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Characterization of an erythrocytic virus in the

family Iridoviridae from a peninsula

ribbon snake (Thamnophis sauritus sackenii)

James F.X. Wellehan Jr.a,*, Nicole I. Strik b, Brian A. Stacy a,April L. Childress a, Elliott R. Jacobson a, Sam R. Telford Jr.c

a Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USAb Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA

c The Florida Museum of Natural History, University of Florida, Gainesville, FL 32610, USA

Received 10 January 2008; received in revised form 3 March 2008; accepted 6 March 2008

Abstract

A wild peninsula ribbon snake (Thamnophis sauritus sackenii) in Florida was found to have hypochromic erythrocytes

containing two different types of inclusions: purple granular inclusions, and pale orange or pink crystalloid inclusions that were

round, oval, rectangular, or hexagonal in shape. Transmission electron microscopy revealed hexagonal or pleomorphic,

homogenous inclusions and enveloped particles morphologically consistent with a member of the Iridoviridae. Histopathology

of the animal revealed necrotizing hepatitis consistent with sepsis. Consensus PCR was used to amplify a 628-bp region of

iridoviral DNA-dependent DNA polymerase. Bayesian and maximum likelihood phylogenetic analysis found that this virus was

distinct from other known iridoviral genera and species, and may represent a novel genus and species.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Peninsula ribbon snake; Thamnophis sauritus; Iridoviridae; Thamnophis sauritus erythrocytic virus; Snake erythrocytic virus;

Pirhemocyton; Toddia

1. Introduction

The family Iridoviridae is divided into five

genera: Chloriridovirus, Iridovirus, Lymphocysti-

virus, Megalocytivirus, and Ranavirus (Chinchar

et al., 2005). Chloriridovirus and Iridovirus are

primarily viruses of invertebrates, although Iridovirus

infection has been reported in lizards (Just et al., 2001;

Weinmann et al., 2007). Lymphocystivirus, Mega-

locytivirus, and Ranavirus are primarily fish viruses,

although members of the genus Ranavirus are also

significant pathogens in reptiles and amphibians

(Hyatt et al., 2002; Marschang et al., 1999; Rojas

et al., 2005).

www.elsevier.com/locate/vetmic

Available online at www.sciencedirect.com

Veterinary Microbiology 131 (2008) 115–122

* Corresponding author at: Zoological Medicine Service, Depart-

ment of Small Animal Clinical Sciences, College of Veterinary

Medicine, University of Florida, Gainesville, FL 32610, USA.

Tel.: +1 352 392 2226; fax: +1 352 392 4877.

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

(J.F.X. Wellehan Jr.).

0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2008.03.003

Author's personal copy

Cytoplasmic inclusions in erythrocytes of squa-

mates that were initially described as Toddia sp. and

Pirhemocyton sp. protozoal hemoparasites were later

shown to be consistent with iridoviral inclusions on

electron microscopy (De Sousa and Weigl, 1976;

Stehbens and Johnson, 1966). It has been proposed

that Pirhemocyton sp. should be referred to as lizard

erythrocytic virus (Telford and Jacobson, 1993).

Erythrocytic inclusions ultrastructurally and bio-

chemically consistent with iridoviruses have also

been identified in amphibians and fish (Bernard et al.,

1968; Gruia-Gray et al., 1989; Reno and Nicholson,

1981). None of these viruses have been studied

genetically, thus phylogenetic relationships with other

iridoviruses remain unknown. This study includes

hematologic and pathologic examination of an

erythrocytic iridovirus-infected snake, ultrastructural

and genetic characterization of the virus, and

phylogenetic comparison with other known irido-

viruses.

2. Materials and methods

2.1. Animal

A wild peninsula ribbon snake (Thamnophis

sauritus sackenii) was collected in Dixie County,

Florida in April 2006 as part of a survey of reptilian

hemoparasites.

2.2. Blood samples

An ante mortem blood sample was collected via

cardiocentesis. Blood films were prepared immedi-

ately without anticoagulant and air-dried. Aliquots

were taken for ultrastructural and PCR analysis.

2.3. Light microscopic examination

Blood films were fixed in methanol and stained

with Wright–Giemsa for morphologic evaluation

of erythrocytes, leukocytes and thrombocytes,

and differential leukocyte counts. A leukocyte

estimate was performed under 50� magnification

by counting all leukocytes in 20 fields. The

average number of cells per field was multiplied

by 2500 to obtain the estimated count. 200

leukocytes were counted for the differential leuko-

cyte determination.

2.4. Transmission electron microscopic

examination

Twenty microlitres of whole blood was fixed

immediately after collection with 40 ml 2.5% glutar-

aldehyde in 0.1 M phosphate buffer (pH 7.4),

centrifuged, and immediately cooled at 4 8C. After

24 h, the sample was washed in 0.1 M phosphate

buffer, post-fixed in 2% osmium tetroxide for 1 h,

dehydrated in acetone and embedded in a Spurr’s

mixture. One-micrometre toluidine blue-stained sec-

tions were evaluated to select areas for thin sectioning.

Ultrathin sections, stained with uranyl acetate and lead

citrate, were examined on a Philips CM 100 electron

microscope and images were acquired with an HR

digital camera (Advanced Microscopy Techniques,

Danvers, MA).

2.5. Gross and histopathological examination

This snake was designated for a museum collec-

tion, thus postmortem examination was limited to

gross and histopathological examination of skin, heart,

lungs, liver, gall bladder, pancreas, kidneys, ovaries,

gastrointestinal tract and adrenal glands. For histo-

pathology, tissues were preserved in 10% neutral

phosphate buffered formalin, processed by routine

methods into paraffin blocks, and 5 mm sections were

stained with hematoxylin and eosin.

2.6. PCR amplification and sequencing

DNA was extracted from a blood sample using the

DNA mini kit (Qiagen, Valencia, CA). PCR ampli-

fication of a partial sequence of the DNA-dependent

DNA polymerase gene was performed using pre-

viously described methods (Hanson et al., 2006). The

target region corresponds to the area between motifs A

and C of the polymerase domain, which are thought to

be involved with binding and positioning DNA within

the active site (Knopf, 1998). A Ranavirus from an

eastern box turtle (Terrapene carolina) was used as the

positive control. As it was expected that blood

contamination of tissues would have resulted in

inability to easily distinguish infection in different cell

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 131 (2008) 115–122116

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types, additional tissues were not tested by PCR.

Products were resolved on 1% agarose gels and bands

in the size range expected for viral polymerases were

excised and purified using the QIAquick gel extraction

kit (Qiagen). Direct sequencing was performed using

the big-dye terminator kit (PerkinElmer, Branchburg,

NJ) and analyzed on ABI 377 automated DNA

sequencers at the University of Florida Center for

Mammalian Genetics DNA Sequencing Facilities.

The product was sequenced in both directions. Primer

sequences were edited out prior to further analyses.

2.7. Phylogenetic analysis

The sequences were compared to those in GenBank

(National Center for Biotechnology Information,

Bethesda, MD), EMBL (Cambridge, United King-

dom), and Data Bank of Japan (Mishima, Shizuoka,

Japan) databases using TBLASTX (Altschul et al.,

1997). Predicted homologous 190–268 amino acid

sequences of viral DNA-dependent DNA polymerase

were aligned using three methods; ClustalW (Thomp-

son et al., 1994), T-Coffee (Notredame et al., 2000),

and MUSCLE (Edgar, 2004).

Bayesian analyses of each alignment were per-

formed using MrBayes 3.1 (Ronquist and Huelsen-

beck, 2003) with gamma distributed rate variation and

a proportion of invariant sites, and mixed amino acid

substitution models. The first 10% of 1,000,000

iterations were discarded as a burn in.

Maximum likelihood (ML) analyses of each

alignment were performed using PHYLIP (Phylogeny

Inference Package, Version 3.66) (Felsenstein, 1989),

running each alignment in proml with amino acid

substitution models JTT (Jones et al., 1992), PMB

(Veerassamy et al., 2003), and PAM (Kosiol and

Goldman, 2005) further set with global rearrange-

ments, five replications of random input order, less

rough, gamma plus invariant rate distributions, and

unrooted. The value for the alpha of the gamma

distribution was taken from the Bayesian analysis.

Spodoptera Ascovirus (GenBank accession no.

AAC54632), a member of the family Ascoviridae,

was designated as the outgroup due to the close

relationship of the Ascoviridae to the Iridoviridae

(Stasiak et al., 2003). The combination of alignment

producing the most likely tree was then used to create

data subsets for bootstrap analysis to test the strength

of the tree topology (200 re-samplings) (Felsenstein,

1985), which was analyzed using the amino acid

substitution model producing the most likely tree.

3. Results

3.1. Light microscopic examination

The most prominent morphologic findings in

peripheral blood were observed in the erythroid cell

line (Fig. 1). Approximately 60% of erythrocytes were

severely hypochromic and exhibited moderate aniso-

cytosis and frequent polychromasia. Occasional

rubricytes and rubriblasts were noted. Ninety-five

percent of hypochromic erythrocytes and occasional

normal erythrocytes contained two types of intracy-

toplasmic inclusions, usually concurrently. One type

was a crystalloid inclusion that was pale orange or

pink, and round, oval, rectangular or hexagonal. Up to

three of these inclusions were observed in individual

erythrocytes. The second type of inclusion was

composed of aggregates of dark purple granular

material suggestive of viral origin embedded in a paler

purple background. The morphology of these two

types of inclusions were highly suspicious of

Iridoviral infection. A count of 500 erythrocytes

found that 61% contained inclusions, of which 53%

contained both crystalloid and granular material, 1%

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 131 (2008) 115–122 117

Fig. 1. Photomicrograph of hypochromic erythrocytes with crystal-

loid inclusions (arrowhead) of variable shapes and with inclusions

composed of aggregates of dark purple granular material (arrow)

embedded in a paler purple surrounding background.

Author's personal copy

contained only granular particles, and 7% only

crystalloid. A low number of erythrocytes also

contained hemogregarine gamonts.

The leukocyte estimated count was 22,500 ml�1,

with the following differential: heterophils 400 ml�1

(2%), lymphocytes 7200 ml�1 (32%), monocytes

3800 ml�1 (17%), basophils 200 ml�1 (1%) and

azurophils 10,900 ml�1 (48%). Frequent lymphocytes,

azurophils and monocytes were reactive. Occasional

azurophils and monocytes contained phagocytized

erythrocytes. Heterophils were severely toxic and left-

shifted. The thrombocytes appeared adequate in

number.

Since the amount of blood was insufficient to

perform a CBC, only morphologic assessment of the

blood film could be used for the following interpreta-

tion: the light microscopic findings were consistent with

a mild to moderate macrocytic hypochromic anemia

with strong evidence of regeneration and possible

secondary immune-mediated destruction of erythro-

cytes. The very low number of hemogregarine gamonts

within erythrocytes was consistent with an incidental

finding. The leukogram, with moderate heteropenia,

toxicity and left shift, azurophilia and monocytosis, was

consistent with severe chronic inflammation.

3.2. Transmission electron microscopic

examination

Ultrastructural examination of erythrocytes

revealed enveloped particles with an approximately

200-nm diameter, an electron dense core, and sharp

icosahedral outlines (Fig. 2). Frequent erythrocytes

also contained one or two large variably sized,

hexagonal or pleomorphic, homogenous inclusions,

consistent with crystalloid inclusions observed by

light microscopy. These inclusions are probably

comprised of cellular and viral byproducts of lipids

and proteins (Johnsrude et al., 1997).

3.3. Gross and histopathological examination

The snake was in poor nutritional condition as

indicated by severe atrophy of skeletal muscle and the

fat bodies. Other gross findings were limited to a focal

area of dermatitis in the ventrum. On histopathology,

rare intracytoplasmic inclusions were observed within

erythrocytes, consistent with those seen in the blood

films, and were not observed in any other cell type.

There was evidence of erythrophagocytosis in many

Kupffer cells and circulating mononuclear cells. Major

histopathological findings included a severe, multi-

focally extensive, subacute, necrotizing hepatitis

characterized by intense infiltration of the liver by

heterophils and fewer macrophages, and early granu-

loma formation. A small intraventricular fibrin throm-

bus also was present. These findings are consistent with

septicemia; however, no bacterial cultures were

collected. Several different parasites were observed,

including intrapulmonary nematodes (Rhabdias sp.),

encysted cestodes and pancreatic and alimentary

nematodes associated with granuloma formation.

3.4. PCR amplification and sequencing

PCR amplification resulted in a 628-bp product

when primer sequences were edited out. This virus

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 131 (2008) 115–122118

Fig. 2. Transmission electron photomicrograph of an erythrocyte

(nucleus on lower left) with numerous intracytoplasmic enveloped

particles of up to 200 nm in diameter. These icosahedral particles

exhibit an electron dense core and sharp borders. The two large

homogenous structures at the top and right are consistent with

crystalloid inclusions. Bar = 500 nm.

Author's personal copy

was distinct from other known viruses, and is hereafter

referred to as Thamnophis sauritus erythrocytic virus

(TsEV). Sequence was submitted to GenBank under

accession number EF608450.

3.5. Phylogenetic analysis

TBLASTX results for ElHV3 and ElHV4 showed

the highest score with a Chinese isolate of Lympho-

cystis disease virus (GenBank accession #

AY380826), an Iridovirus in the genus Lymphocys-

tivirus.

Bayesian phylogenetic analysis showed the great-

est harmonic mean of estimated marginal likelihoods

using the MUSCLE alignment (Fig. 3). Branching

patterns did not differ between analyses of different

alignments. The Blosum62 model of amino acid

substitution was found to be most probable with a

posterior probability of 0.989 (Henikoff and Henikoff,

1992). The Bayesian tree using the MUSCLE

alignment is shown in Fig. 4.

ML analysis found the most likely tree resulted

from the MUSCLE alignment and the PMB model of

amino acid substitution, and these parameters were

used for bootstrap analysis. Branching patterns did not

differ from the Bayesian analysis. Bootstrap values

from ML analysis are shown on the Bayesian tree.

4. Discussion

The clinical significance of squamate erythrocytic

viruses is not well understood. In one case report in a

fer de lance (Bothrops moojeni), infection was

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 131 (2008) 115–122 119

Fig. 3. Alignment of predicted partial viral DNA-dependent DNA polymerase amino acid sequences created using MUSCLE. Genera are

separated by lines, and Spodoptera Ascovirus, the only non-Iridovirus, is shaded. Thamnophis sauritus erythrocytic virus 3 is in bold. Sequences

retrieved from Gen Bank include Lymphocystis disease virus—China isolate (GenBank accession no. YP_073706), Lymphocystis disease

virus—Mississippi isolate (ABA41590), Grouper Iridovirus (AAV91098), Frog virus 3 (YP_031639), Largemouth Bass virus (ABA41591), Red

Sea Bream Iridovirus (O70736), Infectious spleen and kidney necrosis virus (AAL98743), Aedes taeniorhynchus iridescent Virus (ABF82150),

Iridovirus RMIV (CAC84133), Iridovirus IV31 (CAC19196), Chilo Iridescent virus (AAD48150), and Spodoptera Ascovirus (AAC54632).

Author's personal copy

associated with a marked anemia (Johnsrude et al.,

1997). Anemia has also been documented in

Australian geckos, African agamids and a skink

(Paperna and Alves de Matos, 1993). In experimental

infections of Lacerta schreiberi and Iberolacerta

(Lacerta) monticola lizards with an erythrocytic virus,

many infections were not clinically apparent, but I.

monticola infected at colder temperatures (2–15 8C)

also developed leukocytic inclusions and often died

(Alves de Matos et al., 2002). At necropsy, these

animals also had inclusions in endothelial cells and

hepatocytes. Temperature has also been shown to play

a significant role in development of disease in

infections with iridoviruses from the genera Ranavirus

(Rojas et al., 2005) and Megalocytivirus (Oh et al.,

2006). In the case presented here, the infected snake

clearly was not healthy as evidenced by the poor

nutritional state; however, the exact role of iridoviral

infection is uncertain. At the time of year the animal

was collected, the snake had recently been exposed to

cooler temperatures. The character of the blood film

suggested a macrocytic hypochromic anemia with

regeneration, and possible secondary immune-

mediated destruction of erythrocytes, as seen with

clinical disease attributed to erythrocytic iridoviruses

in other reptiles. Probable septicemia, as evidenced by

the hematologic and histopathologic findings, was a

major confounding disease process in this snake.

Erythrocytic viruses of snakes were formerly

known as Toddia sp., and the erythrocytic viruses of

lizards were formerly Pirhemocyton sp. They have

been reported to be distinct from each other on light

microscopy, with the erythrocytic viruses of lizards

reported to form albuminoid vacuoles which stain

differently from the crystalloid structures that are seen

in erythrocytic viruses of snakes (Telford and

Jacobson, 1993). However, there has been no previous

molecular characterization of erythrocytic viruses of

reptiles or amphibians, and it is not clear whether these

characteristics are truly phylogenetically informative.

Evolutionarily, snakes branched off in the middle of

the squamates, so if snakes are considered separate

from other squamata, then lizards are not a mono-

phyletic group (Vidal and Hedges, 2005). Agamid

lizards and snakes are more closely related to each

other than either is to a gecko (Vidal and Hedges,

2005), and all have been shown to be susceptible to

erythrocytic iridoviruses (Paperna and Alves de

Matos, 1993). Lack of distinct host monophyly would

argue against segregation of the former Toddia and

Pirhemocyton. Additionally, while most large DNA

viruses tend to be fairly host restricted, the Iridoviridae

tend to have broader host ranges. Further sequence

characterization of additional erythrocytic irido-

viruses is indicated to determine whether erythrocytic

iridoviruses of snakes and lizards are distinct from

each other and from erythrocytic iridoviruses of

amphibians and fish.

Further work remains to be done on the ecology of

erythrocytic iridoviruses. The location of this virus in

erythrocytes raises the possibility of blood-borne

transmission. Amongst the large DNA viruses, only

the Iridoviridae and Asfarviridae contain viruses that

have been found capable of infecting both arthropods

and tetrapods. It is possible that hematophagous

arthropods may play a significant role in virus

transmission. If so, prevalence of disease is likely to

vary depending on arthropod prevalence as well as

temperature and other factors. Of 432 snakes in

Florida surveyed for hemoparasites by light micro-

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 131 (2008) 115–122120

Fig. 4. Bayesian phylogenetic tree of predicted 190–268 amino acid

partial iridoviral DNA-dependent DNA polymerase sequences

based on MUSCLE alignment. Bayesian posterior probabilities of

branchings as percentages are in bold, and ML bootstrap values for

branchings based on 200 re-samplings are given to the right or

below. Spodoptera Ascovirus was used as the outgroup. Iridoviral

genera are delineated by brackets. Areas of trifurcation are marked

by arcs. Thamnophis sauritus erythrocytic virus is bolded. GenBank

accession numbers are given in the legend to Fig. 3.

Author's personal copy

scopy from 1970 to 2008, only the ribbon snake in this

report was found to be infected. In other studies, up to

58% of some examined populations screened by light

microscopy have been found to be positive (Smith

et al., 1994). Future studies using PCR-based methods

may be more sensitive and have differing results.

Sequence analysis of TsEV finds that it is very

distinct from other species in the Iridoviridae, and

consistent with a novel species. It is not possible

based on this analysis to determine whether TsEV is

most closely related to Lymphocystivirus or Rana-

virus. TsEV is distinct from the recognized genera

of iridoviruses, and may potentially represent a new

genus. Additional sequence of TsEV and other

erythrocytic iridoviruses will help to determine

this.

Sequence-based data is significantly more phylo-

genetically informative than dated methods such as

restriction fragment length polymorphism (RFLP),

which is still in common use in the Iridoviridae (Qiao

et al., 2006). Although the major capsid protein has

often been used for initial characterization of

iridoviruses, viral polymerases have been repeatedly

demonstrated to be good choices for long-range

phylogeny (Attoui et al., 2002; Gonzalez et al., 2003;

Knopf, 1998). Currently, ICTV criteria for species

designation are preliminary, and include analysis of

the major capsid protein, RFLP, dot blot hybridization,

and serology (Chinchar et al., 2005). When looking at

evolutionary relationships of more distantly related

organisms, the continued accrual of mutations

resulting in homoplasy can diminish the ability to

correctly resolve phylogeny, making a rapidly mutat-

ing gene a poor choice. Genes for which there is strong

negative selection will have fewer nucleotides with a

history of multiple changes, making them a better

choice for resolving phylogeny over greater distances.

Genes that are critical for basic organismal functions

and are not under heavy immune selection are often

highly conserved. Polymerases are therefore more

likely to accurately reflect the history of the virus than

capsid proteins. Obtaining full genome sequence data

from all known iridoviruses would obviously be

optimal.

In conclusion, we describe an erythrocytic irido-

virus in a species in which this has previously not been

documented. The pathologic findings were consistent

with those associated with erythrocytic iridoviruses in

other reptiles. Sequence characterization of this agent

supports that it is a novel genus and species. The

techniques used here should be applicable to

identification and initial characterization of additional

erythrocytic iridoviruses for both clinical diagnosis

and phylogeny.

Acknowledgements

The authors would like to thank Paul E. Moler of

the Florida Fish and Wildlife Conservation Commis-

sion for collecting the snake, and Dr. William Clapp

and Linda Wright from the Electron microscopy lab at

the VA Hospital in Gainesville, FL for their support

and for performing ultrastructural processing of the

blood sample.

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