www.elsevier.com/locate/vetmic

Veterinary Microbiology 105 (2005) 83–92

Varanid herpesvirus 1: a novel herpesvirus associated with

proliferative stomatitis in green tree monitors (Varanus prasinus)

James F.X. Wellehana,*, April J. Johnsona, Kenneth S. Latimerb,Douglas P. Whitesidec,1, Graham J. Crawshawc, Carol J. Detrisacd,2,

Scott P. Terrelld, Darryl J. Hearda, April Childressa, Elliott R. Jacobsona

aDepartment of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USAbDepartment of Veterinary Pathology, University of Georgia, Athens, GA 30602, USA

cToronto Zoo, 361A Old Finch Avenue, Scarborough, Ont. M1B 5K7, USAdDepartment of Veterinary Pathobiology, College of Veterinary Medicine, University of Florida,

Gainesville, FL 32610, USA

Received 7 July 2004; received in revised form 11 October 2004; accepted 28 October 2004

Abstract

Stomatitis is a common problem in lizards, and the etiologies of stomatitis in lizards are not well understood. Four green tree

monitor lizards (Varanus prasinus) from two different collections were evaluated because of proliferative stomatitis. Degenerate

PCR primers targeting a conserved region of herpesvirus DNA-dependent DNA polymerase were used to amplify and sequence

a product from gingival tissue of three of four lizards (cases 1, 3, and 4). DNA in situ hybridization of tissues from three lizards

was positive for herpesvirus in the oral mucosa of all three lizards tested (cases 1–3) and the brain of two lizards (cases 1 and 3).

Comparative sequence analysis suggests that this virus is a novel member of the subfamily a-herpesvirinae, and is here termed

varanid herpesvirus 1.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Green tree monitor; Varanus prasinus; Reptiles; Herpesvirus; Proliferative stomatitis; Nucleic acid sequencing

* Corresponding author. Tel.: +1 352 392 4700;

fax: +1 352 392 4877.

E-mail address: wellehanj@mail.vetmed.ufl.edu

(James F.X. Wellehan).1 Present address: Calgary Zoo, 1300 Zoo Road NE, Calgary,

Alberta, T2E 7V6, USA.2 Present address: Pathology Associates, 10 W 35th Street,

Chicago, IL 60616, USA.

0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2004.10.012

1. Introduction

Stomatitis is a common problem in captive reptiles.

The etiologies of stomatitis in reptiles are often

multifactorial. Stomatitis in reptiles is frequently

associated with stress (Mader, 1996). Bacterial agents

associated with stomatitis in reptiles have included

Pseudomonas spp., Aeromonas spp., Klebsiella spp.,

Salmonella spp., and Mycobacterium spp. (Mader,

.

J.F.X. Wellehan et al. / Veterinary Microbiology 105 (2005) 83–9284

1996). Herpesviruses, iridoviruses, picornaviruses,

and reoviruses have been associated with stomatitis

in tortoises (Jacobson et al., 1985; Marschang et al.,

1999; Marschang and Chitty, 2004; Marschang and

Ruemenapf, 2002), and herpesviruses have been

associated with stomatitis in plated lizards (Gerrho-

saurus spp.) (Wellehan et al., 2004). An oral

fibrosarcoma has been previously reported in a green

tree monitor (Done, 1996), but viral diagnostics were

not reported.

The herpesviruses of lizards include lacertid

herpesvirus, iguanid herpesviruses 1 and 2, agamid

herpesvirus, and gerrhosaurid herpesviruses 1–3.

Lacertid herpesvirus has been identified with electron

microscopy associated with cutaneous papillomatous

lesions in green lizards (Lacerta viridis) (Raynaud and

Adrian, 1976). Iguanid herpesvirus 1 was isolated

from green iguana (Iguana iguana) heart cell cultures

(Clark and Karzon, 1972). Cytopathic effects were

seen in cell culture. Inoculation of 12 young iguanas

produced no consistent pattern of lesions, and

although there was a much higher mortality rate in

the inoculated population, a causal relationship was

not established. Iguanid herpesvirus 2 was found in a

San Esteban chuckwalla (Sauromalus varius) that died

with acute hepatic necrosis. Electron microscopy of

eosinophilic intranuclear inclusions revealed herpes-

virus particles and PCR and sequencing confirmed the

presence of a novel herpesvirus (Wellehan et al.,

2003). Agamid herpesvirus was seen by electron

microscopy in liver, lung, and spleen of two red-

headed agamas (Agama agama) that died (Watson,

1993). Gerrhosaurid herpesviruses were seen asso-

ciated with stomatitis lesions in two species of plated

lizards (Gerrhosaurus spp.) and were identified by

PCR and sequencing (Wellehan et al., 2004).

Phylogenetic relationships of herpesviruses are

now formally based on genetic content, as defined by

homology of nucleic acid sequences and identification

of particular genes unique to a virus subset (Minson

et al., 2000). Consensus PCR represents a rapid way to

obtain DNA template from clinical samples of novel

viruses suitable for sequencing (VanDevanter et al.,

1996). To date, all reptile herpesvirus sequences

available in the GenBank (National Center for

Biotechnology Information, Bethesda, MD), EMBL

(Cambridge, UK), and Data Bank of Japan (Mishima,

Shiuoka, Japan) databases appear to belong to the

subfamily a-herpesvirinae (Quackenbush et al., 1998;

Une et al., 2000; Wellehan et al., 2003, 2004).

This study describes proliferative stomatitis seen in

green tree monitors (Varanus prasinus) that was

associated with the presence of a unique herpesvirus

sequence. This is the first report of herpesvirus-

associated disease in varanid lizards.

2. Materials and methods

2.1. Animals

An adult, intact female 250 g (0.55 lb) green tree

monitor (V. prasinus) (case 1) was evaluated at the

University of Florida Veterinary Medical Teaching

Hospital (UFVMTH) because of a proliferative

stomatitis involving the lower right dental arcade.

The animal was kept in an indoor enclosure

maintained at 27–31 8C (81–88 8F) with a basking

area at 43 8C (109 8F). Radiographic evaluation of the

skull showed right mandibular soft tissue swelling

with multifocal osteolysis within the ramus. Bacterial

culture of the lesion produced heavy growth of a

mixed gram-positive and gram-negative aerobic and

anaerobic flora. The animal was treated with

ceftazidime (20 mg/kg, IM, q72 h, Fortaz, GlaxoS-

mithKline, Research Triangle Park, NC) and azithro-

mycin (10 mg/kg, PO, q48h, Zithromax, Pfizer Inc.,

New York, NY).

At recheck one week later, the soft tissue swelling

was significantly decreased and the lesion appeared to

be healing well. The animal was reported to be eating

normally and to have a normal activity level.

Twenty days after initial presentation, the monitor

was reevaluated because of marked depression.

Physical examination revealed pupil dilatation, a

delayed righting response and marked gingival

proliferation involving the entire mandibular dental

arcade (Fig. 1). A gingival biopsy was taken and blood

was collected from the ventral coccygeal vein for a

CBC, plasma biochemical panel, and blood culture.

Normal CBC and plasma biochemical values have not

been published for green tree monitors, and values were

interpreted based on reference intervals for other lizard

species (Wright and Skeba, 1992; Harr et al., 2001).

Clinically significant findings included a leukocytosis

of 24.5 � 109/L, heterophilia (18.1 � 109/L), lympho-

J.F.X. Wellehan et al. / Veterinary Microbiology 105 (2005) 83–92 85

Fig. 1. Proliferative stomatitis in case 1, a green tree monitor (V. prasinus).

penia (1.2 � 109/L), monocytosis (5.1 � 109/L),

elevated aspartate transferase activity (702 IU/L),

hypocalcemia (1.55 mmol/L (6.2 mg/dL)), hyperpho-

sphatemia (5.14 mmol/L (15.9 mg/dL)), hyperglyce-

mia (35.4 mmol/L (638 mg/dL)), hyperuricemia (3575

mmol/L (60.1 mg/dL)), hyperkalemia (7.1 mmol/L),

hypochloremia (79 mmol/L), and a metabolic acidemia

(total carbon dioxide 10 mmol/L). A diagnosis of renal

failure was made based on the biochemical findings and

an intraosseous catheter was placed in the left femur for

administration of fluids.

Coelomic ultrasonography revealed increased

echogenicity consistent with mineralization of heart

valves, hepatic vessels, and renal parenchyma. An

ultrasound-guided fine-needle aspirate of the kidney

revealed peripheral blood and renal epithelial cells

with mineralized material in a few cell clusters. The

lizard died 12 h after admission. Tissues from all

major organ systems were collected, fixed in neutral

buffered 10% formalin and processed for light

microscopic evaluation.

Based on the results of case 1, paraffin embedded

formalin fixed tissues from two historical cases of

proliferative stomatitis and squamous cell carcinomas

in green tree monitors from the Toronto Zoo were

evaluated (cases 2 and 3). Both monitors had

originated from the same source in 1988 as adults,

and were housed together in an indoor exhibit with

Murray River turtles (Emydura macquarrii).

Case 2 was noted to have a focus of gingival

necrosis with adjacent mucosal hyperplasia approxi-

mately 2 years after arrival at the zoo. Over the

course of the next 2 years, the lesion was treated

with topical iodine and topical and systemic anti-

biotics, and recurred periodically. Dental radiographs

revealed no bony involvement. A gingival biopsy was

taken.

The monitor was transferred to the Ontario

Veterinary College, University of Guelph for cobalt

radiation therapy. Ten doses were administered over

18 days. The tumor regressed completely over the next

2 months, but the remaining gingiva was still

J.F.X. Wellehan et al. / Veterinary Microbiology 105 (2005) 83–9286

hyperplastic and bled easily. The tumor returned

within 7 months, rapidly increasing in size with

associated loss of all dentition of the left maxilla. A

similar proliferative 6 mm wide mass was also present

on the left side of the mandible, and the remainder of

the gingiva of the maxilla and mandible was

proliferative. Radiography revealed osteolysis of the

left maxilla and mandible associated with the tumors.

Significant alterations in biochemical parameters

included elevated alkaline phosphatase (425 IU/L),

creatinine phosphokinase (907 IU/L) and lactate

dehydrogenase (916 IU/L) activities, and hypopho-

sphatemia (0.72 mmol/L (2.2 mg/dL)).

Despite the tumors, the monitor had maintained its

body weight and condition, and continued to eat well.

The tumors were excised with electrocautery followed

by cryotherapy via application of liquid nitrogen

saturated cotton tipped applicators. When the tumors

had grown back to their original size within 3 months,

with significant accumulations of necrotic debris

despite weekly debridement, euthanasia was elected.

Tissues from all major organ systems were collected,

fixed in neutral buffered 10% formalin and processed

for light microscopic evaluation.

Case 3 was also noted to have a small pocket of

ulcerative gingivitis of the right mandible, and a larger

focus along the buccal aspect of the left maxilla. The

lesions initially responded well to antibiotic therapy,

as with cases 1 and 2. A recurrence 2 years later again

responded to surgical and medical management.

However, within 1.5 years, general gingival hyper-

plasia was noted on the left maxilla and the mandible,

with caseation, ulceration, and necrosis of both sides

of the mandible. Over the next 3 years the lizard was

treated intermittently for ulcerative gingivitis with

local debridement and antibiotics. Gingival biopsies

were taken. Over the next 3 months, there was a

significant improvement in appetite, an increase in

bodyweight and condition, a decrease in mucosal

erythema and ulceration, and stabilization of the

gingival hyperplasia. The monitor was found dead in

the exhibit’s pool 6 months after diagnosis. Tissues

from all major organ systems were collected, fixed in

neutral buffered 10% formalin and processed for light

microscopic evaluation.

Case 4, from the same collection as case 1, was

presented to the UFVMTH for ulcerative right

maxillary gingivitis. A gingival swab was taken for

PCR amplification and sequencing. Tissue was not

taken for in situ DNA hybridization. The lesions

responded well to antibiotic therapy, as with cases 1–

3. Two months after initial presentation, no recurrence

was seen.

2.2. PCR amplification and sequencing

DNA was extracted from tissues using the DNEasy

Kit (Qiagen, Valencia, CA). Nested PCR amplification

of partial sequence of the herpesvirus DNA-dependent

DNA polymerase gene was performed using methods

described previously (VanDevanter et al., 1996). The

PCR products were resolved in 1% agarose gels,

excised, and purified using the QIAquick gel

extraction kit (Qiagen). Products were sequenced

directly using the Big-Dye Terminator Kit (Perkin-

Elmer, Branchburg, NJ) and analyzed on ABI 377

automated DNA sequencers at the University of

Florida’s Sequencing Center.

2.3. Phylogenetic analysis

The sequences were compared to those in GenBank

(National Center for Biotechnology Information,

Bethesda, MD), EMBL (Cambridge, UK), and Data

Bank of Japan (Mishima, Shiuoka, Japan) databases

using TBLASTX (Altschul et al., 1997).

Predicted homologous 55–61 amino acid

sequences of corresponding herpesviral DNA-depen-

dent DNA polymerase available from GenBank were

aligned. Phylogenetic analyses of the predicted

alignment were performed with the PHYLIP (Phylo-

geny Inference Package, Version 3.573c) program

package (Felsenstein, 1989). Protdist (Dayhoff PAM

001 matrix) followed by Fitch (global rearrangements)

were used. Elephant herpesvirus (GenBank accession

no. AAG41999) was used as the outgroup. The

strength of the tree topology obtained was tested by

using bootstrap analysis (Felsenstein, 1985) starting

with Seqboot with 1000 re-samplings, followed by

distance matrix calculations, and consense to calculate

the bootstrap values.

2.4. DNA in situ hybridization

Unstained histologic sections of tissues were

submitted to the DNA in situ Hybridization Labora-

J.F.X. Wellehan et al. / Veterinary Microbiology 105 (2005) 83–92 87

tory at the University of Georgia. DNA probing was

performed using two digoxigenin-labeled oligonu-

cleotide probes designated FN-49 and FN-65, con-

served herpesvirus sequences originally used to detect

Pacheco’s disease virus in psittacine birds (Ramis

et al., 1994). Probe hybridization foci were detected by

high affinity immunohistochemistry using an anti-

digoxigenin antibody conjugated to alkaline phospha-

tase. In foci of DNA hybridization, conjugated

alkaline phosphatase reduced soluble nitrobluetetra-

zolium dye solution (light yellow color) to insoluble

formazan (blue-black pigment). The slides were

counterstained in fast green FCF dye. Samples were

not taken for in situ hybridization from case 4.

3. Results

3.1. Animals

In case 1, histologic evaluation of the oral tissue

revealed marked mucosal epithelial proliferation (up

to 100 cells) (Fig. 2a). The epithelial cells were tightly

packed and had round to oval nuclei and scant

basophilic cytoplasm. The mitotic index averaged 8

mitoses per 40� field of view. The epithelial cell

proliferation extended along the salivary ducts and

multiple submucosal ducts were up to seven cell layers

thick. The submucosa contained multiple aggregates

of lymphocytes and plasma cells.

Significant gross necropsy findings in case 1

included numerous small white chalky plaques in

the coelomic membrane, within fascial planes of the

thoracic musculature, liver, kidneys, heart, and the

joints, consistent with urate deposits. Histologic

evaluation revealed severe pulmonary, myocardial,

hepatic and renal vascular thrombosis. Hepatic

lipidosis was present. Many renal tubules were

markedly dilated and contained tophi. Tubular

endothelial cells were usually sloughed and had

disrupted basement membranes. Many tubular epithe-

lial cells had large brown cytoplasmic granules.

Glomeruli were often replaced with eosinophilic

material and heterophilic infiltrates were seen in

other glomeruli, tubules and the interstitium. The

esophageal mucosa had infiltrates of lymphocytes,

plasmacytes, and heterophils, and extensive areas of

hemorrhage and edema were present within the

serosa. Small areas of gliosis were present in the

brain.

Histopathology of the gingival biopsy of case 2

revealed chronic proliferative gingivitis and period-

ontitis with a plasma-cell rich inflammatory response

extending deep into the lamina propria. One year later,

a second biopsy of the proliferative gingiva was

consistent with squamous cell carcinoma. At necropsy

of case 2, the left maxillary mass measured 7 mm

deep � 15 mm wide � 12 mm in length, while the left

mandibular mass measured 8 mm � 7 mm � 15 mm.

Diffuse proliferative gingivitis was present. Scattered

throughout the pulmonary parenchyma were multiple,

1–2 mm, tan, miliary pulmonary nodules. Histo-

pathology confirmed diffuse squamous cell carcinoma

of the oral cavity with osteolysis of the maxilla and

mandible, and chronic, mononuclear- and heterophi-

lic-rich proliferative gingivitis.

Histopathology of the gingival biopsy of case 3

revealed diffuse squamous cell carcinoma of the oral

cavity and chronic proliferative stomatitis. An intense

lymphoplasmacytic inflammatory response with lesser

numbers of macrophages and few heterophils was

present in the submucosal and lamina propria. At

necropsy of case 3, there was diffuse villous-like

proliferation of the oral mucosa with patches of focal

erythema. The only other significant gross finding was

fibrinous exudate on the serosa of the gall bladder.

Histopathology revealed diffuse oral squamous cell

carcinoma with chronic lymphoplasmacytic ulcerative

stomatitis and chronic alveolitis, mild non-suppurative

meningitis, chronic cholangiohepatitis, multifocal

ulcerative enteritis, and hepatic and renal amyloidosis.

3.2. PCR amplification

PCR amplification of case 1 resulted in a 175 bp

product when primer sequences were edited out.

Retrospective attempts at PCR amplification of

paraffinized tissues from case 2 did not result in

product suitable for sequencing; this was interpreted

as likely due to DNA degradation after 9 years of

storage of the paraffinized tissue at room temperature.

PCR amplification and sequencing of paraffinized

tissues from cases 3 and 4 resulted in identical

nucleotide sequence that differed by one nucleotide

from case 1, which did not result in a change in the

predicted amino acid sequence.

J.F.X. Wellehan et al. / Veterinary Microbiology 105 (2005) 83–9288

Fig. 2. (a) Histologic appearance of proliferation of gingival epithelium in case 1, a green tree monitor (V. prasinus). Hematoxylin/eosin staining

(b) DNA in situ hybridization of gingiva. Nuclei of scattered cells within the superficial mucosa are positive for herpesvirus nucleic acid.

Bar = 20 mm.

3.3. Phylogenetic analysis

TBLASTX results showed the highest score with

bovine herpesvirus 1 DNA polymerase (GenBank

accession no. Z78205), an a-herpesvirus. Sequence

data was submitted to GenBank (accession no.

AY437559). The phylogenetic tree shows that the

green tree monitor herpesvirus clusters with other

members of the subfamily a-herpesvirinae (Fig. 3).

Although the separation between a-herpesvirinae and

b-herpesvirinae is not statistically clear, all estab-

lished members of the b-herpesvirinae are supported

as a monophyletic group which does not include the

green tree monitor herpesvirus sequence. These

J.F.X. Wellehan et al. / Veterinary Microbiology 105 (2005) 83–92 89

Fig. 3. Phylogenetic tree of partial herpesviral DNA polymerase amino acid sequences. Elephantid HV1 was used as the outgroup (GenBank

accession no. AAG41999). The validity of the tree topology obtained was tested by using bootstrap analysis with 1000 re-samplings. Branchings

with bootstrap values less than 50 are not shown, and areas where these branchings occurred are checkered. Herpesviral genera are boxed with

genus names in italics. Subfamilies are shown with brackets. Varanid herpesvirus 1 is in bold. Other sequences were retrieved from GenBank.

Ateline HV3 (T42925), Bovine HV1 (CAA64335), Bovine HV2 (AAD55134), Callitrichid HV3 (NC004367), Caviid HV2 (AAA43832),

Cercopithecine HV5 (U63460), Cercopithecine HV 8 (AY186194), Columbid HV1 (AAD30145), Equid HV1 (AAB02465), Equid HV2

(P52367), Felid HV1 (CAA12264), Gallid HV1 (AAD56202), Gallid HV2 (AAA79862), Gerrhosaurid HV1 (AF416628), Gerrhosaurid HV2

(AF416629), Gerrhosaurid HV3 (AF416630), Green Turtle HV (AF035004), Human HV1 (P09854), Human HV2 (P079218), Human HV4

(DJBE2L), Human HV6 (AAD49652), Human HV7 (T41940), Human HV8 (AAC57974), Iguanid HV2 (AY236869), Mandrill CMV

(AAG39064), Meleagrid HV1 (AAG30070), Murid HV1 (AAA45940), Passerid HV1 (AF520812), Phocine HV1 (AAB93518), Pongine

HV3 (AAK28018), Psittacid HV1 (AAC55656), Psittacine Papilloma HV (AF456881), Rhinoceros HV (AAK00812), Sea Turtle papilloma HV

(AF035004), Suid HV1 (AAA74383), Suid HV2 (AAF80111), Tortoise HV (BAB40430), Varanid HV1 (AY437559).

results support the classification of this virus in the

subfamily a-herpesvirinae. Further sequence data is

necessary to firmly establish this classification. There

is not bootstrap support for clustering this virus with

the other lizard herpesviruses for which sequence is

available, and this virus appears to be fairly distinct

from these viruses. Based on naming conventions, this

herpesvirus should be named varanid herpesvirus 1

(Minson et al., 2000).

3.4. DNA in situ hybridization

In case 1, nuclei of scattered cells within the

superficial mucosa of the gingiva were positive for

herpesvirus nucleic acid following DNA in situ

hybridization (Fig. 2b). The viral-infected cells

appeared to be superficial epithelial cells. The

superficial nature of the viral-infected cells suggested

that some infected cells might have been sloughed

J.F.X. Wellehan et al. / Veterinary Microbiology 105 (2005) 83–9290

from the surface of the lesion. Nuclei of scattered cells

within the lung were also positive. The viral-infected

cells appeared to be epithelial cells and possibly a few

heterophils. Rare nuclear staining of myocardiocytes

was seen in the heart. Sections of brain had trace

reactivity in a few foci of gliosis.

Retrospective DNA in situ hybridization of tissues

from case 2 was positive for herpesvirus nucleic acid

in sections of oral basal epithelium including positive

staining in some leukocytes, as well as bone marrow,

intestine, pancreas, and skeletal muscle. Sections of

nerve, kidney, liver, heart, brain, and lung were

negative.

Retrospective DNA in situ hybridization of tissues

from case 3 was positive for herpesvirus nucleic acid

with one section of mucosa, while other sections were

negative. Staining was positive for herpesvirus nucleic

acid in the brain stem. The spleen, trachea, lung,

kidney, gall bladder, liver, gastrointestinal tract, testis,

cartilage, bone, skeletal muscle, skin, eye, optic nerve,

and pancreas were negative.

4. Discussion

Stomatitis is a common problem in lizards. In the

four green tree monitors described here, sequences

representing a novel herpesvirus were associated with

a proliferative stomatitis. Although a causal relation-

ship was not demonstrated, the observed pathology is

consistent with disease caused by other members of

the herpesviridae in other host species. Proliferative

gingivitis has been associated with human herpesvirus

(HHV) 5 (Kaur et al., 2003), HHV4, HHV6 (Flaitz and

Hicks, 1998) and cercopithecine herpesvirus 15

(Baskin et al., 1995). Mucosal papillomatous lesions

have been associated with a-herpesviruses in psitta-

cine birds (Johne et al., 2002; Styles et al., 2004).

Proliferative gingivitis was seen in a San Esteban

chuckwalla prior to death from acute hepatic necrosis

associated with a herpesvirus (Wellehan et al., 2003).

Reptilian herpesviruses have also been associated with

stomatitis in tortoises (Jacobson et al., 1985), and

plated lizards (Wellehan et al., 2004). The cause of

renal failure in case 1 is unknown, but may have been

related to dehydration due to either reluctance to drink

due to stomatitis or failure to drink due to neurologic

disease. The lesions and clinical signs observed in

each of the infected lizards were consistent in

appearance with herpesvirus-induced lesions seen in

other species. However, Koch’s postulates were not

tested and a causal relationship between proliferative

stomatitis and this newly identified herpesvirus

remains to be demonstrated.

Comparative sequence analysis of the herpes-

viruses of reptiles should contribute to a further

understanding of viral phylogeny and the evolution of

this important class of viruses. Previous phylogenetic

analyses of mammalian herpesviruses suggest that

many elements in the branching patterns of herpesvir-

idae are congruent with branching patterns for the

corresponding host species (McGeoch and Davison,

1999). The branching of the a-herpesviruses from the

b- and g-herpesviruses has been tentatively estimated

to have occurred approximately 200 million years ago

(McGeoch and Davison, 1999). According to mole-

cular evidence, squamates appear to be the first

modern group of reptiles to have diverged from other

reptiles and birds (Hedges and Poling, 1999; Mannen

et al., 1997), and is estimated to have occurred

approximately 245 million years ago, although the

oldest squamate fossils date from only approximately

156 million years ago (Hedges and Poling, 1999).

Based on this early branching of the squamates from

other reptiles and birds, it is tempting to speculate that

the virus identified in our current investigations may

have arisen very close to the basal branching point of

the a-herpesviruses, and may provide insight into the

genetic structure of the ancestral herpesviruses.

Until recently, diagnosis of herpesviral infection

has often relied on histopathologic identification of

inclusions, followed by further characterization such

as electron microscopy, virus culture, and immuno-

histochemistry. While herpesviral diseases often

produce characteristic intranuclear inclusions, they

are not always present. Diseases such as malignant

catarrhal fever in hoofstock and fibropapillomatosis in

sea turtles have been strongly associated with

herpesviruses, but inclusions are rare (Kleiforth

et al., 2002; Lackovitch et al., 1999). Appropriate

culture conditions have also not been determined for

many herpesviruses. In these cases, although a

herpesvirus was present in the lesions, intranuclear

inclusions were not seen. Consensus nested PCR was

used to identify a novel herpesvirus, and DNA in situ

hybridization was used to localize the presence of the

J.F.X. Wellehan et al. / Veterinary Microbiology 105 (2005) 83–92 91

virus in the tissue. While DNA in situ hybridization

has been previously reported for tortoise herpesvirus

(Teifke et al., 2000), this report documents the first use

of DNA in situ hybridization for the localization of a

novel squamate herpesvirus.

Acknowledgments

We thank Sylvia Tucker and Maud Lafortune at the

University of Florida and Kate Pennick at the

University of Georgia for their assistance with these

cases. On the Toronto Zoo cases, we also thank Dr.

Karrie Rose for her assistance, and Dr. Ian Barker and

Dr. Dale Smith at the Ontario Veterinary College,

University of Guelph for assistance with histopatho-

logical interpretation.

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