50
Journal of Zoo and Wildlife Medicine 35(1): 50?54, 2004
Copyright 2004 by American Association of Zoo Veterinarians
THREE NOVEL HERPESVIRUSES ASSOCIATED WITH
STOMATITIS IN SUDAN PLATED LIZARDS (GERRHOSAURUS
MAJOR) AND A BLACK-LINED PLATED LIZARD
(GERRHOSAURUS NIGROLINEATUS)
James F. X. Wellehan, D.V.M., M.S., Donald K. Nichols, D.V.M., Dipl. A.C.V.P.,
Ling-ling Li, M.S., and Vivek Kapur, B.V.Sc., M.Sc., Ph.D.
Abstract: Glossal stomatitis was observed in a Sudan plated lizard (Gerrhosaurus major) with severe dyspnea. On
necropsy, intranuclear inclusion bodies were seen in the periglottal lingual epithelium. Labial stomatitis was seen in a
second Sudan plated lizard and a black-lined plated lizard (G. nigrolineatus). Degenerate polymerase chain reaction
(PCR) primers targeting a conserved region of herpesvirus DNA?dependent DNA polymerase gene were used to
amplify products from lesions from each lizard. Nucleotide sequencing of the PCR products showed that the sequence
from each lizard was unique. Phylogenetic and comparative sequence analyses suggest that these viruses are novel
members of the subfamily Alphaherpesvirinae, and they are here termed gerrhosaurid herpesviruses 1?3. Results of
our analyses suggest that the genus Gerrhosaurus can be infected by these novel herpesviruses.
Key words: Gerrhosaurus major, Gerrhosaurus nigrolineatus, herpes, stomatitis, virus, plated lizard.
INTRODUCTION
Herpesviridae is a diverse family of enveloped
double-stranded DNA viruses found in many dif-
ferent orders of vertebrates, including fish, amphib-
ians, reptiles, birds, and mammals.15 Herpesviruses
have been further divided into the subfamilies Al-
phaherpesvirinae, Betaherpesvirinae, and Gammah-
erpesvirinae on the basis of properties of infection,
host range, and behavior in culture. Evidence from
DNA analysis has largely reinforced the extant
classification system and suggests the presence of
further subfamilies. All known sequences of fish
and amphibian herpesviruses appear to be distinct
from the subfamilies listed above, and an oyster
herpesvirus may also be distinct.15 To date, pub-
lished analyses of sequences of avian herpesviruses
all appear to belong to the subfamily Alphaherpes-
virinae.15,21
Herpesviruses of reptiles have been described in
chelonians,3,5,6,10,11 snakes,7,12,16 and lizards,2,18,22 as-
sociated with a variety of lesions, including sto-
matitis in tortoises.10 However, with a few excep-
tions, little information is available regarding the
genetic classification of the viruses recovered from
From the Departments of Microbiology and Veterinary
Pathobiology, and Biomedical Genomics Center, Univer-
sity of Minnesota, St. Paul, Minnesota 55108, USA (Wel-
lehan, Li, Kapur); and the Department of Pathology, Na-
tional Zoological Park, Washington, D.C. 20008, USA
(Nichols). Present address (Wellehan): Department of
Small Animal Clinical Sciences, College of Veterinary
Medicine, University of Florida, Gainesville, Florida
32610, USA. Correspondence should be directed to Dr.
Wellehan.
reptiles. All reptile herpesvirus sequences available
in the public databases (GenBank, National Center
for Biotechnology Information, Bethesda, Mary-
land 20894, USA; EMBL, Cambridge, United
Kingdom; and Data Bank of Japan, Mishima, Shi-
uoka, Japan) are from chelonians and appear to be
most closely related to members of the Alphaher-
pesvirinae.17,20
The herpesviruses described in lizards are lac-
ertid herpesvirus, iguana herpesvirus, and agamid
herpesvirus. Lacertid herpesvirus has been seen by
electron microscopy associated with cutaneous pap-
illomatous lesions.18 Iguana herpesvirus was isolat-
ed from iguana heart cell cultures. Cytopathic ef-
fects were seen in cell culture. Inoculation of 12
young iguanas produced no consistent pattern of
lesions, and although there was a much higher mor-
tality rate in the inoculated population, no causal
relationship was established.2 Agamid herpesvirus
was seen by electron microscopy in the liver, lung,
and spleen of two red-headed agamas (Agama aga-
ma) that died.22
This study describes stomatitis in two species of
gerrhosaurid lizards associated with the presence of
three unique herpesviruses. These are the first re-
ported sequences for herpesviruses in lizards, and
the results of our analyses suggest that the genus
Gerrhosaurus can be infected by members of the
Alphaherpesvirinae.
MATERIALS AND METHODS
Study animals
Case 1: A male Sudan plated lizard (G. major),
at least 6 yr old, from a private collection in Min-
51WELLEHAN ET AL.?THREE NOVEL GERRHOSAURID HERPESVIRUSES
Figure 1. a. Sudan plated lizard (Gerrhosaurus major) with periglottal glossal stomatitis (case 1). b. Sudan plated
lizard (G. major) with labial stomatitis (case 3). c. Black-lined plated lizard (G. nigrolineatus) with maxillary labial
stomatitis (case 2).
nesota was presented for necropsy (Fig. 1a) after a
4-mo history of severe dyspnea and periglottal in-
flammation that was not responsive to enrofloxacin
(Baytril, Bayer, Shawnee Mission, Kansas 66201,
USA; 10 mg/kg, p.o., s.i.d.) or piperacillin (Pipra-
cil, Lederle, Carolina, Puerto Rico 00984, USA;
100 mg/kg, s.c., s.i.d.) treatments. No significant
radiographic lesions were identified. No new lizards
had been added to the collection for more than 5 yr.
Case 2: A male black-lined plated lizard (G. ni-
grolineatus) with labial stomatitis was purchased
from a pet store in Minnesota (Fig. 1c), where it
had been housed with a female black-lined plated
lizard without stomatitis. No further history was
available.
Case 3: A second male Sudan plated lizard (case
3), at least 6 yr old, from the same private collec-
tion as case 1 had a history of a chronic labial pro-
liferative growth that periodically became ulcerated
during at least the past 6 yr (Fig. 1b). The lizard
had not been observed traumatizing the rostrum,
and the caging material was not abrasive. No prior
history was available. The two Sudan plated lizards
had been housed separately.
Polymerase chain reaction amplification and
sequencing
DNA was extracted from paraffinized tissue from
case 1, and very small amounts of fresh tissue were
debrided from the lesions for therapeutic purposes
in cases 2 and 3. DNA extraction was performed
with the QIAamp DNA mini kit (Qiagen, Valencia,
California 91355, USA).
Nested PCR amplification of herpesvirus DNA?
dependent DNA polymerase was performed using
the methods described previously.21 The first round
of amplification used forward primers DFA (59-
GAYTTYGCNAGYYTNTAYCC-39, Y 5 pyrimi-
dine, N 5 nucleotide) and ILK (59-TCCTGGA-
CAAGCAGCARNYSGCNMTNAA-39, R 5 pu-
rine, M 5 A or C) and reverse primer KG1 (59-
GTCTTGCTCACCAGNTCNACNCCYTT-39), and
the second round of amplification used forward
primer TGV (59-TGTAACTCGGTGTAYGGNT-
TYACNGGNGT-39) and reverse primer IYG (59-
CACAGAGTCCGTRTCNCCRTADAT-39, D 5 A,
G, or T). For both Sudan plated lizard samples, the
first round was modified to use IYG instead of KG1
as a reverse primer; use of the original protocol did
not result in a product for either Sudan plated lizard
herpesvirus.
The PCR products were purified using the QIA-
quick PCR purification kit (Qiagen), and the prod-
ucts of the PCR reactions were sequenced either
directly or after cloning into pGEM-T plasmid vec-
tors (Promega, Madison, Wisconsin 53711, USA)
using the Big-Dye Terminator kit (Perkin?Elmer,
Branchburg, New Jersey 08876, USA) and ana-
lyzed on ABI 3100 automated DNA sequencers at
the University of Minnesota Advanced Genetic
Analysis Center. All products were sequenced in
sense and antisense directions.
The sequences were compared with known se-
quences in the public genetic databases (GenBank,
EMBL, and Data Bank of Japan) using the
TBLASTX algorithm.1 Predicted protein sequences
of homologous 55- to 61?amino acid segments of
herpes DNA polymerases were aligned by the Jo-
tun?Hein algorithm9 using MegAlign (DNAstar,
Madison, Wisconsin 53711, USA), and phyloge-
52 JOURNAL OF ZOO AND WILDLIFE MEDICINE
Figure 2. Eosinophilic intranuclear inclusions in tongue of case 1. Inclusions are indicated with arrows. H&E. Bar
5 10 mm.
netic analyses were performed with the PHYLIP
3.573c package.4 Trees were out-group rooted using
the corresponding region of the delta polymerase
gene of Plasmodium falciparum (GenBank acces-
sion No. S17330). Gaps of all lengths were counted
as single events. A total of 100 bootstrapped rep-
licates were performed, each data set was shuffled
10 times in random order of input to create most
probable trees, and a majority-rule consensus tree
was then created.
Sequence data were submitted to GenBank and
are available under accession Nos. AF416628?
AF416630.
RESULTS
Necropsy of case 1, a Sudan plated lizard, re-
vealed very little body fat. The periglottal tongue
was raised and tan (Fig. 1a). The lungs appeared
grossly normal; no other gross lesions were ob-
served. The tongue, glottis, and proximal trachea
were fixed in 10% formalin and paraffinized. He-
matoxylin and eosin staining of the fixed tissue re-
vealed areas inside the glottal trachea of both gran-
ulocytic and lymphocytic inflammation with ero-
sion of overlying epithelium. The periglottal lingual
epithelium contained areas with eosinophilic intra-
nuclear inclusions with both granulocytic and lym-
phocytic inflammation of the underlying tissue (Fig.
2). Histology was not done on cases 2 and 3.
Polymerase chain reaction amplification and
sequencing
Amplification of herpesvirus DNA polymerase
gene sequences from DNA extracted from cases 1
and 2 using the primers and amplification condi-
tions described above resulted in a 231?base pair
(bp) product. Amplification of herpesvirus DNA
polymerase gene sequences from case 3 using the
same primers and conditions resulted in a 234-bp
product. TBLASTX searches of the public genetic
databases for herpesvirus DNA polymerase se-
quence from cases 1 and 2 showed the highest
score with gallid herpesvirus 2 DNA polymerase
(GenBank accession No. AAA79862). In contrast,
DNA polymerase sequence from case 3 showed the
highest score with bovine herpesvirus 2 DNA poly-
53WELLEHAN ET AL.?THREE NOVEL GERRHOSAURID HERPESVIRUSES
Figure 3. Phylogenetic tree of predicted protein sequences of homologous 55- to 61?amino acid segments of
herpes DNA polymerases created using the PHYLIP 3.573c package.4 Trees were out-group rooted using polymerase
D of Plasmodium falciparum (GenBank accession No. S17330). Bootstrap values are shown. A. Branchings with
bootstrap values less than 50 are shown as dotted lines. B. Branchings with bootstrap values less than 50 are not
shown. Genbank accession numbers are given in parentheses: alcephaline HV1 (AAC58060), bovine HV2 (AAD55134),
elephantid HV1 (AAG41999), equid HV1 (AAB02465), 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 HV3 (BAB41073), human HV4 (DJBE2L), human HV5
(AAG02102), human HV6 (AAD49652), human HV7 (T41940), human HV8 (AAC57974), leoporid HV2
(AAC55655), murid HV1 (AAA45940), psittacid HV1 (AAC55656), saimiriine HV2 (AAA46165), suid HV2
(AAF80111), tortoise HV (BAB40430), tupaiid HV1 (Q9YUS3).
merase (GenBank accession No. AAD55134). The
majority-rule consensus phylogenetic tree of pre-
dicted protein sequences of homologous 55- to 61?
amino acid segments of herpes DNA polymerases
is shown in Figure 3. These results suggest that
these viruses belong to the subfamily Alphaherpes-
virinae. Based on naming conventions for herpes-
viruses, the herpesvirus from case 1, a Sudan plated
lizard, is given the provisional name of gerrhosaur-
id herpesvirus 1, the black-lined plated lizard her-
pesvirus (case 2) is gerrhosaurid herpesvirus 2, and
that from case 3, the second Sudan plated lizard, is
gerrhosaurid herpesvirus 3.19
DISCUSSION
Previous studies have shown that stomatitis in rep-
tiles is multifactorial and is frequently associated with
stress.13 Bacteria previously associated with stomatitis
in reptiles have included Pseudomonas spp., Aero-
monas spp., Klebsiella spp., Salmonella spp., and My-
cobacteria spp.13 Although herpesviruses have been
associated with stomatitis in tortoises,10 they have not
been previously described in affected squamates. In
the current investigation we describe sequences rep-
resenting three novel members of the herpesvirus
family in gerrhosaurid lizards with stomatitis. The
gross lesions and clinical signs observed in each of
the infected lizards were consistent in appearance
with the alphaherpesvirus-induced lesions seen in oth-
er species including tortoises10 and humans. Histology
of the lesions in case 1 was also consistent with a
herpesvirus-induced lesion; histology was not done in
cases 2 and 3. A causal relationship between the sto-
54 JOURNAL OF ZOO AND WILDLIFE MEDICINE
matitis and these newly identified herpesviruses was
not demonstrated. Members of the Alphaherpesviri-
nae typically exhibit a more variable host range than
the Betaherpesvirinae or Gammaherpesvirinae and
have previously been shown to establish latent infec-
tions in sensory ganglia.19 The lack of recent exposure
to other lizards in the history of at least the two Sudan
plated lizards is consistent with the establishment of
latent infection in these species, although recent ac-
quisition from a nonlizard source cannot be formally
excluded.
Comparative sequence analysis of the herpesvirus-
es of reptiles should contribute to a further under-
standing of viral phylogeny and the evolution of this
important family of viruses. Previous phylogenetic
analyses of mammalian herpesviruses suggest that
many elements in the branching patterns of herpes-
viridae are congruent with branching patterns for the
corresponding host species.15 According to molecular
evidence, squamates appear to be the first modern
group of reptiles to have diverged from other reptiles
and birds.8,14 This is the first sequence information on
the herpesviruses of squamates. Further sequence in-
formation may provide a fuller understanding of the
diversity and evolution of the Herpesviridae.
The results of our investigations have identified
sequences representing three novel alphaherpesvi-
ruses in gerrhosaurid lizards. Further investigations
on the evolutionary history of these viruses and the
causal association, if any, with stomatitis in lizards
are warranted.
Acknowledgments: We thank Dr. Isabelle Aubert
for assistance with language translation and Drs.
Dale Smith and Arno Wunschmann for assistance.
Research in the laboratory of VK was supported by
competitive awards from the National Institutes of
Health, the U.S. Department of Agriculture, and the
Minnesota Agricultural Experiment Station.
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Received for publication 30 January 2003