Bowsprit Tortoise Herpes

7/25/2019 Bowsprit Tortoise Herpes

1/8

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)


2/8


3/8

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 co-

evolution 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 hostsfrom 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 associ-

ation 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 ner-

vous system lesions,13,30,36 necrotizing hepati-

tis,2,10,14 and fibro-epithelial tumors.17,33

Despite the significance of herpesviral diseasein tortoises, there has been limited characteriza-

tion of tortoise herpesviruses. Phylogenetic rela-

tionships 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 herpes-

virus was from a disease outbreak in Russian

tortoises (Agrionemys [Testudo] horsfieldii), pan-

cake 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 characteriza-

tion 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 TheTestudo 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 charac-

terized to date appear to belong within the

subfamily Alphaherpesvirinae.23,33,35,38,4143 The ge-

neric name Chelonivirus has been proposed for

the monophyletic clade containing the character-

ized testudinean herpesviruses.35 Comparativesequence data for reptilian herpesviruses avail-

able in GenBank (National Center for Biotech-

nology Information, Bethesda, Maryland),

EMBL (Cambridge, United Kingdom), and Data

From the Zoological Society of San Diego, P.O. Box

120551, San Diego, California 92112, USA (Bicknese);

and the Zoological Medicine Service, Department ofSmall 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

([email protected]).

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

Copyright 2010 by American Association of Zoo Veterinarians

353


4/8

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 her-

pesviruses may be expected to provide a clearerunderstanding of relationships and therefore viral

behavior through evolution.

A wild-caught sub-adult male entered quaran-

tine 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 wereswabbed 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 polymer-

ase gene was performed using previously de-scribed methods.39 The product was resolved on

a 1% agarose gel and purified using the QIAquick

Gel Extraction Kit (Qiagen). To obtain addition-

al 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 automat-

ed 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 com-

parison 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 testudi-

nean herpesviruses present in the available

databases. The highest score obtained was withTortoise herpesvirus 1 (GenBank Accession

No. AB047545), with 85% predicted amino acid

sequence homology, followed by lung-eye-

trachea 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 Full-length 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. TheWag 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 (Phy-

logeny Inference Package, Version 3.66),8 run-

ning 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 plusinvariant rate distributions, and unrooted. The

values for the gamma distribution were taken

from the Bayesian analysis. Iguanid herpesvirus 2

(GenBank Accession No. AY236869) was desig-

nated as the out-group due to its early divergence

from other herpesviruses.26,43 ML analysis found

the most likely tree using the MUSCLE align-

ment and the JTT model of amino acid substi-

tution. 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 herpesvi-

ruses 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 herpesvirus-

es suggest that many elements in the branching

patterns of Herpesviridae are congruent withbranching patterns for the corresponding host

species.16,27,43 This is consistent with host-virus

codivergence. In humans (Homo sapiens), a well-

studied single host species, there are eight

354 JOURNAL OF ZOO AND WILDLIFE MEDICINE


5/8

endemic herpesvirus species. There are approxi-

mately 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 tohypothesize 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 herpes-

virus-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

Figure 1. Bayesian phylogenetic tree of predicted 136142 amino acid partial herpesviral DNA-dependent-

DNA 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 genital-

respiratory herpesvirus (LGRV) (ABV59128), Loggerhead orocutaneous herpesvirus (LOHV) (ABV59131), Lung-

eye-trachea disease virus (LETV) (ABU93815), Macropodid HV3 (EF467663), Psittacid HV1(AY372243), Suid

HV1 (BK001744), Tortoise HV1 (AB047545), Tortoise HV2 (AY916792), and Tortoise HV3 (ABC70832).

BICKNESE ET AL.NOVEL HERPESVIRUS IN ASYMPTOMATIC TORTOISE 355


6/8

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 withfull 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 herpes-virus 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 her-

pesviruses 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 tortoisepathogens 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 implica-

tions of these viruses in other species. Addition-

ally, it is important to characterize by sequencing

any bands generated by consensus PCR. The

authors have seen that the assay used in thisstudy 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.

LITERATURE CITED

1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J.

Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997.

Gapped BLAST and PSI-BLAST: a new generation of

protein database search programs. Nucl. Acids Res. 25:

33893402.

2. Cox, W. R., W. A. Rapley, and I. K. Barker.

1980. Herpesvirus-like infection in a painted turtle

(Chrysemys picta). J. Wildl. Dis. 16: 445449.

3. Davison, A. J., R. Erberle, G. S. Hayward, D. J.

McGeoch, A. C. Minson, P. E. Pellett, B. Roizman, M.

J. Studdert, and E. Thiry. 2005. Family Herpesviridae.

In: Fauquet, C. M., M. A. Mayo, J. Maniloff, E.

Desselberger, and L. A. Ball (eds.). Virus Taxonomy:

Classification and Nomenclature of Viruses. Academic

Press, San Diego, California. Pp. 193212.

4. Drury S. E., R. E. Gough, S. McArthur, and M.

Jessop. 1998. Detection of herpesvirus-like and papil-

lomavirus-like particles associated with diseases of

tortoises. Vet. Rec. 143: 639.

5. Dunowska M., G. J. Letchworth, J. K. Collins,

and J. C. Demartini. 2001. Ovine herpesvirus-2

glycoprotein B sequences from tissues of ruminant

malignant catarrhal fever cases and healthy sheep are

highly conserved. J. Gen. Virol. 82: 27852790.

6. Edgar, R. C. 2004. MUSCLE: multiple sequence

alignment with high accuracy and high throughput.

Nucl. Acids Res. 32: 17921797.

7. Felsenstein, J., 1985. Confidence limits on phy-

logenies: an approach using bootstrap. Evolution 39:

783791.

8. Felsenstein, J., 1989. PHYLIP-phylogeny infer-

ence package. Cladistics 5: 164166.

9. Flynn, J. J., J. A. Finarelli, S. Zehr, J. Hsu, and

M. A. Nedbal. 2005. Molecular phylogeny of the

carnivora (mammalia): assessing the impact of in-

creased sampling on resolving enigmatic relationships.Syst. Biol. 54: 317337.

10. Frye, F. L., L. S. Oshiro, F. R. Dutra, and J. D.

Carney. 1977. Herpesvirus-like infection in two Pacific

pond turtles. J. Am. Vet. Med. Assoc. 171: 882884.

11. Govett, P. D., C. A. Harms, A. J. Johnson, K. S.

Latimer, J. F. X. Wellehan, M. H. Fatzinger, L. S.

Christian, T. R. Kelly, and G. A. Lewbart. 2005. A

novel herpesvirus associated with lymphoid follicular

cloacal inflammation in juvenile alligators (Alligator

mississippiensis). J. Vet. Diagn. Invest. 17: 474479.

12. Harper, P. A., D. A. Hammond, and W. P.

Heuschele. 1982. A herpesvirus-like agent associatedwith a pharyngeal abscess in a desert tortoise. J. Wildl.

Dis. 18: 491494.

13. Heldstab, A., and G. Bestetti. 1989. Herpesvir-

idae causing glossitis and meningoencephalitis in

land tortoises (Testudo hermanni). Herpetopathologia.

1: 59.

14. Hervas, J., P. J. Sanchez-Cordon, F. Chacon de

Lara, L. Carrasco, and J. C. Gomez-Villamandos. 2002.

Hepatitis associated with herpes viral infection in the

tortoise (Testudo horsfieldii). J. Vet. Med. B Infect. Dis.

Vet. Public Health. 49: 111114.

15. Huelsenbeck, J. P., and F. Ronquist. 2001.MRBAYES: Bayesian inference of phylogenetic trees.

Bioinformat. 17: 754755.

16. Jackson, A. P. 2005. The effect of paralogous

lineages on the application of reconciliation analysis by

cophylogeny mapping. Syst. Biol. 54: 127145.



7/8

17. Jacobson, E. R., C. Buergelt, B. Williams, and

R. K. Harris. 1991. Herpesvirus in cutaneous fibropap-

illomas of the green turtle Chelonia mydas. Dis. Aquat.

Org. 12: 16.

18. Jacobson, E. R., S. Clubb, J. M. Gaskin, and C.

Gardiner. 1985. Herpesvirus-like infection in Argentine

tortoises. J. Am. Vet. Med. Assoc. 187: 12271229.

19. Johnson, A. J., A. P. Pessier, J. F. Wellehan, R.

Brown, and E. R. Jacobson. 2005. Identification of a

novel herpesvirus from a California desert tortoise

(Gopherus agassizii). Vet. Microbiol. 111: 107116.

20. Jones, D. T., W. R. Taylor, and J. M. Thornton.

1992. The rapid generation of mutation data matrices

from protein sequences. Comput. Appl. Biosci. 8: 275

282.

21. Kosiol, C., and N. Goldman. 2005. Different

versions of the Dayhoff rate matrix. Mol. Biol. Evol.

22: 193199.

22. Landolfi, J. A., J. F. X. Wellehan, A. J. Johnson,

and M. Kinsel. 2005. Fatal human herpesvirus type 1

infection in a white-handed gibbon (Hylobates lar). J.

Vet. Diagn. Invest. 17: 369371.

23. Marschang, R. E., C. B. Gleiser, T. Papp, A. J.

Pfitzner, R. Bohm, and B. N. Roth. 2006. Comparison

of 11 herpesvirus isolates from tortoises using partial

sequences from three conserved genes. Vet. Microbiol.

117: 258266.

24. Marschang R. E., J. W. Frost, M. Gravendyck,

and E. F. Kaleta. 2001. Comparison of 16 chelonid

herpesviruses by virus neutralization tests and restric-

tion endonuclease digestion of viral DNA. J. Vet. Med.B Infect. Dis. Vet. Public Health. 48: 393399.

25. McArthur, S., S. Blahak, P. Koelle, E. R.

Jacobson, R. E. Marschang, and F. Origgi. 2002.

Chelonian Herpesvirus. J. Herpetol. Med. Surg. 12: 14

31.

26. McGeoch, D. J., and D. Gatherer. 2005.

Integrating reptilian herpesviruses into the family

Herpesviridae. J. Virol. 79: 725731.

27. McGeoch, D. J., F. J. Rixon, and A. J. Davison.

2006. Topics in herpesvirus genomics and evolution.

Virus Res. 117: 90104.

28. Notredame, C., D. G. Higgins, and J. Heringa.2000. T-Coffee: a novel method for fast and accurate

multiple sequence alignment. J. Mol. Biol. 302: 205

217.

29. Oettle, E. E., Y. G. M. Steytler, and M. C.

Williams. 1990. High mortality in a tortoise colony. S.

Afr. J. Wildl. Res. 20: 2125.

30. Origgi, F. C., C. H. Romero, D. C. Bloom, P. A.

Klein, S. J. Gaskin, S. J. Tucker, and E. R. Jacobson.

2004. Experimental transmission of herpesvirus in

Greek tortoises (Testudo graeca). Vet. Pathol. 41: 50

61.

31. Ostrowski, S. R., M. J. Leslie, T. Parrott, S.Abelt, and P. E. Piercy. 1998. B-virus from pet macaque

monkeys: an emerging threat in the United States?

Emerg. Infect. Dis. 4: 117121.

32. Pinkerton M. E., J. F. X. Wellehan, A. J.

Johnson, A. L. Childress, S. D. Fitzgerald, and M. J.

Kinsel. 2008. Columbid herpesvirus-1 in two Coopers

hawks (Accipiter cooperii) with fatal inclusion body

disease. J. Wildl. Dis. 44: 622628.

33. Quackenbush, S. L., T. M. Work, G. H. Balazs,

R. N. Casey, J. Rovnak, A. Chaves, L. duToit, J. D.

Baines, C. R. Parrish, P. R. Bowser, and J. W. Casey.

1998. Three closely related herpesviruses are associated

with fibropapillomatosis in marine turtles. Virology

246: 392399.

34. Rebell, G., A. Rywlin, and H. Haines. 1975. A

herpesvirus-type agent associated with skin lesions of

green sea turtles in aquaculture. Am. J. Vet. Res. 36:

12211224.

35. Stacy, B. A., J. F. X. Wellehan, A. M. Foley, S.

S. Coberley, C. A. Manire, N. S. Mettee, M. M.

Garner, and E. R. Jacobson. 2008. Two novel

herpesviruses associated with disease in wild Atlantic

loggerhead sea turtles (Caretta caretta). Vet. Microbiol.

126: 6373.

36. Teifke, J. P., C. V. Lohr, R. E. Marschang, N.

Osterreider, and H. Posthaus. 2000. Detection of

Chelonid Herpesvirus DNA by nonradioactive in situ

hybridization in tissues from tortoises suffering from

stomatitis-rhinitis complex in Europe and North

America. Vet. Pathol. 37: 377385.

37. Thompson, J. D., D. G. Higgins, and T. J.

Gibson. 1994. CLUSTAL W: improving the sensitivity

of progressive multiple sequence alignments through

sequence weighting, position specific gap penalties

and weight matrix choice. Nucl. Acids Res. 22: 4673

4680.38. Une, Y., M. Murakami, K. Uemura, H.

Fujitani, T. Ishibashi, and Y. Nomura. 2000. Polymer-

ase chain reaction (PCR) for the detection of herpes-

virus in tortoises. J. Vet. Med. Sci. 62: 905907.

39. Van Devanter, D. R., P. Warrener, L. Bennett,

E. R. Schultz, S. Coulter, R. L. Garber, and T. M.

Rose. 1996. Detection and analysis of diverse herpes-

viral species by consensus primer PCR. J. Clin.

Microbiol. 34: 16661671.

40. Veerassamy, S., A. Smith, and E. R. Tillier.

2003. A transition probability model for amino acid

substitutions from blocks. J. Comput. Biol. 10: 9971010.

41. Wellehan, J. F. X., A. J. Johnson, K. S. Latimer,

D. P. Whiteside, G. J. Crawshaw, C. J. Detrisac, S. P.

Terrell, D. J. Heard, A. Childress, and E. R. Jacobson.

2005. Varanid herpesvirus 1: a novel herpesvirus

associated with proliferative stomatitis in green tree

monitors (Varanus prasinus). Vet. Microbiol. 105: 83

92.

42. Wellehan, J. F. X., D. K. Nichols, L. L. Li,

and V. Kapur. 2004. Three novel herpesviruses

associated with stomatitis in Sudan plated lizards

(Gerrhosaurus major) a nd a b la ck -lin ed p la te dlizard (Gerrhosaurus nigrolineatus). J. Zoo Wildl. Med.

35: 5054.

43. Wellehan, J. F. X., J. L. Jarchow, C. Reggiardo,

and E. R. Jacobson. 2003. A novel herpesvirus

associated with hepatic necrosis in a San Esteban

BICKNESE ET AL.NOVEL HERPESVIRUS IN ASYMPTOMATIC TORTOISE 357


8/8

chuckwalla (Sauromalus varius). J. Herpetol. Med.

Surg. 13: 1519.

44. Whelan, S., and N. Goldman. 2001. A general

empirical model of protein evolution derived from

multiple protein families using a maximum-likelihood

approach. Mol. Biol. Evol. 18: 691699.

Received for publication 30 October 2009


Documents

Bowsprit Tortoise Herpes