PCR-sequence characterization of new adenoviruses found in reptiles and the first successful isolation of a lizard adenovirus

Accepted Manuscript

Title: PCR-sequence characterisation of new adenovirusesfound in reptiles and the first successful isolation of a lizardadenovirus

Authors: Tibor Papp, Beth Fledelius, Volker Schmidt, GyozoL. Kajan, Rachel E. Marschang

PII: S0378-1135(08)00338-6DOI: doi:10.1016/j.vetmic.2008.08.003Reference: VETMIC 4124

To appear in: VETMIC

Received date: 13-5-2008Revised date: 24-7-2008Accepted date: 14-8-2008

Please cite this article as: Papp, T., Fledelius, B., Schmidt, V., Kajan, G.L., Marschang,R.E., PCR-sequence characterisation of new adenoviruses found in reptiles and thefirst successful isolation of a lizard adenovirus, Veterinary Microbiology (2007),doi:10.1016/j.vetmic.2008.08.003

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DOI : 10.1016/j.vetmic.2008.08.003

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PCR-sequence characterisation of new adenoviruses found in 1

reptiles and the first successful isolation of a lizard adenovirus. 2

3

4

Tibor Papp1, Beth Fledelius2, Volker Schmidt3, Gyızı L. Kaján 4 and Rachel E. 5

Marschang1* 6

7

1 Institut für Umwelt- und Tierhygiene, Hohenheim University, Garbenstr. 30, 70599 8

Stuttgart, Germany 9

2 Small Animal Clinic „Hjortekaer”, 2800 Kgs Lyngby, Denmark 10

3 Bird and Reptile Klinik of Leipzig University, 04103 Leipzig, Germany 11

4 Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Hungária 12

krt. 21., 1143 Budapest, Hungary 13

14

*Corresponding author: Tel.: +49 711 459 22468; fax: +49 711 459 22431. E-mail address: 15

[email protected] 16

17

Abstract 18

A consensus nested PCR was used to screen diagnostic samples from approximately 70 19

reptiles for the presence of adenoviruses (AdV) in the years 2006-2007. Classical virus 20

isolation methods were also used with all samples. After adenoviruses were detected in a 21

group of helodermatid lizards in a Danish zoo, a follow up study was also carried out on 22

lizards from this group (10 Mexican beaded lizards and 24 Gila monsters) over the period of 23

a year. Adenoviruses were detected in a total of 26 lizards and snakes by PCR. The PCR 24

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amplicons from all positive animals were sequenced and the resulting polymerase gene 25

sequences were used for phylogenetic analysis. Altogether six Agamid AdVs were amplified, 26

with a minimal sequence variation between one another and between these and GenBank 27

Agamid AdVs. The sequence obtained from one of the Gila monsters is identical with the 28

GenBank Helodermatid AdV. In a snake collection we have detected a new AdV from an 29

Asp viper. All of the above mentioned adenoviruses cluster in the Atadenovirus genus. 30

However, the sequence from a new Varanid AdV detected in this study clusters outside this 31

genus. On cell culture, viruses were isolated from three of the AdV positive helodermatid 32

lizards (one Mexican beaded lizard and two Gila monsters) and identified as AdVs based on 33

electron microscopy and PCR and sequencing using cell culture supernatant. This is the first 34

report of the successful isolation of a lizard AdV. 35

36

Key words: Adenovirus, Atadenovirus, lizard, PCR, reptile, virus isolation 37

38

Introduction 39

Adenoviruses (AdVs) occur worldwide and have been described from representatives of five 40

classes of the group Vertebrata (Russell & Benkı, 1999). Current taxonomy of the family 41

Adenoviridae (Benkı et al., 2005) suggests a coevolutionary lineage of the viruses with their 42

hosts, and additional host switches. According to this theory classes of vertebrates can be 43

assigned to different genera of the virus family. For mammals this would be the genus 44

Mastadenovirus, for birds the genus Aviadenovirus, for reptiles the genus Atadenovirus, for 45

amphibians the genus Siadenovirus and for fish the proposed genus “Ichtadenovirus”. The 46

genus classification criteria are mainly based on genomic and genetic characteristics of these 47

viruses, and host specificity, other than that described above, has been postulated to indicate 48

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host-switches in the evolutionary past (Harrach, 2000). All reptilian AdVs described so far 49

are members of the genus Atadenovirus (Wellehan et al., 2004; Benkı et al., 2006). 50

In reptiles, AdV infections have been detected by light and electron microscopy (EM) 51

examination or by in situ hybridization (ISH) (Ramis et al., 2000; Perkins et al., 2001) of 52

histopathological sections in a number of different species of the Diapsida class, including 53

one crocodile species from the Archosauria subclass (Jacobson et al., 1984), and several 54

agamid, varanid and chameleonid species as well as 10 snake species from the Squamata 55

order of the Lepidosauria subclass (Essbauer & Ahne, 2001; Wellehan et al., 2004). 56

Associated pathological lesions varied from enterohepatic inflammation (hepatitis, 57

oesophagitis, enteritis,) to splenitis, nephritis, pneumonia or encephalopathy. The primary 58

pathogenic role of these viruses was questioned in many cases in which they were detected 59

without signs of concurrent disease (Jacobson & Kollias, 1986; Jacobson & Gardiner, 1990; 60

Ogawa et al. 1992; Schumacher et al., 1994). However, the pathogenicity of an AdV for 61

reptiles was demonstrated in one case by an experimental transmission study (Jacobson et al., 62

1985). 63

In spite of the numerous detections of reptilian AdV infection by EM, ISH or, more recently, 64

by PCR there are very few reported cases in which the virus was successfully isolated. 65

Jacobson et al. (1985) obtained AdV from a boa constrictor (Boa constrictor) while Ahne and 66

his co-workers isolated an AdV strain from a royal python (Python regius) (Ogawa et al., 67

1992) and from a moribund corn snake (Elaphe guttata) showing clinical signs of pneumonia 68

(Juhasz & Ahne, 1992). This corn snake isolate was later randomly cloned and completely 69

sequenced (Farkas et al. 2002, 2008) and thus serves as a prototype for reptilian AdVs. A 70

sequence comparison of partial IVa2 and polymerase gene sequences of this prototype virus 71

with those of three AdV isolates from other German snakes showed that they were identical 72

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(Marschang et al., 2003). Although adenovirus infections are frequently described in lizards, 73

no virus has been isolated from a lizard in cell culture to date. 74

Wellehan et al. (2004) designed two degenerate primer pairs based on the consensus 75

sequence of the polymerase genes of different adenovirus types from three genera. This 76

nested PCR system has been shown to be an efficient tool for surveying adenovirus infections 77

of all genera in a wide range of animals, among them reptiles (Zsivanovits et al., 2006; Benkı 78

et al., 2006). In spite of the degenerate primers, direct sequencing of the products is possible, 79

and phylogenetic analysis of the sequences can help to determine the virus type. Wellehan 80

and co-workers have used this system to describe 6 novel lizard adenoviruses from seven 81

host species. In both of the above mentioned studies the phylogenetic analysis of the short 82

(ca. 300 bp) polymerase segments clearly clustered all reptilian AdVs within the 83

Atadenovirus genus, giving further support for the coevolution theory (Harrach, 2000). 84

However, the sequence recently obtained from a Sulawesi tortoise using the same PCR 85

(Wellehan, unpublished; GenBank Accession No: EU056826) clusters in the Siadenovirus 86

genus, showing that Testudines AdVs may differ from this scheme. 87

In our diagnostic laboratory we use this consensus nested PCR, together with classical virus 88

isolation methods for the detection of reptilian adenoviruses. The present report describes the 89

use of these methods to detect and characterize adenoviruses from different lizards and 90

snakes, resulting in the description of new reptilian AdVs and the first isolation of a lizard 91

AdV in cell culture. 92

93

Materials and Methods 94

Samples 95

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Routine diagnostic samples from a total of about 60 lizards and snakes with case histories 96

considered suspicious for AdV infection were sent to our laboratory during 2006 and the 97

beginning of 2007. Twelve additional reptilian samples sent for other virus tests were 98

surveyed “blind” for the presence of AdVs as well, using the PCR protocol described later. 99

DNA or RNA extracts of different organs or swabs were pooled for the same animal or same 100

batch and treated as one. The animals that were positive for AdVs are listed in Table 1. 101

In the case of the AdV positive Gila monsters (Heloderma suspectum) and Mexican beaded 102

lizards (Heloderma horidum) (Nos. 7-11 in Table 1), follow-up tests were performed at the 103

Danish zoo in which these animals were kept after the detection of AdV. Five consecutive 104

tests were performed in the group consisting of Gila monsters, Mexican beaded lizards and 105

leopard tortoises (Geochelone pardalis). First intestine, heart, liver and liver-swab samples 106

from a dead Mexican beaded lizard (No.7) were tested when an outbreak started among a 107

group of juveniles. Two weeks later oral and cloacal swabs were taken from some survivors. 108

This time two beaded lizards (No.8) and a Gila monster (No.9) were tested. Another two 109

months later a Gila monster and two leopard tortoises from the same animal group were 110

swabbed (these samples are not listed in Table 1). Six months after the outbreak, two 111

asymptomatic young Gila monsters from a separate enclosure were swabed for a follow up 112

study (No.10). Finally, another half year later 21 newly introduced Gila monsters and 7 fresh 113

Mexican beaded lizards were orally swabbed, before introducing them to the facility (No. 114

11). 115

Virus isolation 116

Isolation of viruses was attempted from all samples on the iguana heart cell line (IgH-2, 117

ATCC: CCL-108) and/or on Russell’s viper heart cells (VH-2, ATCC: CCL-140) and/or 118

Terrapene heart cells (TH-1, ATCC: CCL-50). Small pieces of tissues or the cotton heads of 119

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swabs were sonicated in 3ml Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom AG, 120

Berlin, Germany) supplemented with antibiotics. The samples were centrifuged at low speed 121

(2000xg, 10 min) for the removal of cell-debris and bacteria, then 200 µl of the homogenate 122

was inoculated onto approximately 70% confluent cell monolayers in 30 mm diameter 123

Cellstar® tissue culture dishes (Greiner Bio-One GmbH, Frickenhausen, Germany). After 124

incubating for 2 hours at 28 ºC, 2ml nutrient medium (DMEM supplemented with 2% foetal 125

calf serum, 1 % non-essential amino acids and antibiotics) was added to each dish. Cells were 126

examined for cytopathic effects (CPE) approximately every 3 days with an inverted light 127

microscope (Wilovet, Wetzlar, Germany), and dishes were frozen when extended CPE was 128

seen. Dishes showing no CPE were frozen after 2 weeks of incubation for blind passaging. 129

Two additional passages were performed from each dish after a freeze and thaw cycle and 130

low speed centrifugation. 131

Electron microscopy 132

Cell culture supernatant was negative-stained with 2% potassium phosphotungstate (pH 7.3) 133

on 3.05 mm copper grids (Plano GmbH, Wetzlar, Germany), and examined with a JEM-1011 134

transmission electron microscope (JEOL, Japan) for the presence of viral particles. Electron 135

microscopic examinations were carried out by Valerij Akimkin at the Chemische und 136

Veterinäruntersuchungsamt (CVUA) Stuttgart, Germany. 137

PCR and sequencing 138

DNA was extracted from sample homogenates using the DNAeasy® kit (Qiagen GmbH, 139

Hilden, Germany). Nested PCR was done in 25 µl reaction mixtures, containing 1x 140

concentration of Taq Buffer with (NH4)2SO4, 1.5 mM of MgCl2, 200 µM of each dNTP, and 141

0.6 U of Taq polymerase (all from MBI Fermantas, St Leon-Rot, Germany). 1 µM from both 142

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forward and reverse primers of the outer or inner pairs (Wellehan et al., 2004) were used in 143

the first or second rounds of the reaction respectively. In each round, 45 cycles were carried 144

out. In the first round 1 µl DNA extract, in the second round 2.5 µl first round amplicon 145

served as a template. Products were separated on 1.5 % agarose gels (Bioenzym, Oldendorf, 146

Germany) in TAE puffer containing 0.5 µg/ml ethidium-bromide and visualised under 320 147

nm UV light. 148

Gel purified PCR amplicons (Invisorb Spin DNA Extraction Kit; Ivitek GmbH, Berlin, 149

Germany) were sequenced directly using a Big-Dye terminator kit v.1.1 and analysed on an 150

ABI prism 310 automated DNA sequencer (both Applied Biosystems, Foster City, USA). 151

Analysis of sequences 152

Raw sequences were processed by the ABI Sequence Analysis Programme 5.1.1 (Applied 153

Biosystems, Foster City, USA) then edited, assembled and compared using the STADEN 154

Package version 2003.0 Pregap4 and Gap4 programmes (Bonfield et al., 1995). The 155

sequences were compared to the data in GenBank (National Center for Biotechnology 156

Information, Bethesda, USA) online (www.ncbi.nih.gov) using BLASTN and BLASTX 157

options. Homologous sequences were retrieved from GenBank through the non-redundant 158

AdV database of the Molecular Virology Group at the Veterinary Medical Research Institute, 159

Budapest (www.vmri.hu/~harrach). Multiple alignment of sequences was performed with 160

ClustalW algorithm of the BioEdit Sequence Alignment Editor programme (Hall, 1999) 161

using default settings. This alignment was further used for phylogenetic calculations in the 162

PHYLIP program Package version 3.6. (Felsenstein, 1989) applying various methods 163

(maximum likelihood and distance based) to obtain an optimal tree. With a bootstrap of 100 164

replicates, the Dayhoff matrix algorithm for the protein distance calculations and the Fitch-165

Margoliash method for the tree calculations provided the most robust tree. 166

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167

Results 168

Isolation of the viruses 169

Viruses were isolated from three animals out of the total of 26 that tested adenovirus positive 170

by PCR during the course of the study (Tab. 1). All three of these were Heloderma spp. (one 171

Mexican beaded lizard and two Gila monsters) from a single zoo. During this study and the 172

follow-up investigation at this zoo, a total of 34 Heloderma spp. specimens (10 Mexican 173

beaded lizards and 24 Gila monsters) were tested for adenoviruses. The isolated viruses 174

caused a CPE with rounding and detachment of some cells. The samples from the dead 175

Mexican beaded lizard (No.7) were strongly positive on IgH-2 cells in the first passage. 176

Liver, intestine, liver-swab and heart samples all showed extensive CPE after 4, 14, 24 and 177

30 days post inoculation, respectively (Fig. 1). Oral and clocal swabs of a Gila monster tested 178

6 months later (sample No.10) and one year later (sample No.11) showed a similar CPE on 179

IgH-2 cells. All of the isolates could be passaged. The isolated viruses were identified as 180

AdVs by PCR amplification of virus from the cell culture supernatant as described above as 181

well as by electron microscopic examination of the cell culture supernatants. Non-enveloped 182

icosahedral particles of approximately 80 nm were detected with negative staining in both the 183

Mexican beaded lizard and the Gila monster isolates (Fig. 2). 184

Consensus nested PCR 185

We have found AdVs in a total of 26 diagnostic samples during 2006 and beginning of 2007. 186

AdVs were detected from a total of 6 bearded dragons. In three cases (No.1, No.2 and No.6) 187

virus was detected from both oral and cloacal swabs, in one case (No.5) only from a cloacal 188

swab, in one case (No.3) from mixed tissue and faeces, and in one case (No.4) from several 189

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different tissues (lung, liver and kidney) (Table 1). In the case of one dead animal (No.4) and 190

a chronically sick one (No.5) we also tested oral and cloacal swabs of the partner dragons 191

from the same enclosure, but they were all negative in the AdV PCR. 192

In the Danish zoo cases, among the Mexican beaded lizards all of the tissues and swabs tested 193

from No.7, and the swabs from No.8 were positive for AdVs by PCR. Oral swabs as solitary 194

probes taken from several Gila monsters from the same enclosure collected some time after 195

the original detection were also all positive (No.8 & No.9). However, the companion beaded 196

lizard and two leopard tortoises from the same enclosure tested two months after the original 197

detection gave negative PCR results (data not shown). The latest sampling, of 28 new 198

quarantined Heloderma spp. specimens 1 year after the outbreak detected 12 positives among 199

the Gila monsters (No.11). 200

The kidney and spleen tissue samples from the emerald monitor (Varanus prasinus) that died 201

with a hepatitis were PCR positive although the liver was negative (No.12). 202

AdVs were also detected in a snake collection with no specific signs indicative of AdV 203

infection. No.13 was a batch of samples from snakes at a collection containing nine 204

specimens from two asp viper subspecies (Vipera aspis aspis, Vipera a. francisciredi) and 205

two striped Aesculapian rat snakes (Elaphe lineata). The solitary AdV positive sample from 206

this batch was an asp viper which died suddenly and organ samples of intestine, liver, kidney 207

and lung were sent in for paramyxovirus (PMV) testing, along with oral and cloacal swabs 208

from each living companion. PMV was detected in three of these survivors but not in the 209

tissues of the dead snake. However, a pooled organ sample of this animal was positive in the 210

AdV consensus nested PCR. 211

In many cases, multiple bands of nonspecific amplicons were visible above the approx. 320 212

bp long second round specific product, making the evaluation more difficult. In the case of 213

No.8, these bands were equally strong as the specific one. 214

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Sequence analysis 215

The second round PCR products were all 272 nt long after editing out the primer sequences. 216

These sequences were used for further analysis. An identity matrix of the sequences is shown 217

in Fig. 3, an alignment of the amino acid sequences in Fig. 4 and a phylogenetic tree based on 218

this alignment is shown in Fig.5. The sequences were submitted to GenBank and have been 219

assigned the accession numbers EU914202 to EU914209 (Table 1). 220

The six agamid AdVs detected are most similar to one another. There is a maximum of 6 nt 221

differences between the sequences, with at most 2 non-silent mutations. In the data set of the 222

follow-up study from the Heloderma spp., the sequences from the Mexican beaded lizards 223

were always distinctly different from those of the Gila monsters. All Gila sequences in the 224

batches No.10 and No.11 were identical with the corresponding GenBank Helodermatid AdV 225

sequence. Similarly, the sequences of all of the Mexican beaded lizard samples were identical 226

with one another, but show 24 nt differences compared to the Helodermatid AdV sequence. 227

Yet only 3 of these were non-silent mutations. The partial sequence obtained from the 228

emerald monitor AdV No.12 has the highest BLASTX values with the snake AdV sequence, 229

whereas the identity matrix of the nucleotide sequences shows the highest value with the 230

Agamid AdVs. The asp viper AdV (sample No.13) has the greatest similarity to the 231

Eublepharid AdV and the Helodermatid AdV with approx. 65-68 % and 70-72 % similarity 232

values on the nucleotide and amino acid level respectively. The partial polymerase sequence 233

from the AdV isolate from a Boa constrictor used as a positive control in all PCRs (No. 14) 234

was identical to the corresponding region of the SnAdV-1. 235

236

Discussion 237

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This is the first report of the successful isolation of a lizard AdV. Although there have been 238

several papers describing the occurrence of AdVs in eublepharid, geckonid, varanid, 239

chameleonid and agamid Squamates (Essbauer and Ahne, 2001; Farkas et al., 2002; 240

Wellehan et al,. 2004), these viruses have never been isolated. All reptilian isolates to date 241

are from snakes, and there is evidence that these might represent the same virus (Marschang 242

et al., 2003). Isolation of the two different Helodermatid AdVs, in one case from a Mexican 243

beaded lizard and two cases from Gila monsters is therefore an important step in the further 244

study of AdVs of reptiles. PCR amplified partial sequence comparison is possible with field 245

samples, but virus isolation is essential for physico-chemical and ultrastructural 246

characterisation as well as for pathogenicity studies. 247

The comparison of the partial polymerase sequences of our helodermatid sequences with that 248

in GenBank has revealed that the Gila monsters and Mexican beaded lizards posess two 249

different AdVs. The GenBank helodermatid AdV sequence from a Gila monster in the USA 250

is identical to our Danish Gila sequences, whereas our beaded lizard isolate and sample 251

sequences from the same enclosure differ from these. This finding supports the host-252

specificity coevolution-cospeciation theory of AdVs (Harrach, 2000). However, this strict 253

host-specificity is not supported by the identical sequences found in isolates from different 254

snakes (Boa sample No.14 and SnAdV-1 from a corn snake; also in Marschang et al., 2003; 255

python, corn snake & rat snake samples in Benkı et al., 2006). The difference (8.9% aa and 256

3.3% nt) between the gene portions of the two helodermatid viruses are relatively small. 257

Compared to those between other AdVs, these viruses seem to represent the same species. 258

Further sequencing and/or serological testing are needed for further characterization of these 259

isolates. 260

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Our six bearded dragon AdV sequences possess less than 2.5% nt variation in the examined 261

region, and thus, together with data from GenBank and additional sequences from Hungary 262

(Benkı et al, 2006; Farkas, personal communication) they provide further support that a 263

single agamid AdV is circulating in the bearded dragon collections across Europe and the 264

USA. 265

There were several interesting aspects of the AdV found in the emerald monitor (No.12). 266

Although the histological examination of this animal showed hepatitis, no AdV was detected 267

in the liver by PCR. The spleen and kidney were both PCR positive and the sequence 268

obtained from these amplicons revealed that this varanid AdV is very different from every 269

earlier described reptilian AdVs. Using either nucleotide or amino acid sequence in the 270

distance matrix analysis the virus clustered not in but beside the Atadenovirus genus, equally 271

distant to the proposed “Ichtadenovirus” genus and the Mastadenovirus genus. Yet the 272

sequence is short and the bootstrap values at this branching of the tree are under 50 (Fig. 5). 273

Further sequencing is necessary to determine the exact taxonomic position of this virus. 274

The asp viper sample (No.13) is a good example for the successful detection of new AdVs in 275

blind surveys. This snake was the only animal that died in a collection of snakes in which a 276

PMV was detected although only the companion animals were positive in a PMV RT-PCR. 277

The phylogenetic analysis of the polymerase gene portion of the AdV in this animal has 278

clustered this virus into the Atadenovirus genus, but representing a new species: Viperid 279

AdV. 280

Each of the viruses presented above have a balanced A+T content (41.7% - 57.5%) in the 281

sequenced region which might indicate that they have coevolved over a longer period of time 282

together with their hosts (Farkas et al., 2002; Wellehan et al., 2004). 283

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In conclusion, while the majority of our sequence data support the coevolution-cospeciation 284

theory and the reptilian origin of atadenoviruses, a close look at individual reptilian AdV 285

shows that this theory may not explain all of the AdV found in reptiles. One open question is 286

the occurance of the same AdV in different snake hosts. The other is posed by the Varanid 287

AdV which does not seem to belong to the Atadenovirus genus. Additional work is 288

necessary, both to obtain additional taxonomic information on the AdVs described here as 289

well as to characterize further reptilian AdV. 290

291

Acknowledgements 292

We would like to thank Silvia Speck and Christa Shäfer for their excellent technical 293

assistance. Samples were sent to our laboratory by Dr. Marc Hoferer from the Chemisches- 294

und Veterinär Untersuchungsamt (CVUA), Stuttgart-Fellbach; Dr. Karina Mathes and Ms. 295

Günther, Small Animal Clinic of the Veterinary College of Hannover; Dr. Jutta Wiechert, 296

Veterinary Praxis, Mainz; Dr. Bernd Dörsch, Small Animal Praxis, Kiel, and Dr. Michael 297

Pees, Bird and Reptile Klinik of Leipzig University, all in Germany. Their help is gratefully 298

acknowledged. We also thank Valerij Akimkin at the CVUA, Stuttgart-Fellbach, Germany, 299

for his help with the electron microscopic examination and Dr. Balázs Harrach at the 300

Veterinary Medical Research Institute, Budapest, Hungary for his advice in taxonomical 301

questions. Dr. Kaján’s work was partially supported by the National Office for Research and 302

Technology, József Öveges programme, and the Hungarian Scientific Research Fund, grant 303

OTKA K 67781. 304

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guttata) imply a common origin with members of the proposed new genus 326

Atadenovirus. J. Gen. Virol. 83, 2403-2410. 327

Farkas, S.L., Harrach, B., Benkı, M., 2008. Completion of the genome analysis of snake 328

adenovirus type 1, a representative of the reptilian lineage within the novel genus 329

Atadenovirus. Vir. Res. 132, 132-139. 330

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373

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Table 1. Adenovirus positive samples with short case histories, the results of virus isolation 374

on cell-culture and GenBank accession numbers of the obtained partial DNA polymerase 375

gene sequences. Only PCR positive animals are listed. 376

377 Species Ident.

No. Age Case history Sample

type Virus

isolation AdV PCR

Investigation for other agents

GenBank Acc. No.

1 ?

Imported from the USA, CNS symptoms, opisthotonus, spasms

o./cl. swabs __ all pos.

continuous problems with coccidiosis, IIV-PCR negativ

EU914202

2 ? No report o./cl. swabs

__

all pos.

__

EU914203

3 ? No report organ, feaces

__

all pos. IIV-PCR negativ EU914203

4 adult

Abnormal blinking; inactivity, apathy, hiding, anorexia, dark colouration, rounded back with fallen tail, tachycardia, dyspnoea, exitus.

liver, kidney, lung

__ all pos.

Lung of this animal, as well as swab of partner animal was positive in IIV-PCR

EU914204

5 adult

Chronic gastrointestinal problems

blood, cl. swab __ swab

pos. __

EU914205

Bearded dragon Pogona vitticeps

6 adult

Paralysis of the hind limbs and tail, trembling, hyperesthesia: root of the tail and the back

blood, o./cl. swabs

__ swabs pos. IIV-PCR negativ

EU914206

7* juv.

Kept together with Gila monsters and leopard tortoises. Outbreak with several deaths among the Heloderma spp.

intestine, liver, heart

all + (IgH2 cells)

all pos. Rana- and Herpesvirus-PCR negativ

EU914207

Mexican beaded lizard Heloderma horridum

8* juv.

Swab taken 2 weeks later from the two survivors of the above group (both pos.)

o./cl. swabs

__

all pos.

__

EU914207

9* juv.

Swab taken 2 weeks later from a survivor Gila in the same enclosure

oral swab

__

all pos. __

AY576680†

10* juv.

Swabs taken half a year p.ob., from 2 new animals which had no contact with the others (both positive)

oral swabs

1 swab + (IgH2 cells)

all pos. __

AY576680†

Gila monster Heloderma suspectum

11* juv.

Swab taken a year p. ob., from 21 Gila and 7 beaded lizard, all new animals, no contact with the others

oral swabs

1 swab + (IgH2 cells)

12 Gila pos.

__

AY576680†

Emerald 12 ? Died, hepatitis liver, __ spleen, __ EU914208

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monitor Varanus prasinus

spleen, kidney

kidney pos.

Asp viper Vipera a. aspis

13 ? Group of nine snakes, this one died

internal organs

__ pos.

3 of the survivors positive for PMV in RT-PCR, this one not

EU914209

Common boa Boa constrictor

14 ?

Isolate from an earlier study, served as a positive control

VH2 cell passage + pos.

__

DQ106414†

378 cl.= cloacal, CNS= central nervous system, Ident. No.= identification number, IgH2= iguana 379

heart cell line (ATCC: CCL-108), IIV = invertebrate iridovirus, juv.= juvenile, o.= oral, 380

PMV= paramyxovirus, p.ob.= post outbreak, RT-PCR= reverse trancriptase polymerase 381

chain reaction, USA= United States of America, * = from the same owner, †Corresponding 382

sequence identical to that of the existing GenBank entry 383

384

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Figure captions: 385

386

Fig. 1. Adenovirus isolate from a Gila monster (Heloderma suspectum). Cytopathic effect 387

(CPE) five days after inoculation. A- Negative control IgH-2. B- 3rd passage of an isolate 388

from an oral swab from case No.11 on IgH2 cells 5 days post inoculation. (Magnification: 389

400x) 390

Fig. 2. Electron micrographs of icosahedral non-enveloped virus particles in cell culture 391

supernatant from two Helodermatid isolates. (Both 3rd passage on IgH2 cells 5 days post 392

inoculation). A- Mexican beaded lizard isolate with identification No.7. B- Isolate from an 393

oral swab from Gila monster with identification No.10 394

Fig. 3. Identity matrix of the nucleotide (above diagonal, with white letters) and the deduced 395

amino acid (below diagonal, with black letters) sequences of the amplified DNA-polymerase 396

gene region. Accepted and proposed members of the Atadenovirus genus from GenBank are 397

marked with lighter background colouring compared to the sequences newly reported here 398

with darker grey background. Highest values for the new sequences compared to GenBank 399

sequences are shown in bold. For accession numbers see legend of Fig. 5; Agamid AdV, 400

Austria (AAY83284), Agamid AdV, USA (AY576678). 401

Fig. 4. Alignment of predicted partial DNA-polymerase sequences of the amplified region 402

from the atadenoviruses and the sturgeon adenovirus. (Identical sequences are represented 403

by one selected virus. Start of alignment corresponds to the codon at position 6458 of 404

HAdV-1 whole genomic nucleotide sequence, Accession No.: AF534906.) Sequences of 405

reptilian viruses are printed with capital letters, sequences new to science are highlighted 406

with bold printed names. OAdV-7 type species was taken as a null-sequence for the 407

comparison, identical amino acids are marked with dots in the rest of the alignment. Virus 408

species with more than one serotype are separated with lines and indicated with grey 409

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background shading of the names. Vertical background shading of sequences in black and 410

grey refers to 80% identity and similarity of conserved amino acid positions respectively, 411

regarding the alignment of all available sequences for this region. (helodermatid and 412

agamid isolates show a maximum of 3 amino acid variations between one another, and are 413

therefore also grouped together as they most probably belong to one species.) For 414

abbreviations and GenBank accession numbers see legend of Fig. 5. 415

Fig 5. Phylogenetic distance tree of partial adenovirus DNA-polymerase amino acid 416

sequences. The tree was generated using the Protdist program with Dayhoff matrix, followed 417

by Fitch program with global rearrangements. Bootstrap values above 50 (for 100 418

replications) are shown beside the branches, lower value branchings are shown with dotted 419

lines. Accepted and presumed virus species are indicated with grey circles, genera with half 420

circles. Names of reptilian viruses are printed in italics, and those representing sequences 421

from our study (too), are in bold italics. 422

Abbreviations and GenBank accession numbers (in brackets): B=Bovine, C=Canine, 423

D=Duck, F=Fowl, H=Human, M=Murine, O=Ovine, P=Porcine, Rus=BadV-4 strain Rus, 424

S=Simian, T=Turkey, TS=Tree shrew 425

Agamid AdV (AY576678 and AAY83284), BAdV-1 (YP_094032), BAdV-2 (AP_000006), 426

BAdV-3 (AP_000026), BAdV-4 (AAK13183), BAdV-4 Rus (not yet released), Bat AdV 427

(AB303301), CAdV-1 (AAB05434), CAdV-2 (AAB38716), Chameleonid AdV 428

(AY576679), DAdV-1 (AP_000080), Eublepharid AdV (AY576677), FAdV-1 (AP_000410), 429

FAdV-5 (DQ159938), FAdV-9 (AC_000013), Frog (AAF86924), Gekkonid AdV 430

(AY576681), HAdV-9 (CAI05960), HAdV-12 (CAA51882), HAdV-17 (AP_000143), 431

HAdV-40 (AAC13953), HAdV-52 (ABK35035), HAdV-B=HAdV-3 (ABB17778) + HAdV-432

7 (AP_000539) + HAdV-35 (AAN17476), HAdV-E=HAdV-4 (AAS66917) + SAdV-25 433

(AP_000304), Helodermatid AdV (AAS89696), MAdV-1 (AP_000342), OAdV-7 434

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(AAD45950), PAdV-3 (AB026117), PAdV-5 (AAK26504), Parakeet AdV (EU056825), 435

SAdV-1 (AAX19399), SAdV-3 (AAT84618), Scincid AdV (AY576682), Snake AdV 436

(AAL89790), Sturgeon AdV (not yet released), TAdV-3 (AAC64523), Tortoise AdV 437

(EU056826), TSAdV (YP_068060), 438

439

440

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D1 O287 B4 Sn1 Cham. Gekko Scinc. Helod. Eubl. Ag. U Ag. A No.1 No.2+3 No.4 No.5 No.6 No.7+8 No.9-11 No.12 No.13 No.14 DadV-1 0,601 0,664 0,597 0,597 0,583 0,616 0,634 0,583 0,645 0,645 0,649 0,645 0,645 0,645 0,645 0,638 0,634 0,572 0,627 0,597OAV-287 0,588 0,678 0,579 0,535 0,591 0,542 0,601 0,564 0,535 0,538 0,535 0,527 0,538 0,531 0,535 0,583 0,601 0,544 0,590 0,579BAdV-4 0,666 0,733 0,601 0,542 0,591 0,575 0,583 0,564 0,568 0,571 0,568 0,568 0,568 0,564 0,560 0,594 0,583 0,556 0,619 0,601SnAdV-1 0,622 0,611 0,655 0,627 0,565 0,645 0,697 0,671 0,649 0,653 0,649 0,645 0,645 0,645 0,649 0,701 0,697 0,604 0,649 1,000Chameleonid 0,644 0,577 0,577 0,655 0,543 0,557 0,553 0,597 0,605 0,597 0,601 0,608 0,597 0,605 0,605 0,560 0,553 0,588 0,601 0,627Gekkonid 0,593 0,593 0,593 0,571 0,582 0,562 0,594 0,532 0,572 0,576 0,580 0,580 0,576 0,580 0,572 0,587 0,594 0,513 0,609 0,565Scincid 0,677 0,588 0,655 0,688 0,577 0,604 0,608 0,571 0,612 0,608 0,605 0,608 0,601 0,605 0,605 0,601 0,608 0,560 0,597 0,645Helodermatid 0,711 0,588 0,622 0,744 0,622 0,549 0,633 0,686 0,653 0,660 0,656 0,653 0,653 0,653 0,656 0,911 1,000 0,544 0,682 0,697Eublepharid 0,633 0,566 0,611 0,744 0,611 0,538 0,633 0,811 0,590 0,594 0,597 0,594 0,601 0,594 0,590 0,667 0,686 0,588 0,649 0,671Agamid-USA 0,722 0,622 0,644 0,666 0,655 0,604 0,688 0,733 0,644 0,988 0,985 0,992 0,981 0,981 0,988 0,645 0,653 0,612 0,623 0,649Agamid-A ustria 0,722 0,622 0,644 0,666 0,655 0,604 0,688 0,733 0,644 1,000 0,996 0,981 0,992 0,992 0,977 0,649 0,660 0,612 0,627 0,653No.1 0,722 0,622 0,644 0,666 0,655 0,604 0,688 0,733 0,644 1,000 1,000 0,985 0,996 0,996 0,981 0,645 0,656 0,612 0,623 0,649No.2+3 0,722 0,622 0,644 0,666 0,655 0,604 0,688 0,733 0,644 1,000 1,000 1,000 0,981 0,981 0,988 0,645 0,653 0,612 0,623 0,645No.4 0,722 0,622 0,644 0,666 0,655 0,604 0,688 0,733 0,644 1,000 1,000 1,000 1,000 0,992 0,977 0,642 0,653 0,608 0,619 0,645No.5 0,722 0,622 0,644 0,666 0,655 0,604 0,688 0,733 0,644 0,988 0,988 0,988 0,988 0,988 0,977 0,642 0,653 0,612 0,619 0,645No.6 0,711 0,622 0,644 0,677 0,655 0,593 0,677 0,744 0,644 0,988 0,988 0,988 0,988 0,988 0,977 0,649 0,656 0,608 0,616 0,649No.7+8 0,700 0,577 0,600 0,744 0,611 0,538 0,633 0,966 0,800 0,711 0,711 0,711 0,711 0,711 0,711 0,722 0,911 0,540 0,671 0,701No.9-11 0,711 0,588 0,622 0,744 0,622 0,549 0,633 1,000 0,811 0,733 0,733 0,733 0,733 0,733 0,733 0,744 0,966 0,544 0,682 0,697No.12 0,577 0,577 0,555 0,644 0,633 0,593 0,588 0,600 0,622 0,600 0,600 0,600 0,600 0,600 0,600 0,588 0,600 0,600 0,626 0,604No.13 0,622 0,566 0,566 0,677 0,622 0,604 0,600 0,700 0,722 0,655 0,655 0,655 0,655 0,655 0,655 0,644 0,688 0,700 0,655 0,649No.14 0,622 0,611 0,655 1,000 0,655 0,571 0,688 0,744 0,744 0,666 0,666 0,666 0,666 0,666 0,666 0,677 0,744 0,744 0,644 0,677

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10 20 30 40 50 60 70 80 90 | | | | | | | | | | | | | | | | | | Ovine 7 salthplpygktlnafeanaqidyfqellqr-kekidyfdnsikpmivvadceppsldyldvlpplcskksgklcwsnetlinevltsidlDuck 1 ......m.f.r.edplt.sis.kt..dk.ds-pa.ls..ge.......y...y..p.ehv........r...r...t..p.lg..v.t...Bovine 4 ......m...r...p....ts..em.nm.ds-s.vls...pr..a..........t.e..................t..p....tv.....isol. Rus ......m...r...p....ia..el.sm.ds-svils...sg..a.v........t.e..................t.gp.v..tv.....SNAKE 1 ...S..M...P..SP.DSAVA.AE..RK.DG-QSELS...PD.F.......AF....HC..........R......T..P.LG....TV..CHAMELEON ...S..M.S.T.ESPTD.ALS.A...D..DK-PDQ.S..S-QV.....L...Y..A.AR.........RR......T..V.TA.A..TV..EUBLEPARID ...S..M...T..SP.DSSKAMAS..A..DG-.DCLS...PR.L....KV..F..P.YH..T..........R...T..P.LG..I.TV.IGEKKONID ......M.F.LPCEP.T..IH.RQ..Y..DEVGKP.S...ER......A...F...IKE......M.TR.G.....T..S.HM.I...V..SCINCID ......M.S.IP.DP.TSSIA.RK..NK.DE-PST.S...PD.F..V.....S..P.EQ.............R...T..P.EV.T..T...GILA M. ...S..M...L..SPLD.SVAMAR..DK.DS-T..LSF..KN.L....K...F..P.YH.............R...T..P.LG....TV.IHELOD.No8 ...S..M...L..SPLD.SVAMAR..DK.ES-TG.LSF..KD.L....K...F..P.YH.............R...T..P.LG....TV.IAGAMID ...S..M.C.R..PPLD.SIE.RR..DK.DK-PH..S...PNL.....A...I..P.NE..........A..R...T..P.VG........AGAMA No5 ...S..M.C.R..PPLD.SIE.RR..DK.DK-PH..S...PNL.....A...I..P.NE..........A..R...T..P.AG........AGAMA No6 ...S..M.C.R..PPLD.SVE.RR..DK.DK-PH..S...PNL.....A...I..P.NE..........A..R...T..P.VG........VIPER No13 ...S..M...R..GP.D.AIAMRH..A..DT-QK..S..EPT.N...ITV..F..DILR.........R...R...T..A.LG....TV.IVARAN.No12 ...?..M.S.NPESP.D.AIS.EN.RR..EQ-I.P.....PR......TV.AD...D.Q.........RH..R...T..P.RK....TV.I

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No.12 Varanus

Sturgeon

GekkonidNo.13 Vipera

Scincid

Chameleonid

Snake-1

Euble-pharidHelo-

dermatid (Gila)

D1Agamid

O7

Rus

B4

T3

Parakeet

Tortoise

Frog

Parrot

F1

F9 F5

Bat

M1

P3

B3

C2C1 TS

P5

S3 H40 H52S1

H12

H17H9B1

B2

No. 7+8 Heloderma

100 100

75 89

100

71

57

100

100

99

54

52

66100

100

97

100

100

73

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