia mydas. J.Clinical MicrobioL, 39,
3572-3577, 2001.
3. Coberley, S.S., Herbst, L.H, Brown, D.R., Ehrhart, L.M., Bagley, D.A, Hirama, S.,
Jacobson, E.R., and P.A. Klein. Survey of Florida Green Turtles for Exposure to a
Disease-associated Herpesvirus. Diseases of Aquatic Organisms, 2001. In press.
Figure 1. Two Dimensional Gel Electrophoresis and Western Blot of LETV
Proteins
A. Coomassie Stained 2D Gel B. Western Blot
250
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Further strategies for evaluating the etiological role of a

tumor-associated herpesvirus in marine turtle fibropapillomatosis

http://aquaticcommons.org/1088/1/OCRKlein%2C_P._2001..pdf

Further strategies for evaluating the etiological role of a tumor-associated herpesvirus in marine turtle fibropapillomatosis

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