Journal of Zoo and Wildlife Medicine 41(2): 353358, 2010

Copyright 2010 by American Association of Zoo Veterinarians

A NOVEL HERPESVIRUS OF THE PROPOSED GENUS

CHELONIVIRUS FROM AN ASYMPTOMATIC BOWSPRIT
TORTOISE (CHERSINA ANGULATA)
Elizabeth J. Bicknese, M.P.V.M., D.V.M., April L. Childress, and James F. X. Wellehan, Jr., M.S.,
D.V.M., Dipl. A.C.Z.M., Dipl. A.C.V.M. (Virology, Bacteriology/Mycology)

Abstract: A wild-caught Bowsprit tortoise (Chersina angulata) was received into quarantine and appeared
clinically normal. Oral swabs for consensus herpesvirus polymerase chain reaction (PCR) and sequencing were
obtained during routine quarantine, and a novel herpesvirus was identified. Comparative sequence analysis shows
that this virus is a member of the subfamily Alphaherpesvirinae in the proposed genus Chelonivirus. Host/virus coevolution appears to be common amongst herpesviruses and their hosts, and the most significant disease is typically
seen when herpesviruses jump to related host species. Previous studies have found some diversity of herpesviruses in
tortoises. This report expands the number of known herpesviruses of tortoises. It is reasonable to expect that there
will be significantly different clinical consequences of different tortoise herpesviruses in different species, and that
identification of host/virus relationships will aid in clinical management of tortoise collections. Further work is
needed to determine the clinical implications of this and other tortoise herpesviruses in different tortoise species.
Key words: Alphaherpesvirinae, bowsprit tortoise, Chersina angulata, consensus PCR, polymerase chain
reaction, Herpesviridae, tortoise herpesvirus 4.

BRIEF COMMUNICATION
Herpesviruses were first reported from hosts in
the order Testudines (turtles and tortoises) in
1975, when herpesvirus-like particles were seen
on electron microscopy in cutaneous lesions from
green turtles (Chelonia mydas).34 Within hosts
from the family Testudinidae (tortoises), the
presence of herpesvirus-like particles was first
reported in 1982 from a California desert tortoise
(Gopherus agassizii).12 Lesions reported in association with herpesviruses in Testudines include
proliferative and ulcerative stomatitis,12,18,19,24,35
respiratory tract infections,19,35 conjunctivitis,30,35
dermatitis,34,35 genital ulcerations,35 central nervous system lesions,13,30,36 necrotizing hepatitis,2,10,14 and fibro-epithelial tumors.17,33
Despite the significance of herpesviral disease
in tortoises, there has been limited characterization of tortoise herpesviruses. 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.3 The
first genetic characterization of a tortoise herpesFrom the Zoological Society of San Diego, P.O. Box
120551, San Diego, California 92112, USA (Bicknese);
and the Zoological Medicine Service, Department of
Small Animal Clinical Sciences, College of Veterinary
Medicine, University of Florida, P.O. Box 100126,
Gainesville, Florida 32610, USA (Childress, Wellehan).
Correspondence should be directed to Dr. Bicknese
(bbicknese@sandiegozoo.org).

virus was from a disease outbreak in Russian

tortoises (Agrionemys [Testudo] horsfieldii), pancake tortoises (Malacochersus tornieri), and
Greek tortoises (Testudo graeca).24 This virus is
hereafter referred to as Tortoise herpesvirus 1
(THV1). A California desert tortoise isolate was
later shown to be distinct to a degree seen
between herpesvirus species, and is hereafter
referred to as Tortoise herpesvirus 2 (THV2).19
Serologic and restriction digestion differences
were shown between an A. horsfieldii isolate
and 15 other isolates from tortoises in the genera
Testudo and Agrionemys.24 Genetic characterization found the isolates seen in the majority of
Testudo were distinct to a degree seen between
herpesvirus species, and the A. horsfieldii isolate
was identical to the Une et al. isolate as well as
another A. horsfieldii isolate.23 The Testudo virus
is hereafter referred to as Tortoise herpesvirus 3
(THV3). Previous recovery of sequence identical
to THV3 from an American alligator (Alligator
mississippiensis) may have represented laboratory
contamination.11
All reptilian herpesviruses sufficiently characterized to date appear to belong within the
subfamily Alphaherpesvirinae.23,33,35,38,4143 The generic name Chelonivirus has been proposed for
the monophyletic clade containing the characterized testudinean herpesviruses.35 Comparative
sequence data for reptilian herpesviruses available in GenBank (National Center for Biotechnology Information, Bethesda, Maryland),
EMBL (Cambridge, United Kingdom), and Data

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Bank of Japan (Mishima, Shizuoka, Japan) is

very limited. The availability of more complete
data sets for comparison results in greater
phylogenetic resolution,9 so identification and
characterization of additional testudinean herpesviruses may be expected to provide a clearer
understanding of relationships and therefore viral
behavior through evolution.
A wild-caught sub-adult male entered quarantine at the San Diego Zoo (SDZ) in July 2007
from a South African source. Bowsprit tortoises
(Chersina angulata) are native to southwestern
South Africa and the extreme southern edge of
Namibia. The animal was in apparent good
health and body condition and adapted quickly
to the captive diet. Four wk after entry, his
oropharyngeal and choanal slit areas were
swabbed with a sterile rayon-tipped applicator,
placed in a sterile cryo-tube, and shipped to
the University of Florida for herpesvirus
polymerase chain reaction (PCR) testing. No
lesions or oral pathology were noted at the time
of sampling.
DNA was extracted from the sample using the
DNeasy Kit (Qiagen, Valencia, California, 91355,
USA). Nested PCR amplification of a partial
sequence of the DNA-dependent-DNA polymerase gene was performed using previously described methods.39 The product was resolved on
a 1% agarose gel and purified using the QIAquick
Gel Extraction Kit (Qiagen). To obtain additional sequence, the second round was altered to use
primers DFA and IYG.39 Direct sequencing was
performed using the Big-Dye Terminator Kit
(Applied Biosystems, Foster City, California
94404, USA) and analyzed on ABI 377 automated DNA sequencers. Primer sequences were
edited out prior to further analyses. Initial
PCR amplification of partial sequence of the
DNA-dependent-DNA polymerase gene yielded
a 181 base pair (bp) product (after editing).
Additional sequence for phylogenetic comparison brought this to 423 bp. Sequences were
submitted to GenBank (GenBank Accession
No. GQ222415).
The sequence was compared with those in the
databases of GenBank, EMBL, and the Data
Bank of Japan using TBLASTX.1 The sequence
was similar to, but distinct from, other testudinean herpesviruses present in the available
databases. The highest score obtained was with
Tortoise herpesvirus 1 (GenBank Accession
No. AB047545), with 85% predicted amino acid
sequence homology, followed by lung-eyetrachea disease-associated herpesvirus (GenBank

Accession No. EU006876), with 74% predicted

amino acid sequence homology.
Predicted homologous 136142 amino acid
sequences of herpesviral DNA-dependent-DNA
polymerase were aligned using three methods:
ClustalW,37 T-Coffee,28 and MUSCLE.6 Fulllength sequences were not available for THV2
and THV3, so the available 60 homologous
amino acids were used along with ambiguities.
Bayesian analyses of each alignment were
performed using MrBayes 3.115 with gamma
distributed rate variation and a proportion of
invariant sites, and mixed amino acid substitution
models. The first 10% of 1 million iterations were
discarded as a burn in. The analysis showed the
greatest harmonic mean of estimated marginal
likelihoods using the MUSCLE alignment. The
Wag model of amino acid substitution was found
to be most probable with a posterior probability
of 1.00.44 Figure 1 shows the Bayesian tree using
the MUSCLE alignment.
Maximum likelihood (ML) analyses of each
alignment were performed using PHYLIP (Phylogeny Inference Package, Version 3.66),8 running each alignment in proML with amino acid
substitution models JTT,20 PMB,40 and PAM21
further set with global rearrangements, five
replications of random input order, gamma plus
invariant rate distributions, and unrooted. The
values for the gamma distribution were taken
from the Bayesian analysis. Iguanid herpesvirus 2
(GenBank Accession No. AY236869) was designated as the out-group due to its early divergence
from other herpesviruses.26,43 ML analysis found
the most likely tree using the MUSCLE alignment and the JTT model of amino acid substitution. These parameters were then used for
bootstrap analysis to test the strength of the tree
topology (200 resamplings).7 The bootstrap
values from ML analysis are shown on the
Bayesian tree in Figure 1.
The genetic distance seen between the bowsprit
tortoise virus and other characterized herpesviruses is consistent with placement of this virus as
a novel species and will hereafter be referred to as
Tortoise herpesvirus 4 (THV4). The phylogenetic
analysis shows that THV4 clusters within the
proposed genus Chelonivirus.
Previous phylogenetic analyses of herpesviruses suggest that many elements in the branching
patterns of Herpesviridae are congruent with
branching patterns for the corresponding host
species.16,27,43 This is consistent with host-virus
codivergence. In humans (Homo sapiens), a wellstudied single host species, there are eight

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355

Figure 1. Bayesian phylogenetic tree of predicted 136142 amino acid partial herpesviral DNA-dependentDNA polymerase sequences based on MUSCLE alignment. Bayesian posterior probabilities of branchings as
percentages are in bold, and maximum likelihood (ML) bootstrap values for branchings based on 200 re-samplings
are given below. Iguanid HV2 (GenBank Accession No. AY236869) was used as the outgroup. Herpesviral genera
are delineated by thin brackets, and subfamilies are delineated by thick brackets. A multifurcation is marked with
an arc. Tortoise herpesvirus 4 is bolded. Sequences retrieved from Gen Bank include Callitrichine HV3
(AF319782), Cercopithecine HV1 (AF533768), Cercopithecine HV5 (AY117754), Fibropapillomatosis HV
(AY644454), Equid HV1 (AY665713), Gallid HV1 (AF168792 ), Gallid HV2 (DQ530348), Gallid HV3
(AB049735), Human HV1 (X14112), Human HV2 (CAB06755), Human HV6 (X83413), Loggerhead genitalrespiratory herpesvirus (LGRV) (ABV59128), Loggerhead orocutaneous herpesvirus (LOHV) (ABV59131), Lungeye-trachea disease virus (LETV) (ABU93815), Macropodid HV3 (EF467663), Psittacid HV1(AY372243), Suid
HV1 (BK001744), Tortoise HV1 (AB047545), Tortoise HV2 (AY916792), and Tortoise HV3 (ABC70832).

endemic herpesvirus species. There are approximately 300 extant species in the order Testudines,
and approximately 45 extant species in the family
Testudinidae. Given the apparent prevalence of
herpesviral/host codivergence, it is reasonable to
hypothesize that the four characterized tortoise
herpesviruses represent a small fraction of
tortoise herpesviral diversity. Uncharacterized
herpesvirus infections have been found in Chaco

tortoises (Chelonoidis [Geochelone] chiliensis) and

leopard tortoises (Psammobates [Geochelone]
pardalis).4,18 In addition, uncharacterized herpesvirus-like inclusions were seen in a padloper
(Homopus areolatus) that died with stomatitis,
hepatosis, and pneumonia; this animal was part
of a mortality event in a mixed-species collection
of South African tortoises that included bowsprit
tortoises.29

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The clinical implications of THV4 in Bowsprit

tortoises and other species are currently not
known but may be significant. For this reason,
this male bowsprit tortoise was not incorporated
into the SDZ collection but placed with a private
collector with extensive tortoise experience with
full disclosure. The animal was alive and healthy
at the time of this manuscript preparation.
Herpesvirus infections often cause subclinical or
mild disease in the natural host species and fatal
disease in aberrant species. There are numerous
examples of herpesviruses causing more severe
disease in aberrant hosts.5,22,31,32 There is greater
divergence seen among the tortoise herpesviruses
than among the members of the genus Simplex
virus included in this analysis. Of the simplex
viruses included here, in humans, human herpesvirus 1 primarily causes mild cold sores, human
herpesvirus 2 primarily causes genital lesions, and
Cercopithecine herpesvirus 1 is rapidly fatal. It is
reasonable to hypothesize that similar clinical
differences may exist for different tortoise herpesviruses in different species.
Due to the knowledge gaps with tortoise
herpesviruses, testing tortoises for herpesviruses
is recommended followed by the subsequent
characterization of any viruses found. From an
individual animal perspective, several tortoise
pathogens have clinical signs that overlap, so it
is important to perform appropriate laboratory
testing to differentiate tortoise herpesviruses,
iridoviruses, and mycoplasma infections.25 From
a population management perspective, it is
crucial to know which viruses are endemic in
each tortoise species and the pathogenic implications of these viruses in other species. Additionally, it is important to characterize by sequencing
any bands generated by consensus PCR. The
authors have seen that the assay used in this
study amplify non-target DNA, which would be
misinterpreted as a positive without sequencing.
Furthermore, there is very limited clinical utility
to knowing that there is a herpesvirus present
without knowing which herpesvirus it is. Until
further data are available on the diversity and
clinical significance of the tortoise herpesviruses,
the mixing of species should be minimized.
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Received for publication 30 October 2009