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Journal of Virological Methods 136 (2006) 261–266

Short communication

Identification of novel alpha- and gammaherpesviruses from cutaneous


and mucosal lesions of dolphins and whales
Kara A. Smolarek Benson a , Charles A. Manire b , Ruth Y. Ewing c , Jeremiah T. Saliki d ,
Forrest I. Townsend e , Bernhard Ehlers f , Carlos H. Romero a,∗
a Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA
b Dolphin and Whale Hospital, Mote Marine Laboratory and Aquarium, Sarasota, FL 34236, USA
c National Marine Fisheries Service, NOAA, Miami, FL 33149, USA
d Athens Veterinary Diagnostic Laboratory, University of Georgia, Athens, GA 30602, USA
e Bayside Hospital for Animals, Fort Walton Beach, FL 32547, USA
f Robert Koch-Institut, Nordufer 20, D-13353, Berlin, Germany

Received 12 December 2005; received in revised form 7 March 2006; accepted 13 March 2006
Available online 19 June 2006

Abstract
Total DNA extracted from mucosal and skin lesions of captive and stranded cetaceans was analyzed for herpesvirus DNA by nested and direct
polymerase chain reactions (PCR). The targeted sequences corresponded to a region of the DNA polymerase gene containing multiple conserved
amino acid motifs. Herpesvirus genomic DNA fragments (222–244 bp) were amplified from 11 lesions by nested PCR and from eight lesions
(∼730 bp) using direct PCR from US cetaceans. Fragments of various sizes were also amplified from skin, spleen and blood of a German dolphin.
Sequencing and BLAST analysis of these DNA fragments indicated that alpha- or gammaherpesviruses were present in the cetacean lesions.
Alphaherpesviruses were associated with skin lesions of three Atlantic bottlenose dolphins (Tursiops truncatus), while gammaherpesviruses were
present in genital lesions of five Atlantic bottlenose dolphins, one Risso’s dolphin (Grampus griseus), one dwarf sperm whale (Kogia sima) and
one Blainville’s beaked whale (Mesoplodon densirostris), as well as in one oral lesion from an Atlantic bottlenose dolphin. Phylogenetic analysis
of deduced amino acid sequences showed that the cetacean alphaherpesviruses were most closely related to human alphaherpesviruses, namely,
herpes simplex-1 and -2. On the other hand, cetacean gammaherpesviruses were most closely related to Rhadinoviruses. These novel cetacean
herpesviruses appeared to be distinct from known herpesviruses of marine and terrestrial vertebrates. The sequencing data strongly suggest that
these viruses are most likely cetacean specific and possibly have coevolved with their cetacean hosts.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Herpesvirus; Cetaceae; Alphaherpesvirinae; Gammaherpesvirinae; Molecular identification

Herpesvirus infections have been known to occur for at members of the Alphaherpesvirinae in terrestrial mammals.
least three decades in three families of cetaceans, namely Pho- Herpesvirus-like particles have been visualized by transmis-
coenidae, Monodontidae and Delphinidae (Van Bressem et al., sion electron microscopy (TEM) in epithelial cells in sections of
1999) and have been associated with localized infections of the necrotizing dermatitis from two beluga whales (Delphinapterus
skin and mucosae as well as systemic infections. Skin lesions, leucas); one captured off the coast of Manitoba, Canada (Barr
most likely of herpesvirus etiology, have been reported in a et al., 1989); the other stranded in the St. Lawrence Estuary,
killer whale (Orcinus orca) (Greenwood et al., 1974), a striped Québec, Canada (Martineau et al., 1988). Skin lesions asso-
dolphin (Stenella coeruleoalba) (Baker, 1992) and three harbor ciated with herpesvirus-like particles have also been reported
porpoises (Phocoena phocoena) (Baker and Martin, 1992). Clin- in dusky dolphins (Lagenorhynchus obscurus) from Peruvian
ical disease was described as resembling that induced by some coastal waters (Van Bressem et al., 1994) and in association
with a plaque-like lesion on the penile mucosa of a harbor
porpoise (Lipscomb et al., 1996). A herpesvirus has also been
∗ Corresponding author. Tel.: +1 352 392 4700x5871; fax: +1 352 392 1910. responsible for more serious disease in the form of encephalitis
E-mail address: romeroc@mail.vetmed.ufl.edu (C.H. Romero). diagnosed in a harbor porpoise that stranded off the coast of Swe-

0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jviromet.2006.03.033
262 K.A. Smolarek Benson et al. / Journal of Virological Methods 136 (2006) 261–266

den (Kennedy et al., 1992). In that case, herpesvirus infection 500 ng of sample DNA were used as amplification template. The
was diagnosed using TEM that allowed for the visualization of first PCR mixture contained 400 nM of each primer, 100 mM of
herpesvirus-like particles in the nuclei of neurons. This finding each dNTP, 10 mM KCl, 10 mM (NH4 )2 SO4 , 20 mM Tris–HCl,
was complemented by demonstrating Cowdry type A intranu- 2 mM MgSO4 , 0.1% Triton X-100 at pH 8.8 and 2 units of
clear inclusions in the neurons and herpesvirus antigen in the Taq DNA polymerase (New England BioLabs, Beverly, MA).
cytoplasmic-nuclear junction of neurons in the cerebral cor- All polymerase chain reactions were run in a PTC-100 Pro-
tex. Molecular evidence of alphaherpesviruses associated with grammable Thermal Cycler (MJ Research, Inc., Waltham, MA).
fatal systemic infections of cetaceans was obtained for the first Cycling conditions for the first and second PCR were: Initial
time in the case of two Atlantic bottlenose dolphins (Tursiops incubation at 94 ◦ C for 2 min followed by 55 cycles at 94 ◦ C for
truncatus) that stranded on two separate occasions along the 20 s, 46 ◦ C for 30 s, and 72 ◦ C for 30 s. A final extension step
Atlantic coast of the US (Blanchard et al., 2001). Molecular at 72 ◦ C for 10 min finished the cycling. In the second PCR,
assays in the form of polymerase chain reactions (PCR) that mixtures were identical to those in the first PCR but contained
targeted the herpesvirus DNA polymerase and DNA terminase 400 nM of each of the second reaction primers and 2 ␮l of the
genes confirmed that the fatal infections were associated with first PCR product as DNA template. Approximately, 20 ␮l from
two different alphaherpesviruses. To date, herpesviruses have the second PCR were resolved by horizontal gel electrophore-
not been isolated from any species of cetaceans and little is sis in 1.0% agarose containing ethidium bromide (0.5 ␮g/ml)
known about the existence of herpesviruses other than alpha- and the DNA fragments were visualized by UV light transillu-
herpesviruses. In the present study, novel gammaherpesviruses mination and photographed using a gel documentation system.
were identified in dolphins and whales associated with lesions Amplified DNA fragments of the predicted size were purified
in genital and oral mucosae. A novel alphaherpesvirus was also using the MinElute PCR Purification Kit (Qiagen Inc., Valencia,
detected in skin lesions of a dolphin and confirmed the associ- CA).
ation of previously described alphaherpesviruses with localized To obtain additional nucleotide sequences, samples that were
and generalized skin lesions of dolphins. positive for herpesvirus DNA by the nested PCR were retested
Lesion scrapings or biopsies were obtained from stranded to try to amplify a longer fragment (∼700 nucleotides) of the
cetaceans from Florida, Georgia and North and South Carolina. DNA polymerase gene using primers FP1 and RP1 and 3.3 units
Similar clinical samples were also collected from live cetaceans of Expand High Fidelity enzyme mix (Roche Applied Science,
at wildlife parks and rehabilitation centers at various locations Indianapolis, IN). Cycling conditions were: initial denaturing
in California, Florida, Mississippi and Texas. In addition, skin, at 94 ◦ C for 2 min followed by 40 cycles of 94 ◦ C for 30 s to
blood and organ samples were collected from an Atlantic bot- denature the DNA template, annealing at 42 ◦ C for 30 s and
tlenose dolphin that died in 2001 in the Zoological Gardens extension at 72 ◦ C for 2 min. A final extension step at 72 ◦ C for
of Duisburg, Germany. In total, 128 lesion samples obtained 10 min finished the cycling. The annealing temperature varied
between 2001 and 2005 and encompassing 12 cetacean species between 42◦ and 47 ◦ C depending on the species of the DNA
were analyzed for herpesvirus sequences; 88 from skin lesions, template being assayed. Amplified DNA fragments of the right
32 from mucosal lesions (oral and genital) and eight from blood size were purified after PCR using the MinElute PCR Purifica-
and different organs. Total DNA was extracted from all samples tion Kit or excised after electrophoresis in 1.2% low-melting-
using the DNeasy Tissue Kit (Qiagen Inc., Valencia, CA) and point agarose and purified using the MinElute Gel Extraction
the quantity and quality were determined by spectrophotome- Kit (Qiagen Inc.). Purified DNA fragments were cloned into the
try (Ultrospec 3000, Amersham Pharmacia Biotech, Piscataway, plasmid vector pCR2.1-TOPO T/A (Invitrogen, Carlsbad, CA)
NJ). Herpesvirus DNA was detected by PCR using degenerate and both strands sequenced in duplicate using the CEQ 2000
primers (VanDevanter et al., 1996; Ehlers et al., 1999) designed XL (Beckman Coulter Inc., Fullerton, CA) sequencing instru-
to amplify a region of the DNA polymerase gene that contains ment. Exported chromatograms were manually reviewed using
highly conserved amino acid motifs within the Herpesviridae. the Chromas 2.3 software (Technelysium Pty Ltd., Tewantin,
These primers are known to direct the amplification of DNA Queensland, Australia) and the sequences were analyzed using
polymerase gene fragments 215–235 base pairs (bp) in length for the programs Seqed, Gap, Translate, Lineup, Pileup and Pretty
most herpesviruses and 315 bp for cytomegaloviruses. A nested of the University of Wisconsin Package Version 10.2, Genetics
PCR assay was performed with two forward and one reverse Computer Group (GCG), Madison, WI.
primers in the first reaction and one forward and one reverse All German dolphin samples were amplified with degenerate
primer in the second reaction. Primer sequences for the first primers in a nested PCR approach as described by Chmielewicz
reaction were: FP1-5 - GAY TTY GCI AGY YTI TAY CC - et al. (2001). The sequence of the initial 235-bp PCR product
3 , FP2-5 - TCC TGG ACA AGC AGC ARI YSG CIM TIA was extended into the 5 - direction with the help of semi-nested
A -3 , RP1-5 - GTC TTG CTC ACC AGI TCI ACI CCY TT PCR using the degenerate FP1-primer and two sequence spe-
-3 . Primers for the second reaction were: FP3-5 - TGT AAC cific backward primers for generation of a 490 bp fragment as
TCG GTG TAY GGI TTY ACI GGI GT -3 , RP2-5 - CAC performed by Ehlers et al. (2003).
AGA GTC CGT RTC ICC RTA IAT -3 . Total DNA extracted The consensus primer nested PCR approach was successful
from cell monolayers of Madin Darby canine kidney (MDCK) in the amplification of herpesvirus DNA polymerase gene frag-
cultures infected with canine herpesvirus-1 (CHV-1) was used ments from total DNA extracted from nine mucosal (Fig. 1a.
as a positive control for PCR. In the first PCR, approximately Lanes 1–7; PCR fragments for Gg V2129 and ABND V1938
K.A. Smolarek Benson et al. / Journal of Virological Methods 136 (2006) 261–266 263

ments from eight lesions that upon sequencing were shown to


be 725–731 bp in length (Fig. 1b. Lanes 1–6; PCR fragments for
Gg V2129 and ABND V1938 not shown) and translated into
proteins of 241–243 amino acids. From the skin sample of the
German dolphin, a total of 490 bp was amplified (not shown),
encoding a protein of 163 amino acids.
After initial Blast analysis, phylogenetic trees were con-
structed with the Neighbor-Joining method (MacVectorTM ,
version 8.0) and the Maximum-Likelihood method (TREE-
PUZZLE, version 5.0) using multiple alignments of amino acid
sequences deduced from homologous gene fragments of mem-
Fig. 1. Agarose gel electrophoresis of PCR amplified fragments of the her-
bers of the Herpesviridae. The analyses revealed that the cuta-
pesvirus DNA polymerase gene. Panel A. Small fragments (222–244 bp) neous lesions from three Atlantic bottlenose dolphins (K167,
from nested PCR. Lane 1, from penile lesion of Atlantic bottlenose dolphin K231, GER1) were associated with alphaherpesviruses. In addi-
ABND K311 (γ herpesvirus); Lane 2, from vaginal lesion of ABND K263 (γ); tion, sequences derived from the spleen (GER2) and blood
Lane 3, from penile lesion of ABND K310 (γ); Lane 4, from tongue lesion of (GER3) from the German dolphin were also associated with
ABND K308 (γ); Lane 5, from penile lesion of ABND K264; Lane 6, from
vaginal lesion of dwarf sperm whale DSW K265 (γ); Lane 7, from penile
those of alphaherpesviruses. In contrast, eight genital lesions
lesion of Blainville’s beaked whale BBW K285 (γ); Lane 8, from skin lesion of (K263, K264, K265, K285, K310, K311, V1938, V2129) and
ABND K167 (α); Lane 9, from skin lesion of ABND K231 (α); Lane 10, from one oral lesion (K308) contained gammaherpesvirus sequences
green turtle fibropapilloma (α); Lane 11, from Kemp’s ridley turtle fibropapil- (Fig. 2, Fig. 3a and b). Oral lesion K308 and penile lesion K310
loma (α); Lane 12, negative PCR control; Lane 13, from positive PCR control were derived from the same animal and identical short (222 bp)
canine herpes virus (α). Panel B. Larger fragments (731 bp) amplified by direct
PCR. Lane 1, from vaginal lesion of ABND K263 (γ); Lane 2, from penile
gammaherpesvirus sequences were obtained from both lesions.
lesion of ABND K311 (γ); Lane 3, from penile lesion of ABND K310 (γ); However, additional DNA was not available from oral lesion
Lane 4, from penile lesion of ABND K264 (γ); Lane 5, from penile lesion of K308 to amplify the 731 bp fragment and this sequence was
BBW K285 (γ); Lane 6, from vaginal lesion of DSW K265 (γ); Lane 7, neg- excluded from further phylogenetic analysis. Alphaherpesvirus
ative PCR control; Lane 8, from positive PCR control canine herpes virus (α). DNA from lesions K167 and K231 could not be amplified using
bp: molecular size markers in base pairs.
primers FP1 and RP1 and the sequences previously obtained
from the 243 bp fragments were used for comparisons. The
not shown) and two cutaneous lesions (Fig. 1a. Lanes 8–9) from alphaherpesviruses amplified from GER2 and GER3 (spleen and
US cetaceans. In addition, skin, blood and spleen from the Ger- blood, respectively) were identical in amino acid sequence to
man dolphin were positive (not shown). Sequencing of the DNA ABND K167 and ABND SC95, and therefore not subjected to
fragments showed that they ranged in size from 222 to 244 additional PCR experiments.
nucleotides in length which translated into proteins of 54–56 All sequences generated in this study have been
amino acids (the primer binding sites were excluded). Sequence deposited in the GenBank database under accession num-
variability was observed in both the nucleotide and amino acid bers: AY949828 (Blainville’s beaked whale [Mesoplodon
sequences, and multiple sequence alignments with the Clustal W densirostris] BBW K285 penile lesion), AY949830 (dwarf
module of MacVectorTM indicated that these sequences clearly sperm whale [Kogia sima] DSW K265 vaginal slit lesion),
fell into two groups (Fig. 2). Amplification with primers FP1 and AY949831 (Atlantic bottlenose dolphin-ABND K311 penile
RP1 and Expand High Fidelity enzyme generated DNA frag- lesion), AY949832 (ABND K231 skin lesion), AY952776

Fig. 2. Multiple alignment of cetacean herpesvirus DNA polymerase sequences. The deduced amino acid sequences of DNA polymerase fragments of all known and
sequenced cetacean herpesviruses, described in this paper or obtained from the GenBank repository, were aligned with Clustal W (LasergeneTM , MegAlign module).
ABND: Atlantic bottlenose dolphin; BBW: Blainville’s beaked whale; DSW: dwarf sperm whale; Gg: Risso’s dolphin; =: amino acid identical to ABND K263; -:
gap.
264 K.A. Smolarek Benson et al. / Journal of Virological Methods 136 (2006) 261–266
K.A. Smolarek Benson et al. / Journal of Virological Methods 136 (2006) 261–266 265

(ABND K264 penile lesion), AY952777 (ABND K263 vaginal ilar alphaherpesviruses may be responsible for both cutaneous
lesion), AY952778 (ABND K310 penile lesion), AY952779 and fatal systemic infections in Atlantic bottlenose dolphins. It
(ABND K308 tongue lesion), AY757301 (ABND K167 is clear that more sequence data, including those from different
skin lesion), DQ288666 (Risso’s dolphin [Grampus griseus] genes are needed to further characterize these cetacean alpha-
Gg V2129 vaginal lesion), DQ288667 (ABND V1938 penile herpesviruses.
lesion), AY608707 (ABND GER1 skin lesion), DQ295063 Gammaherpesviruses had not previously been reported in
(ABND GER2 spleen), and DQ295064 (ABND GER3 blood). cetaceans and in this study were found associated with localized
The alphaherpesvirus sequences K167 and K231 only shared lesions in oral and genital mucosae. The nucleotide and amino
nucleotide and amino acid identities of 78.8% and 77.2%, acid sequences of gammaherpesviruses from Atlantic bottlenose
respectively, and therefore most likely indicate the existence dolphins (K263, K264, K310, K311, V1938) ranged in identities
of different species of alphaherpesviruses in Atlantic bottlenose from 91.6% to 98.2% and 93.8% to 98.8%, respectively, indi-
dolphins. This concept is further supported by the finding that the cating higher conservation than those of homologous sequences
nucleotide and amino acid sequences derived from the alphaher- from dolphin alphaherpesviruses. A similar comparison of the
pesvirus from skin lesion K167 (AY757301) were only 73.0% nucleotide and amino acid sequences from the Risso’s dolphin
and 71.4% identical to those from a lung lesion (ABND SC95; to those from Atlantic bottlenose dolphins showed, respectively,
AF196646) and 55.6% and 65.0% identical to those from a heart identities of 87.9–89.8% and 91.3–94.2%. The DNA polymerase
lesion (ABND DE99; AF245443), as reported by Blanchard et gene fragments from the Blainville’s beaked whale (K285) and
al. (2001). the dwarf sperm whale (K265) had nucleotide and amino acid
Clinically, the two alphaherpesviruses K167 and K231 that identities of 76.3% and 86.4%, indicating higher genetic vari-
were identified in skin lesions of Atlantic bottlenose dolphins ability of gammaherpesviruses in different species of whales.
from the US produced only transient cutaneous infections that The multiple sequence alignment and phylogenetic analysis also
eventually resolved. Evidence of fatal systemic alphaherpesvirus showed that the cetacean herpesviruses so far identified are
infection as previously described by Blanchard et al. (2001), genetically distinct from herpesviruses of pinnipeds, sea turtles
with acute necrotizing lesions in multiple organ systems was and fish. Alpha- and gammaherpesviruses from North American
not found in these dolphins, even though one of the alpha- and European pinnipeds (Harder et al., 1996; King et al., 1998)
herpesviruses found in lesion K231 had nucleotide and amino have been previously reported and characterized. More recently,
acid identities of 98.9% and 96.9%, respectively, to homologous a novel pinniped gammaherpesvirus, Otarine herpesvirus-1, was
sequences from an alphaherpesvirus (ABND SC95; AF196646) found associated with urogenital carcinoma in California sea
identified in lung tissue of a bottlenose dolphin that stranded in lions, Zalophus californianus (King et al., 2002). In sea turtles,
South Carolina in 1995 (Blanchard et al., 2001). Phylogenet- alphaherpesviruses have been implicated as causative agents
ically, the alphaherpesviruses detected in this study and those of fibropapillomatosis, a debilitating and often fatal disease of
described by Blanchard et al. (2001), although heterogeneous, green (Chelonia mydas), loggerhead (Caretta caretta), olive rid-
formed a single clade and three subclades (Fig. 3b) revealing ley (Lepidochelys olivacea) and Kemps’s ridley (Lepidochelys
pairwise amino acid identities from 57% to 100%. The mem- kempi) turtles (Quackenbush et al., 1998; Lackovich et al., 1999;
bers of this group of cetacean alphaherpesviruses were most Lu et al., 2000; Herbst et al., 2004). Multiple sequence com-
closely related to the Simplexviruses of humans (HSV-1, HSV- parisons and phylogenetic analyses of the alpha- and gamma-
2) (Fig. 3a and b) with which they shared amino acid identities cetacean herpesviruses with herpesviruses of pinnipeds, marine
of 52–61%. These findings suggest that identical or very sim- turtles and fish, unambiguously showed that they are genetically

Fig. 3. Phylogenetic analysis of cetacean herpesviruses. A Neighbor-Joining phylogenetic tree was constructed with 56–64 aa sequences, available for all cetacean
herpesviruses. The human representatives of the alpha-, beta- and gammaherpesviruses (HSV-1 and VZV; HCMV; EBV and HHV-8) and the fish herpesvirus CCV
(as outgroup) were included for comparison (A). The cetacean herpesviruses for which 155–163 aa were available were analyzed in comparison with several human
and animal herpesviruses. The fish viruses KoiHV1 and CCV were used as outgroup (B). The branch lengths are proportional to evolutionary distance (scale bar). At
the branch nodes, values before the vertical divider indicate the percentage confidence out of 1000 bootstrap replications. Values behind the vertical divider represent
support values generated by the maximum-likelihood method of the program TREE-PUZZLE (Version 5.0), and were estimated by the quartet puzzling (QP) tree
search. They express the QP reliability in percent. The cetacean HV are marked with rectangular brackets. The three herpesvirus subfamilies and the fish herpesviruses
are indicated. The GenBank accession numbers of the DNA polymerase gene fragments used for the construction of the trees are, Alphaherpesvirinae subfamily:
AY949832-Atlantic bottlenose dolphin or ABND K231, AF245443-ABND SC95, AY757301-ABND K167, AF245443-ABND DE99, AY608707-ABND GER1,
DQ295063-ABND GER2, DQ295064-ABND GER3, NC 001847-Bovine HV-1 (BoHV1), NC 006151-Pseudorabies virus (PRV), AJ224971-Feline HV-1 (FHV1),
U92269-Phocid HV-1 (PhocHV1), AY949827-Canine HV-1 (CanHV1), AY464052-Equine HV-1 (EHV1), AB070848-Herpes simplex virus-1 (HSV1), AY038367-
Herpes simplex virus -2 (HSV2), AF147806-Marek’s disease virus (MDV), NC 001348-Varicella-zoster virus (VZV), AB047545-Tortoise HV-1 (TortHV1),
AY646888-Loggerhead turtle HV (LogghTHV), AY646894-Kemp’s ridley turtle HV (KempsrHV), AF239684-Hawaiian green turtle HV (GreenTHV). Gammaher-
pesvirinae subfamily: AF236050-Otarine HV-1 (OtariHV1), AJ507799-Epstein-Barr virus (EBV), NC 003409-Human herpesvirus-8 (HHV8), AY949830-Dwarf
sperm whale or DSW K265, AY949828- Blainville’s beaked whale or BBW K285, AY952776-ABND K264, AY952779-ABND K310, AY952777-ABND K263,
AY949831-ABND K311, DQ288666-Risso’s dolphin or Gg V2129, DQ288667-ABND V1938, AF083424-Ateline HV-3 (HVA), NC 001350-Herpesvirus saimiri-2
(HVS), AF029302-Rhesus monkey rhadinovirus (RRV), NC 002665-Bovine HV-4 (BoHV4), AF376034-Mustelid HV (MustHV), NC 001650-Equine HV-2 (EHV2),
AF327830-Bovine lymphotropic HV (BLHV), AF118399-Porcine lymphotropic HV-1 (PLHV1), AAC58060-Alcelaphine HV-1 (AlHV1). Betaherpesvirinae sub-
family: AF268040-Porcine cytomegalovirus (PCMV), NC 001664-Human cytomegalovirus (HCMV), NC 004065-Murine cytomegalovirus (MCMV). Unclassified
viruses: AY572853-Koi HV-1 (KoiHV1), NC 001493-Channel catfish HV (CCV).
266 K.A. Smolarek Benson et al. / Journal of Virological Methods 136 (2006) 261–266

distinct and only distantly related. The phylogenetic analysis Ehlers, B., Ochs, A., Leendertz, F., Goltz, M., Boesch, C., Matz-Rensing,
also indicated that both the alpha- and gamma-cetacean her- K., 2003. Novel simian homologues of Epstein–Barr virus. J. Virol. 77,
pesviruses are most closely related to members of the genus 10695–10699.
Greenwood, A.G., Harrison, R.J., Whitting, H.W., 1974. Functional and
Simplexvirus and Rhadinovirus, respectively. Although more pathological aspects of the skin of marine mammals. In: Harrison, R.J.
sequence data, preferably including sequences from different (Ed.), Functional Anatomy of Marine Mammals. Academic Press, Inc.,
genes, are needed from these cetacean herpesviruses to confirm London, pp. 73–111.
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polymerase gene fragments (VanDevanter et al., 1996; Ehlers et Herbst, L., Ene, A., Su, M., Desalle, R., Lenz, J., 2004. Tumor outbreaks
al., 1999; Blanchard et al., 2001). Our results indicate that the in marine turtles are not due to recent herpesvirus mutations. Curr. Biol.
cetacean herpesviruses are most likely species specific and have 14, R697–R699.
probably coevolved with their hosts. Kennedy, S., Lindstedt, I.J., McAliskey, M.M., McConnell, S.A., McCul-
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Acknowledgments King, D.P., Hure, M.C., Goldstein, T., Aldridge, B.M., Gulland, F.M.D.,
Saliki, J.T., Buckles, E.L., Lowenstine, L.J., Stott, J.L., 2002. Otarine
We acknowledge the contributing members of the United herpesvirus-1: a novel gammaherpesvirus associated with urogenital car-
States Southeast Regional Marine Mammal Stranding Networks cinoma in California sea lions (Zalophus californianus). Vet. Microbiol.
86, 131–137.
for their efforts in the collection of specimens provided to this
King, D.P., Parselles, R., Gulland, F.M., Lapointe, J.M., Lowenstine, L.J.,
study. We kindly thank Manuel Garcia-Hartmann, Zoological Ferrick, D.A., Stott, J.L., 1998. Antigenic and nucleotide characteriza-
Gardens of Duisburg, Germany, for providing samples from the tion of a herpesvirus isolated from Pacific harbor seals (Phoca vitulina
German dolphin. This research was supported by funding pro- richardsii). Arch. Virol. 143, 2021–2027.
vided by the Florida Fish and Wildlife Commission through Lackovich, J.K., Brown, D.R., Homer, B.L., Garber, R.L., Mader, D.R.,
Moretti, R.H., Patterson, A.D., Herbst, L.H., Oros, J., Jacobson, E.R.,
the Training Program in the Care of Marine Mammals, Col-
Curry, S.S., Klein, P.A., 1999. Association of herpesvirus with fibropa-
lege of Veterinary Medicine, University of Florida, and the Save pillomatosis of the green turtle Chelonia mydas and the loggerhead turtle
the Wild Dolphin Grant Program from Harbor Branch Oceano- Caretta caretta in Florida. Dis. Aquat. Org. 37, 89–97.
graphic Institution, Florida. The Institutional Animal Care and Lipscomb, T.P., Habecker, P.L., Damback, D.M., Schoelkopf, R., 1996. Gen-
Use Committee of the University of Florida approved the use ital herpesvirus in a male harbor porpoise (Phocoena phocoena). Proc.
Intl. Assoc. Aquat. Anim. Med., 17.
of animal tissues in this study. Opinions or assertions presented
Lu, Y., Wang, Y., Yu, Q., Aguirre, A.A., Balazs, G.H., Nerurkar, V.R., Yanag-
are the private views of the authors and are not to be construed ihara, R., 2000. Detection of herpesviral sequences in tissues of green
as the official position of the Department of Commerce. turtles with fibropapilloma by polymerase chain reaction. Arch. Virol.
145, 1885–1893.
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