Short communication

Identification of a novel herpesvirus from a California desert

tortoise (Gopherus agassizii)

April J. Johnson a,*, Allan P. Pessier b, James F. X. Wellehan a,Roseanne Brown c, Elliott R. Jacobson a

a Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, P.O. Box 100126,

Gainesville, FL 32610, USAb Department of Pathology, Center for Reproduction of Endangered Species, Zoological Society of San Diego,

San Diego, CA 92112-0551, USAc Rancho San Diego Animal Hospital, 2988 Jamacha Rd., Suite 176, El Cajon, CA 92013, USA

Received 12 May 2005; received in revised form 22 August 2005; accepted 14 September 2005

Abstract

Herpesviruses are significant pathogens of tortoises, causing upper respiratory tract disease and necrotizing stomatitis, with

infections often associated with high mortality rates. Herpesvirus infection in a captive California desert tortoise (Gopherus

agassizii) was detected by light microscopic observation of intranuclear inclusion bodies in various tissues followed by

transmission electron microscopic observation of herpesvirus-like particles, and amplification of herpesvirus nucleic acid

sequences using polymerase chain reaction. Using an indirect enzyme linked immunosorbent assay, anti-tortoise herpesvirus

antibodies were detected one month after initial onset of clinical signs. This novel herpesvirus is distinct from the previously

described tortoise herpesvirus (tortoise herpesvirus-1, THV-1) sharing 83% sequence identity of 60 amino acids of a portion of

the DNA polymerase gene and 79% sequence identity across 120 amino acids of a portion of the ribonucleotide reductase gene.

Similar to THV-1, this novel herpesvirus, tortoise herpesvirus-2 (THV-2), also clusters with the alphaherpesviruses.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Desert tortoise; Gopherus agassizii; Herpesvirus; Reptiles

www.elsevier.com/locate/vetmic

Veterinary Microbiology 111 (2005) 107–116

1. Introduction

Herpesvirus infections were first described in

tortoises in the early 1980s (Harper et al., 1982).

* Corresponding author. Tel.: +1 352 392 4700x5256;

fax: +1 352 392 5464.

E-mail address: JohnsonA@mail.vetmed.ufl.edu (A.J. Johnson).

0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2005.09.008

They have since been reported in many species with

varying clinical signs and degrees of severity

(Jacobson et al., 1985; Heldstab and Bestetti, 1989;

Drury et al., 1998; Muro et al., 1998). Infection is most

often associated with the oral cavity and respiratory

tract, with necrotizing stomatitis, glossitis, tracheitis,

pharyngitis, and rhinitis having been repeatedly

described (Jacobson et al., 1985; Muro et al., 1998;

.

A.J. Johnson et al. / Veterinary Microbiology 111 (2005) 107–116108

Fig. 1. Caseous yellow plaques in the oral cavity of the desert

tortoise on January 14, 2003.

Drury et al., 1998). Encephalitis (Heldstab and

Bestetti, 1989) and hepatitis (Hervas et al., 2002)

have also been observed.

Diagnosis of herpesvirus infections in tortoises

have been made by observation of intranuclear

inclusion bodies by light microscopy followed by

visualization of virus particles by transmission

electron microscopy (TEM) (Jacobson et al., 1985;

Muro et al., 1998), DNA in situ hybridization (Teifke

et al., 2000), and immunohistochemistry (Origgi et al.,

2003). Virus isolation on susceptible cell lines can be

performed (Marschang et al., 2001) followed by

negative staining electron microscopy. Amplification

of herpesvirus specific DNA segments can be

performed using consensus (VanDevanter et al.,

1996; Une et al., 2000) or tortoise herpesvirus specific

(Teifke et al., 2000; Origgi et al., 2004) polymerase

chain reaction (PCR) primers. Virus neutralization and

indirect enzyme linked immunosobent assay (ELISA)

have been used to detect anti-herpesvirus antibodies

(Marschang et al., 2001; Origgi et al., 2001). Three

tortoise herpesvirus sequences exist in GenBank for

three genes including the UL5 helicase primase

(Accession #AY188757), UL39 (Accession

#AY338245) and the DNA polymerase (Accession

#AB047545) genes.

The desert tortoise (Gopherus agassizii) has been

experiencing dramatic declines throughout parts of its

range and populations north and west of the Colorado

River are listed as threatened by the US federal

government (USFWS, 1994). Upper respiratory tract

disease (URTD) has been implicated as one of the

causes of declining numbers (USFWS, 1994). Captive

tortoises with URTD were first observed in the late

1970s (Fowler, 1980; Snipes and Biberstein, 1982)

and in wild desert tortoises in the late 1980s (Jacobson

et al., 1991). Mycoplasma agassizii was subsequently

identified and demonstrated to be a causative agent of

URTD (Brown et al., 1994). Clinical signs of

herpesvirus infection overlap with those of myco-

plasmosis including nasal and ocular discharge and

palpebral and periocular edema. The possibility that

herpesviruses may also cause URTD in desert tortoises

has not been thoroughly investigated. Using an

indirect ELISA and herpesvirus isolated from Med-

iterranean tortoises (Testudo graeca) as the coating

antigen, a recent survey of 109 captive desert tortoises

in southern California showed 26.6% to be seropo-

sitive for exposure to tortoise herpesvirus (Johnson

et al., In press). While there are currently three

published reports of herpesvirus-like particles being

detected by TEM in desert tortoises (Harper et al.,

1982; Pettan-Brewer et al., 1996; Martınez-Silvestre

et al., 1999), the phylogenetic relationship between

these viruses is unknown since no sequencing data is

available. Here we describe a novel herpesvirus from a

captive California desert tortoise with a necrotizing

stomatitis.

2. Materials and methods

2.1. Case summary

An adult female captive desert tortoise of unknown

age was presented to a veterinary clinic in El Cajon,

California on January 6, 2003, after an unusually early

exit from hibernation and exhibiting clinical signs of

anorexia and lethargy. She had been obtained through

an adoption agency in August of 2002 and placed in a

homewith three desert tortoises and one Texas tortoise

(Gopherus berlandieri). Physical examination

revealed yellow-white caseous plaques on the tongue

and palate (Fig. 1). A biopsy of the tongue was taken

on January 14 for light microscopic examination, PCR

and virus isolation. Plasma samples were collected on

A.J. Johnson et al. / Veterinary Microbiology 111 (2005) 107–116 109

Fig. 2. Caseous yellow and gray plaques in the oral cavity of the

desert tortoise at necropsy on February 11, 2003.

January 14, January 27 and February 6 for detection of

anti-herpesvirus antibodies by ELISA and plasma

samples were also collected for ELISA on February 4

from the four tortoises housedwith the ill desert tortoise.

Supportive therapy consisting of enrofloxacin, doxycy-

line, fluid therapy, and tube feeding was instituted. Oral

plaques progressed to cover the entire oral cavity (Fig. 2)

and the tortoise died on February 10. A necropsy was

performed and tissues from all major organs were

collected for histopathology, virus isolation and PCR.

2.2. Light and electron microscopy

A portion of the tongue biopsy and tissues collected

at necropsy were fixed in 10% neutral buffered

formalin and processed for histopathology. Paraffin

embedded tissues were cut into 6 mm sections and

stained with hematoxylin and eosin. Portions of the

paraffin embedded tissue from the tongue biopsy were

deparaffinized in xylene, embedded in Spurr’s resin,

and sectioned for TEM at the Electron Microscopy

Core Laboratory, University of Florida.

2.3. Virus isolation

A portion of the tongue biopsy and liver and tongue

collected at necropsy were homogenized and inocu-

lated onto 25 cm2 flasks (Costar, Corning, NY)

containing monolayers of turtle heart cells (TH1

cells; ATCC-CCL 50, American Type Culture

Collection, Rockville, MD) in Dulbecco’s Modified

Eagle Medium (DMEM, Gibco, Carlsbad, CA)

supplemented with 5% fetal bovine serum (Gibco),

gentamicin (60 mg/L; Sigma, St. Louis, MO),

penicillin G (120,000 U/L; Sigma), streptomycin

(120,000 U/L; Sigma) and amphotericin B (300 mg/

L; Sigma). Cells were incubated at 28 8C in the

presence of 5%CO2 and flasks were observed daily for

signs of cytopathic effects.

2.4. ELISA

Plasma samples were evaluated for the presence of

anti-herpesvirus antibodies by indirect enzyme linked

immunosorbent assay (ELISA). A previously

described method was used (Origgi et al., 2001) but

was adapted for a desert tortoise by using mouse-anti-

desert tortoise monoclonal biotinylated antibodies

(Schumacher et al., 1993) in place of mouse-anti-

Greek tortoise monoclonal biotinylated antibodies.

The plates were coated with THV-1 isolated and

purified from a Mediterranean tortoise, which was

used in the original ELISA; the positive optical density

cut off value used was that previously determined in

Mediterranean tortoises (Origgi et al., 2001).

2.5. PCR and DNA sequencing

DNA was extracted from a portion of the tongue

biopsy and brain, tongue, spleen, liver, kidney,

bladder, and adrenal gland collected at necropsy

using the Qiagen DNeasy kit (Qiagen, Valencia, CA).

Nested PCR amplification of partial sequence of the

herpesvirus DNA-dependent DNA polymerase gene

and direct PCR utilizing tortoise herpesvirus specific

primers targeting the ribonucleotide reductase gene

was performed using methods previously described

(VanDevanter et al., 1996; Origgi et al., 2004). The

PCR products were resolved in 1% agarose gels,

excised, and purified using the QIAquick gel

extraction kit (Qiagen). Products were sequenced

directly in both directions using the Big-Dye

Terminator Kit (Perkin-Elmer, Branchburg, NJ) and

analyzed on an ABI 377 automated DNA sequencer at

the University of Florida’s Sequencing Center.

A.J. Johnson et al. / Veterinary Microbiology 111 (2005) 107–116110

Fig. 3. Light microscopic photograph of the tongue biopsy taken on

January 14, 2003 demonstrating many large amphophilic intra-

nuclear inclusion bodies in superficial epithelial cells.

Fig. 4. Electron microscopic photograph demonstrating herpes-

virus-like particles from the paraffin embedded biopsy shown in

Fig. 3.

2.6. Phylogenetic analysis

The sequences were compared to known sequences

in GenBank (National Center for Biotechnology

Information, Bethesda, Maryland), EMBL (Cam-

bridge, United Kingdom), and Data Bank of Japan

(Mishima, Shiuoka, Japan) databases using

TBLASTX (Altschul et al., 1997). Predicted homo-

logous amino acid sequences of corresponding

alphaherpesviral DNA dependent DNA polymerase

Fig. 5. Alignment of partial herpesviral DNA polymerase amino acid sequ

subfamily alphaherpesviriniae: U, unassigned; S, Simplexvirus; V, Varice

and ribonucleotide reductase genes available from

GenBank were aligned from each of the genera

(Simplexvirus, Varicellovirus, Mardivirus, and Ilto-

virus) in addition to other unassigned reptile

herpesviruses using T-Coffee (Notredame et al.,

2000). Analyses of the predicted alignments were

performed with the PHYLIP (Phylogeny Inference

Package, Version 3.61) program package (Felsenstein,

1989) using Proml (Jones–Taylor–Thornton probabil-

ity model, global rearrangements). Elephantid her-

ences. Letters in parentheses denote outgroup (OG) or genera within

llovirus; I, Iltovirus; M, Mardivirus.

A.J. Johnson et al. / Veterinary Microbiology 111 (2005) 107–116 111

p(O

G)orgenerawithin

subfamily

pesvirus-1 (GenBank Accession #AF322977), a

betaherpesvirus, was used as the outgroup for the

polymerase gene and human herpesvirus-4 (GenBank

Accession #NC_001345), a gammaherpesvirus, was

used as the outgroup for the ribonucleotide reductase

gene. The strength of both tree topologies obtained

were tested by bootstrap analysis (Felsenstein, 1985)

starting with Seqboot with 100 resamplings, followed

by maximum likelihood calculations. Consense was

used to calculate the bootstrap values.

Fig.6.Alignmentofpartial

herpesviral

ribonucleotidereductasegeneam

i no

acid

sequences.

Letters

inparentheses

denote

outgrou

alphaherpesviriniae:

U,unassigned;S,

Sim

ple

xvir

us;V,

Vari

cell

ovi

rus;

I,Il

tovi

rus;M,

Mar d

ivir

us.

3. Results

3.1. Light and electron microscopy

Histologic examination of the tongue biopsy

demonstrated many large amphophilic intranuclear

inclusion bodies in superficial epithelial cells (Fig. 3).

Histologic examination of tissues collected at

necropsy revealed a chronic and active necrotizing

stomatitis, pharyngitis, and tracheitis. Epithelial

surfaces in these areas were covered with a thick

coagulum composed of sloughed epithelial cells,

heterophils and bacterial colonies. Sloughed and

viable epithelial cells frequently had eosinophilic to

amphophilic intranuclear inclusion bodies. The sub-

mucosa in affected regions had variable mixed

infiltrates of heterophils, lymphocytes, plasma cells,

and macrophages. In some areas of the oropharynx,

there was complete epithelial loss and replacement by

granulation tissue. The only other significant histo-

logic finding was a granulomatous bronchopneumonia

with intralesional bacterial colonies. Sections of the

brain, liver, spleen, pancreas, small intestine, kidney,

urinary bladder, adrenal gland, thyroid gland, ovary,

and heart were unremarkable.

Transmission electron microscopy of the paraffin

embedded tongue biopsy revealed non-enveloped

virus particles within intranuclear inclusions in

epithelial cells. Virions were approximately 120 nm

in diameter and consistent in size, site of replication,

and morphology with herpesvirus (Fig. 4).

3.2. Virus isolation

All attempts at virus isolation failed to show any

cytopathic effects up to 30 days after inoculation.

A.J. Johnson et al. / Veterinary Microbiology 111 (2005) 107–116112

3.3. ELISA

ELISA performed on the desert tortoise plasma

samples were negative on January 14 and 27 but

positive on February 6. The ELISA performed on the

plasma samples collected from the other four

tortoises housed with the desert tortoise collected

on February 4 showed that two desert tortoises and

one Texas tortoise were positive for anti-tortoise

Fig. 7. Maximum likelihood phylogenetic tree of partial alphaherpesviral

(GenBank Accession #AF322977), a betaherpesvirus, was used as the outgr

by bootstrap analysis with 100 resamplings. Bootstrap values are shown. Br

where these branchings occurred are checkered. Tortoise herpesvirus-2 is

accession numbers include Canid HV (U63459), Cercopithecine HV1 (NC

Gallid HV1 (AY741134), Gallid HV2 (NC_002229), Gallid HV3 (N

(AF416628), Gerrhosaurid HV3 (AF416630), Chelonia mydas HV (AF120

HV2 (AY114171), Iguanid HV2 (AY236869), Meleagrid HV1 (NC_0026

HV1 (AY623128), Psittacid HV2 (AY623121) Suid HV1 (NC_006151),

herpesvirus antibodies, while one desert tortoise was

negative.

3.4. PCR and DNA sequencing

Liver, tongue, spleen, brain, urinary bladder, and

adrenal gland all produced amplicons consistent in

size with THV-1. No amplification product was

obtained from the kidney. Amplification of the

DNA polymerase amino acid sequences. Elephantid herpesvirus-1

oup of this unrooted tree. The validity of the tree topology was tested

anchings with bootstrap values less than 50 are not shown, and areas

shown in bold. Other sequences retrieved from GenBank and their

_004812), Crocodylid HV1 (AY913769), Equid HV1 (NC_001491),

C_002577), Gerrhosaurid HV1 (AF416629), Gerrhosaurid HV2

209), Equid HV4 (NP_045247), Human HV1 (AY705985), Human

41), Passerid HV1 (AF520812), Phocine HV1 (U92269), Psittacid

Tortoise HV1 (AB047545), Varanid HV1 (AY437559).

A.J. Johnson et al. / Veterinary Microbiology 111 (2005) 107–116 113

polymerase gene resulted in a product that was 181

base pairs when primer sequences were edited out and

amplification of the ribonucleotide reductase gene

resulted in a product that was 361 base pairs.

3.5. Phylogenetic analysis

TBLASTX results for the nested PCR sequence

showed the highest score with tortoise herpesvirus

Fig. 8. Maximum likelihood phylogenetic tree of partial alphaherpesvi

herpesvirus-4 (GenBank Accession #P03190), a gammaherpesvirus, was

topology was tested by bootstrap analysis with 100 resamplings. Bootstrap

not shown, and areas where these branchings occurred are checkered. Oth

include Bovine HV1 (CAA9029), Bovine HV2 (AAK56211), Bovine H

(YP_053066), Equid HV4 (CAA53100), Gallid HV1 (AAD56211), Ga

(NP_044509), Meleagrid HV1 (AAG30079), Psittacid HV1 (AAQ73718)

DNA polymerase (GenBank Accession #AB047545),

sharing 83% sequence identity while the direct PCR

showed highest score with the tortoise ribonucleotide

reductase gene (GenBank Accession #AY338245)

sharing 79% sequence identity. Novel sequence data

was submitted to GenBank; the accession number for

the partial polymerase sequence is AY916792 and for

the partial ribonucleotide reductase sequence is

DQ027825. The alignment of partial herpesvirus

ral ribonucleotide reductase gene amino acid sequences. Human

used as the outgroup of this unrooted tree. The validity of the tree

values are shown. Branchings with bootstrap values less than 50 are

er sequences retrieved from GenBank and their accession numbers

V5 (NP_954908), Cercopithecine HV1 (AAP41457), Equid HV1

llid HV2 (AAA80556), Human HV1 (NP_044641), Human HV2

, Suid HV1 (YP_068342), and Tortoise HV1 (AAQ73541).

A.J. Johnson et al. / Veterinary Microbiology 111 (2005) 107–116114

DNA polymerase and ribonucleotide reductase gene

amino acid sequences are shown in Figs. 5 and 6,

respectively. The phylogenetic tree comparing poly-

merase amino acid sequences is shown in Fig. 7 and

the ribonucleotide reductase gene sequences in Fig. 8.

4. Discussion

The lesions seen in the desert tortoise in this study

are consistent with previous reports of herpesvirus

infection in desert tortoises (Harper et al., 1982;

Pettan-Brewer et al., 1996; Martınez-Silvestre et al.,

1999). For the first time, sequencing data was

obtained for a desert tortoise herpesvirus. In a study

sequencing a portion of the gene encoding the DNA

polymerase gene in 5 strains of human herpesvirus-2,

17 strains of human herpesvirus-6 and 5 strains of

human herpesvirus-7, only single base variations that

did not result in alteration of the amino acid sequence

were seen within species (VanDevanter et al., 1996).

Tortoise herpesvirus-2 amino acid sequence differs

by 17% from the previously described tortoise

herpesvirus (Une et al., 2000), supporting the

interpretation that this is a novel virus, and not a

variant of THV-1. The designation THV-2 will

hereafter be used when referring to this virus. The

partial polymerase gene phylogenetic tree (Fig. 7)

demonstrates that human herpesvirus-1 and 2 are

more closely related to each other than THV-1 is

related to THV-2.

It is unknown whether THV-2 is native to North

American tortoises and whether it is affecting wild

populations of desert tortoises. Evidence of herpes-

virus exposure in wild desert tortoises based on

serology with indirect ELISA has been documented,

although until now, herpesvirus infection had not been

confirmed in a seropositive tortoise. Surveys have

shown a seroprevalence of herpesvirus exposure in

wild desert tortoises ranging from 30 to 51%

(Jacobson et al., 2001; Berry et al., 2003). This

suggests that there is a herpesvirus native to wild

desert tortoises or that wild desert tortoises have been

exposed to herpesvirus from non-native tortoises.

However, herpesvirus infection has not been demon-

strated in a wild desert tortoise and therefore cannot be

characterized as THV-1, THV-2, or another novel

herpesvirus. This desert tortoise was likely infected

during captivity because the tortoise did not exhibit

antibodies on ELISA initially, but seroconverted

within a month of clinical evidence of herpesvirus

infection. This tortoise was exposed to three other

desert tortoises and one Texas tortoise (Gopherus

berlandieri) for five months prior to developing

clinical signs of disease. On ELISA, two of the desert

tortoises and the one Texas tortoise were positive for

anti-tortoise herpesvirus antibodies. It is unknown

whether these tortoises had herpesvirus prior to being

taken into captivity, if they were exposed to other

tortoises at some point during captivity or if they had

the same point source as the infected tortoise.

This study shows that the previously described

ELISA (Origgi et al., 2001) can be adapted to detect

herpesvirus exposure in desert tortoises. While certain

herpesviruses are known to permanently infect their

hosts, we do not know if infected tortoises that recover

remain infected for life. In experimental infection

studies, herpesvirus was demonstrated in the central

nervous system of tortoises that were inoculated with

virus and recovered following development of clinical

signs of infection (Origgi et al., 2001).

The data strongly suggests that THV-1 and THV-2

are closely antigenically related, and the currently

used ELISA does not distinguish well between the

two tortoise herpesviruses. Antibodies to this her-

pesvirus cross-reacted to THV-1, the virus used as the

antigen in the indirect ELISA, indicating that

although differing in sequence, both herpesviruses

have similar antigenic sites to which the tortoise can

develop antibodies. A previous transmission study

with Greek tortoises (T. graeca) demonstrated

seroconversion within 4–7 weeks of experimental

infection (Origgi et al., 2001), which was consistent

with the time fromwhen lesions were first observed to

seroconversion.

5. Conclusions

A novel herpesvirus, tortoise herpesvirus-2, was

identified in a California desert tortoise. It is unknown

how this virus differs serologically and biologically to

tortoise herpesvirus-1, although antibodies to THV-2

cross-reacted to THV-1 on ELISA. The previously

described ELISA can be adapted to detect herpesvirus

in desert tortoises.

A.J. Johnson et al. / Veterinary Microbiology 111 (2005) 107–116 115

Acknowledgements

Funding was awarded by Department of Cultural

Natural Resources, Directorate of Public Works at the

National Training Center, Ft. Irwin, CA. We thank the

Cultural and Natural Resources Manager at Ft. Irwin,

Mr. Mickey Quillman, for support of this project,

Sylvia Tucker at the University of Florida and

Marianne Zeitz at the Rancho San Diego Animal

Hospital for their assistance.

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