PCR-sequence characterisation of new adenoviruses found in reptiles and the first successful isolation ofa lizard adenovirus Tibor Papp, Beth Fledelius, Volker Schmidt, Gyözö L. Kaján, Rachel E. Marschang
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Tibor Papp, Beth Fledelius, Volker Schmidt, Gyözö L. Kaján, Rachel E. Marschang. PCR-sequence characterisation of new adenoviruses found in reptiles and the first successful isolation of a lizard adenovirus. Veterinary Microbiology, Elsevier, 2009, 134 (3-4), pp.233. 10.1016/j.vetmic.2008.08.003. hal-00532460
HAL Id: hal-00532460 https://hal.archives-ouvertes.fr/hal-00532460 Submitted on 4 Nov 2010
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Title: PCR-sequence characterisation of new adenoviruses found in reptiles and the first successful isolation of a lizard adenovirus
Authors: Tibor Papp, Beth Fledelius, Volker Schmidt, Gyoz˝ o˝ L. Kajan,´ Rachel E. Marschang
PII: S0378-1135(08)00338-6 DOI: doi:10.1016/j.vetmic.2008.08.003 Reference: VETMIC 4124
To appear in: VETMIC
Received date: 13-5-2008 Revised date: 24-7-2008 Accepted 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 the first successful isolation of a lizard adenovirus, Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2008.08.003
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1 PCR-sequence characterisation of new adenoviruses found in
2 reptiles and the first successful isolation of a lizard adenovirus.
3
4
5 Tibor Papp 1, Beth Fledelius 2, Volker Schmidt 3, Gy ızı L. Kaján 4 and Rachel E.
6 Marschang 1*
7
8 1 Institut für Umwelt- und Tierhygiene, Hohenheim University, Garbenstr. 30, 70599
9 Stuttgart, Germany
10 2 Small Animal Clinic „Hjortekaer”, 2800 Kgs Lyngby, Denmark
11 3 Bird and Reptile Klinik of Leipzig University, 04103 Leipzig, Germany
12 4 Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Hungária
13 krt. 21., 1143 Budapest, Hungary
14
15 *Corresponding author: Tel.: +49 711 459 22468; fax: +49 711 459 22431. E-mail address:
17
18 Abstract
19 A consensus Accepted nested PCR was used to screen diagnosticManuscript samples from approximately 70 20 reptiles for the presence of adenoviruses (AdV) in the years 2006-2007. Classical virus
21 isolation methods were also used with all samples. After adenoviruses were detected in a
22 group of helodermatid lizards in a Danish zoo, a follow up study was also carried out on
23 lizards from this group (10 Mexican beaded lizards and 24 Gila monsters) over the period of
24 a year. Adenoviruses were detected in a total of 26 lizards and snakes by PCR. The PCR
1 Page 1 of 27 25 amplicons from all positive animals were sequenced and the resulting polymerase gene
26 sequences were used for phylogenetic analysis. Altogether six Agamid AdVs were amplified,
27 with a minimal sequence variation between one another and between these and GenBank
28 Agamid AdVs. The sequence obtained from one of the Gila monsters is identical with the
29 GenBank Helodermatid AdV. In a snake collection we have detected a new AdV from an
30 Asp viper. All of the above mentioned adenoviruses cluster in the Atadenovirus genus.
31 However, the sequence from a new Varanid AdV detected in this study clusters outside this
32 genus. On cell culture, viruses were isolated from three of the AdV positive helodermatid
33 lizards (one Mexican beaded lizard and two Gila monsters) and identified as AdVs based on
34 electron microscopy and PCR and sequencing using cell culture supernatant. This is the first
35 report of the successful isolation of a lizard AdV.
36
37 Key words : Adenovirus, Atadenovirus, lizard, PCR, reptile, virus isolation
38
39 Introduction
40 Adenoviruses (AdVs) occur worldwide and have been described from representatives of five
41 classes of the group Vertebrata (Russell & Benk ı, 1999). Current taxonomy of the family
42 Adenoviridae (Benk ı et al., 2005) suggests a coevolutionary lineage of the viruses with their
43 hosts, and additional host switches. According to this theory classes of vertebrates can be
44 assigned to differentAccepted genera of the virus family. Manuscript For mammals this would be the genus
45 Mastadenovirus , for birds the genus Aviadenovirus , for reptiles the genus Atadenovirus , for
46 amphibians the genus Siadenovirus and for fish the proposed genus “Ichtadenovirus”. The
47 genus classification criteria are mainly based on genomic and genetic characteristics of these
48 viruses, and host specificity, other than that described above, has been postulated to indicate
2 Page 2 of 27 49 host-switches in the evolutionary past (Harrach, 2000). All reptilian AdVs described so far
50 are members of the genus Atadenovirus (Wellehan et al., 2004; Benk ı et al., 2006).
51 In reptiles, AdV infections have been detected by light and electron microscopy (EM)
52 examination or by in situ hybridization (ISH) (Ramis et al., 2000; Perkins et al., 2001) of
53 histopathological sections in a number of different species of the Diapsida class, including
54 one crocodile species from the Archosauria subclass (Jacobson et al., 1984), and several
55 agamid, varanid and chameleonid species as well as 10 snake species from the Squamata
56 order of the Lepidosauria subclass (Essbauer & Ahne, 2001; Wellehan et al., 2004).
57 Associated pathological lesions varied from enterohepatic inflammation (hepatitis,
58 oesophagitis, enteritis,) to splenitis, nephritis, pneumonia or encephalopathy. The primary
59 pathogenic role of these viruses was questioned in many cases in which they were detected
60 without signs of concurrent disease (Jacobson & Kollias, 1986; Jacobson & Gardiner, 1990;
61 Ogawa et al. 1992; Schumacher et al., 1994). However, the pathogenicity of an AdV for
62 reptiles was demonstrated in one case by an experimental transmission study (Jacobson et al.,
63 1985).
64 In spite of the numerous detections of reptilian AdV infection by EM, ISH or, more recently,
65 by PCR there are very few reported cases in which the virus was successfully isolated.
66 Jacobson et al . (1985) obtained AdV from a boa constrictor ( Boa constrictor ) while Ahne and
67 his co-workers isolated an AdV strain from a royal python (Python regius ) (Ogawa et al .,
68 1992) and fromAccepted a moribund corn snake ( Elaphe guttata Manuscript) showing clinical signs of pneumonia
69 (Juhasz & Ahne, 1992). This corn snake isolate was later randomly cloned and completely
70 sequenced (Farkas et al. 2002, 2008) and thus serves as a prototype for reptilian AdVs. A
71 sequence comparison of partial IVa2 and polymerase gene sequences of this prototype virus
72 with those of three AdV isolates from other German snakes showed that they were identical
3 Page 3 of 27 73 (Marschang et al., 2003). Although adenovirus infections are frequently described in lizards,
74 no virus has been isolated from a lizard in cell culture to date.
75 Wellehan et al. (2004) designed two degenerate primer pairs based on the consensus
76 sequence of the polymerase genes of different adenovirus types from three genera. This
77 nested PCR system has been shown to be an efficient tool for surveying adenovirus infections
78 of all genera in a wide range of animals, among them reptiles (Zsivanovits et al., 2006; Benk ı
79 et al., 2006). In spite of the degenerate primers, direct sequencing of the products is possible,
80 and phylogenetic analysis of the sequences can help to determine the virus type. Wellehan
81 and co-workers have used this system to describe 6 novel lizard adenoviruses from seven
82 host species. In both of the above mentioned studies the phylogenetic analysis of the short
83 (ca. 300 bp) polymerase segments clearly clustered all reptilian AdVs within the
84 Atadenovirus genus, giving further support for the coevolution theory (Harrach, 2000).
85 However, the sequence recently obtained from a Sulawesi tortoise using the same PCR
86 (Wellehan, unpublished; GenBank Accession No: EU056826) clusters in the Siadenovirus
87 genus, showing that Testudines AdVs may differ from this scheme.
88 In our diagnostic laboratory we use this consensus nested PCR, together with classical virus
89 isolation methods for the detection of reptilian adenoviruses. The present report describes the
90 use of these methods to detect and characterize adenoviruses from different lizards and
91 snakes, resulting in the description of new reptilian AdVs and the first isolation of a lizard
92 AdV in cell culture.Accepted Manuscript
93
94 Materials and Methods
95 Samples
4 Page 4 of 27 96 Routine diagnostic samples from a total of about 60 lizards and snakes with case histories
97 considered suspicious for AdV infection were sent to our laboratory during 2006 and the
98 beginning of 2007. Twelve additional reptilian samples sent for other virus tests were
99 surveyed “blind” for the presence of AdVs as well, using the PCR protocol described later.
100 DNA or RNA extracts of different organs or swabs were pooled for the same animal or same
101 batch and treated as one. The animals that were positive for AdVs are listed in Table 1.
102 In the case of the AdV positive Gila monsters ( Heloderma suspectum ) and Mexican beaded
103 lizards ( Heloderma horidum ) (Nos. 7-11 in Table 1), follow-up tests were performed at the
104 Danish zoo in which these animals were kept after the detection of AdV. Five consecutive
105 tests were performed in the group consisting of Gila monsters, Mexican beaded lizards and
106 leopard tortoises ( Geochelone pardalis ). First intestine, heart, liver and liver-swab samples
107 from a dead Mexican beaded lizard (No.7) were tested when an outbreak started among a
108 group of juveniles. Two weeks later oral and cloacal swabs were taken from some survivors.
109 This time two beaded lizards (No.8) and a Gila monster (No.9) were tested. Another two
110 months later a Gila monster and two leopard tortoises from the same animal group were
111 swabbed (these samples are not listed in Table 1). Six months after the outbreak, two
112 asymptomatic young Gila monsters from a separate enclosure were swabed for a follow up
113 study (No.10). Finally, another half year later 21 newly introduced Gila monsters and 7 fresh
114 Mexican beaded lizards were orally swabbed, before introducing them to the facility (No. 115 11). Accepted Manuscript
116 Virus isolation
117 Isolation of viruses was attempted from all samples on the iguana heart cell line (IgH-2,
118 ATCC: CCL-108) and/or on Russell’s viper heart cells (VH-2, ATCC: CCL-140) and/or
119 Terrapene heart cells (TH-1, ATCC: CCL-50). Small pieces of tissues or the cotton heads of
5 Page 5 of 27 120 swabs were sonicated in 3ml Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom AG,
121 Berlin, Germany) supplemented with antibiotics. The samples were centrifuged at low speed
122 (2000 xg, 10 min) for the removal of cell-debris and bacteria, then 200 µl of the homogenate
123 was inoculated onto approximately 70% confluent cell monolayers in 30 mm diameter
124 Cellstar® tissue culture dishes (Greiner Bio-One GmbH, Frickenhausen, Germany). After
125 incubating for 2 hours at 28 ºC, 2ml nutrient medium (DMEM supplemented with 2% foetal
126 calf serum, 1 % non-essential amino acids and antibiotics) was added to each dish. Cells were
127 examined for cytopathic effects (CPE) approximately every 3 days with an inverted light
128 microscope (Wilovet, Wetzlar, Germany), and dishes were frozen when extended CPE was
129 seen. Dishes showing no CPE were frozen after 2 weeks of incubation for blind passaging.
130 Two additional passages were performed from each dish after a freeze and thaw cycle and
131 low speed centrifugation.
132 Electron microscopy
133 Cell culture supernatant was negative-stained with 2% potassium phosphotungstate (pH 7.3)
134 on 3.05 mm copper grids (Plano GmbH, Wetzlar, Germany), and examined with a JEM-1011
135 transmission electron microscope (JEOL, Japan) for the presence of viral particles. Electron
136 microscopic examinations were carried out by Valerij Akimkin at the Chemische und
137 Veterinäruntersuchungsamt (CVUA) Stuttgart, Germany.
138 PCR and sequencingAccepted Manuscript
139 DNA was extracted from sample homogenates using the DNAeasy® kit (Qiagen GmbH,
140 Hilden, Germany). Nested PCR was done in 25 µl reaction mixtures, containing 1x
141 concentration of Taq Buffer with (NH 4)2SO 4, 1.5 mM of MgCl 2, 200 µM of each dNTP, and
142 0.6 U of Taq polymerase (all from MBI Fermantas, St Leon-Rot, Germany). 1 µM from both
6 Page 6 of 27 143 forward and reverse primers of the outer or inner pairs (Wellehan et al., 2004) were used in
144 the first or second rounds of the reaction respectively. In each round, 45 cycles were carried
145 out. In the first round 1 µl DNA extract, in the second round 2.5 µl first round amplicon
146 served as a template. Products were separated on 1.5 % agarose gels (Bioenzym, Oldendorf,
147 Germany) in TAE puffer containing 0.5 µg/ml ethidium-bromide and visualised under 320
148 nm UV light.
149 Gel purified PCR amplicons (Invisorb Spin DNA Extraction Kit; Ivitek GmbH, Berlin,
150 Germany) were sequenced directly using a Big-Dye terminator kit v.1.1 and analysed on an
151 ABI prism 310 automated DNA sequencer (both Applied Biosystems, Foster City, USA).
152 Analysis of sequences
153 Raw sequences were processed by the ABI Sequence Analysis Programme 5.1.1 (Applied
154 Biosystems, Foster City, USA) then edited, assembled and compared using the STADEN
155 Package version 2003.0 Pregap4 and Gap4 programmes (Bonfield et al., 1995). The
156 sequences were compared to the data in GenBank (National Center for Biotechnology
157 Information, Bethesda, USA) online (www.ncbi.nih.gov) using BLASTN and BLASTX
158 options. Homologous sequences were retrieved from GenBank through the non-redundant
159 AdV database of the Molecular Virology Group at the Veterinary Medical Research Institute,
160 Budapest (www.vmri.hu/~harrach). Multiple alignment of sequences was performed with 161 ClustalW algorithmAccepted of the BioEdit Sequence AlignmentManuscript Editor programme (Hall, 1999) 162 using default settings. This alignment was further used for phylogenetic calculations in the
163 PHYLIP program Package version 3.6. (Felsenstein, 1989) applying various methods
164 (maximum likelihood and distance based) to obtain an optimal tree. With a bootstrap of 100
165 replicates, the Dayhoff matrix algorithm for the protein distance calculations and the Fitch-
166 Margoliash method for the tree calculations provided the most robust tree.
7 Page 7 of 27 167
168 Results
169 Isolation of the viruses
170 Viruses were isolated from three animals out of the total of 26 that tested adenovirus positive
171 by PCR during the course of the study (Tab. 1). All three of these were Heloderma spp . (one
172 Mexican beaded lizard and two Gila monsters) from a single zoo. During this study and the
173 follow-up investigation at this zoo, a total of 34 Heloderma spp. specimens (10 Mexican
174 beaded lizards and 24 Gila monsters) were tested for adenoviruses. The isolated viruses
175 caused a CPE with rounding and detachment of some cells. The samples from the dead
176 Mexican beaded lizard (No.7) were strongly positive on IgH-2 cells in the first passage.
177 Liver, intestine, liver-swab and heart samples all showed extensive CPE after 4, 14, 24 and
178 30 days post inoculation, respectively (Fig. 1). Oral and clocal swabs of a Gila monster tested
179 6 months later (sample No.10) and one year later (sample No.11) showed a similar CPE on
180 IgH-2 cells. All of the isolates could be passaged. The isolated viruses were identified as
181 AdVs by PCR amplification of virus from the cell culture supernatant as described above as
182 well as by electron microscopic examination of the cell culture supernatants. Non-enveloped
183 icosahedral particles of approximately 80 nm were detected with negative staining in both the
184 Mexican beaded lizard and the Gila monster isolates (Fig. 2).
185 Consensus nestedAccepted PCR Manuscript
186 We have found AdVs in a total of 26 diagnostic samples during 2006 and beginning of 2007.
187 AdVs were detected from a total of 6 bearded dragons. In three cases (No.1, No.2 and No.6)
188 virus was detected from both oral and cloacal swabs, in one case (No.5) only from a cloacal
189 swab, in one case (No.3) from mixed tissue and faeces, and in one case (No.4) from several
8 Page 8 of 27 190 different tissues (lung, liver and kidney) (Table 1). In the case of one dead animal (No.4) and
191 a chronically sick one (No.5) we also tested oral and cloacal swabs of the partner dragons
192 from the same enclosure, but they were all negative in the AdV PCR.
193 In the Danish zoo cases, among the Mexican beaded lizards all of the tissues and swabs tested
194 from No.7, and the swabs from No.8 were positive for AdVs by PCR. Oral swabs as solitary
195 probes taken from several Gila monsters from the same enclosure collected some time after
196 the original detection were also all positive (No.8 & No.9). However, the companion beaded
197 lizard and two leopard tortoises from the same enclosure tested two months after the original
198 detection gave negative PCR results (data not shown). The latest sampling, of 28 new
199 quarantined Heloderma spp. specimens 1 year after the outbreak detected 12 positives among
200 the Gila monsters (No.11).
201 The kidney and spleen tissue samples from the emerald monitor ( Varanus prasinus ) that died
202 with a hepatitis were PCR positive although the liver was negative (No.12).
203 AdVs were also detected in a snake collection with no specific signs indicative of AdV
204 infection. No.13 was a batch of samples from snakes at a collection containing nine
205 specimens from two asp viper subspecies ( Vipera aspis aspis, Vipera a. francisciredi ) and
206 two striped Aesculapian rat snakes ( Elaphe lineata ). The solitary AdV positive sample from
207 this batch was an asp viper which died suddenly and organ samples of intestine, liver, kidney
208 and lung were sent in for paramyxovirus (PMV) testing, along with oral and cloacal swabs
209 from each living companion. PMV was detected in three of these survivors but not in the
210 tissues of theAccepted dead snake. However, a pooled organ Manuscript sample of this animal was positive in the
211 AdV consensus nested PCR.
212 In many cases, multiple bands of nonspecific amplicons were visible above the approx. 320
213 bp long second round specific product, making the evaluation more difficult. In the case of
214 No.8, these bands were equally strong as the specific one.
9 Page 9 of 27 215 Sequence analysis
216 The second round PCR products were all 272 nt long after editing out the primer sequences.
217 These sequences were used for further analysis. An identity matrix of the sequences is shown
218 in Fig. 3, an alignment of the amino acid sequences in Fig. 4 and a phylogenetic tree based on
219 this alignment is shown in Fig.5. The sequences were submitted to GenBank and have been
220 assigned the accession numbers EU914202 to EU914209 (Table 1).
221 The six agamid AdVs detected are most similar to one another. There is a maximum of 6 nt
222 differences between the sequences, with at most 2 non-silent mutations. In the data set of the
223 follow-up study from the Heloderma spp. , the sequences from the Mexican beaded lizards
224 were always distinctly different from those of the Gila monsters. All Gila sequences in the
225 batches No.10 and No.11 were identical with the corresponding GenBank Helodermatid AdV
226 sequence. Similarly, the sequences of all of the Mexican beaded lizard samples were identical
227 with one another, but show 24 nt differences compared to the Helodermatid AdV sequence.
228 Yet only 3 of these were non-silent mutations. The partial sequence obtained from the
229 emerald monitor AdV No.12 has the highest BLASTX values with the snake AdV sequence,
230 whereas the identity matrix of the nucleotide sequences shows the highest value with the
231 Agamid AdVs. The asp viper AdV (sample No.13) has the greatest similarity to the
232 Eublepharid AdV and the Helodermatid AdV with approx. 65-68 % and 70-72 % similarity
233 values on the nucleotide and amino acid level respectively. The partial polymerase sequence 234 from the AdVAccepted isolate from a Boa constrictor used Manuscriptas a positive control in all PCRs (No. 14) 235 was identical to the corresponding region of the SnAdV-1.
236
237 Discussion
10 Page 10 of 27 238 This is the first report of the successful isolation of a lizard AdV. Although there have been
239 several papers describing the occurrence of AdVs in eublepharid, geckonid, varanid,
240 chameleonid and agamid Squamates (Essbauer and Ahne, 2001; Farkas et al., 2002;
241 Wellehan et al,. 2004), these viruses have never been isolated. All reptilian isolates to date
242 are from snakes, and there is evidence that these might represent the same virus (Marschang
243 et al., 2003). Isolation of the two different Helodermatid AdVs, in one case from a Mexican
244 beaded lizard and two cases from Gila monsters is therefore an important step in the further
245 study of AdVs of reptiles. PCR amplified partial sequence comparison is possible with field
246 samples, but virus isolation is essential for physico-chemical and ultrastructural
247 characterisation as well as for pathogenicity studies.
248 The comparison of the partial polymerase sequences of our helodermatid sequences with that
249 in GenBank has revealed that the Gila monsters and Mexican beaded lizards posess two
250 different AdVs. The GenBank helodermatid AdV sequence from a Gila monster in the USA
251 is identical to our Danish Gila sequences, whereas our beaded lizard isolate and sample
252 sequences from the same enclosure differ from these. This finding supports the host-
253 specificity coevolution-cospeciation theory of AdVs (Harrach, 2000). However, this strict
254 host-specificity is not supported by the identical sequences found in isolates from different
255 snakes (Boa sample No.14 and SnAdV-1 from a corn snake; also in Marschang et al., 2003;
256 python, corn snake & rat snake samples in Benk ı et al., 2006). The difference (8.9% aa and 257 3.3% nt) betweenAccepted the gene portions of the two helodermatidManuscript viruses are relatively small. 258 Compared to those between other AdVs, these viruses seem to represent the same species.
259 Further sequencing and/or serological testing are needed for further characterization of these
260 isolates.
11 Page 11 of 27 261 Our six bearded dragon AdV sequences possess less than 2.5% nt variation in the examined
262 region, and thus, together with data from GenBank and additional sequences from Hungary
263 (Benk ı et al, 2006; Farkas, personal communication) they provide further support that a
264 single agamid AdV is circulating in the bearded dragon collections across Europe and the
265 USA.
266 There were several interesting aspects of the AdV found in the emerald monitor (No.12).
267 Although the histological examination of this animal showed hepatitis, no AdV was detected
268 in the liver by PCR. The spleen and kidney were both PCR positive and the sequence
269 obtained from these amplicons revealed that this varanid AdV is very different from every
270 earlier described reptilian AdVs. Using either nucleotide or amino acid sequence in the
271 distance matrix analysis the virus clustered not in but beside the Atadenovirus genus, equally
272 distant to the proposed “Ichtadenovirus” genus and the Mastadenovirus genus. Yet the
273 sequence is short and the bootstrap values at this branching of the tree are under 50 (Fig. 5).
274 Further sequencing is necessary to determine the exact taxonomic position of this virus.
275 The asp viper sample (No.13) is a good example for the successful detection of new AdVs in
276 blind surveys. This snake was the only animal that died in a collection of snakes in which a
277 PMV was detected although only the companion animals were positive in a PMV RT-PCR.
278 The phylogenetic analysis of the polymerase gene portion of the AdV in this animal has
279 clustered this virus into the Atadenovirus genus, but representing a new species: Viperid
280 AdV. Accepted Manuscript
281 Each of the viruses presented above have a balanced A+T content (41.7% - 57.5%) in the
282 sequenced region which might indicate that they have coevolved over a longer period of time
283 together with their hosts (Farkas et al., 2002; Wellehan et al., 2004).
12 Page 12 of 27 284 In conclusion, while the majority of our sequence data support the coevolution-cospeciation
285 theory and the reptilian origin of atadenoviruses, a close look at individual reptilian AdV
286 shows that this theory may not explain all of the AdV found in reptiles. One open question is
287 the occurance of the same AdV in different snake hosts. The other is posed by the Varanid
288 AdV which does not seem to belong to the Atadenovirus genus. Additional work is
289 necessary, both to obtain additional taxonomic information on the AdVs described here as
290 well as to characterize further reptilian AdV.
291
292 Acknowledgements
293 We would like to thank Silvia Speck and Christa Shäfer for their excellent technical
294 assistance. Samples were sent to our laboratory by Dr. Marc Hoferer from the Chemisches-
295 und Veterinär Untersuchungsamt (CVUA), Stuttgart-Fellbach; Dr. Karina Mathes and Ms.
296 Günther, Small Animal Clinic of the Veterinary College of Hannover; Dr. Jutta Wiechert,
297 Veterinary Praxis, Mainz; Dr. Bernd Dörsch, Small Animal Praxis, Kiel, and Dr. Michael
298 Pees, Bird and Reptile Klinik of Leipzig University, all in Germany. Their help is gratefully
299 acknowledged. We also thank Valerij Akimkin at the CVUA, Stuttgart-Fellbach, Germany,
300 for his help with the electron microscopic examination and Dr. Balázs Harrach at the
301 Veterinary Medical Research Institute, Budapest, Hungary for his advice in taxonomical 302 questions. Dr.Accepted Kaján’s work was partially supported Manuscript by the National Office for Research and 303 Technology, József Öveges programme, and the Hungarian Scientific Research Fund, grant
304 OTKA K 67781.
305
13 Page 13 of 27 306 References
307 Benk ı, M., Él ı, P., Ursu, K., Ahne, W., LaPatra, S.E., Thomson, D., Harrach, B., 2002. First
308 molecular evidence for the existence of distinct fish and snake adenoviruses. J. Virol.
309 76, 10056-9.
310 Benk ı, M., Harrach, B., Both, G.W., Russell, W.C., Adair, B.M., Ádám, É., de Jong, J.C.,
311 Hess, M., Johnson, M., Kajon, A., Kidd, A.H., Lehmkuhl, H.D., Li, Q.G., Mautner,
312 V., Pring-Akerblom, P., Wadell, G., 2005. Family Adenoviridae. In: Fauquet, C.M.
313 (Ed.), Virus Taxonomy. Eighth Report of the International Committee on Taxonomy
314 of Viruses, Academic Press, New York, pp. 213-228.
315 Benk ı, M., Kaján, G.L., Jánoska, M., Kovács, E.R., Wellehan, J.F.X., Zsivanovits, P.,
316 Cough, R.E., Blahak, S., Schirrmeier, H., Bakonyi, T., Harrach, B., 2006. How to find
317 “new” adenoviruses. In: Leitão, A., Martins, C. (Eds.), Proceedings of the 7 th
318 International Congress of Veterinary Virology. Ministério da Agricultura, Lisboa,
319 Portugal, p. 103.
320 Bonfield, J.K., Smith, K.F., Staden, R., 1995. A new DNA sequence assembly program.
321 Nucleic Acids Res. 24, 4992-4999.
322 Essbauer, S., Ahne, W., 2001. Viruses of lower vertebrates. J. Vet. Med. B, Infectious
323 Diseases and Veterinary Public Health 48, 403-475.
324 Farkas, S.L., Benk ı, M., Él ı, P., Ursu, K., Dán, Á., Ahne, W., Harrach, B., 2002. Genomic 325 and phylogeneticAccepted analyses of an adenovirus Manuscript isolated from a corn snake ( Elaphe 326 guttata ) imply a common origin with members of the proposed new genus
327 Atadenovirus. J. Gen. Virol. 83, 2403-2410.
328 Farkas, S.L., Harrach, B., Benk ı, M., 2008. Completion of the genome analysis of snake
329 adenovirus type 1, a representative of the reptilian lineage within the novel genus
330 Atadenovirus . Vir. Res. 132, 132-139.
14 Page 14 of 27 331 Felsenstein, J., 1989. PHYLIP - Phylogeny Inference Package. Cladistics 5, 164-166.
332 Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis
333 program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41, 95-98.
334 Harrach, B., 2000. Reptile adenoviruses in cattle? Acta Vet. Hung. 48, 484-490.
335 Jacobson, E.R., Kollias, G.V., 1986. Adenovirus-like infection in a savannah monitor. J. Zoo
336 Anim. Med. 17, 149-151.
337 Jacobson, E.R., Gardiner, C.H., 1990. Adeno-like virus in esophageal and tracheal mucosa of
338 a Jackson’s chameleon ( Chamaeleo jacksoni ). Vet. Path. 27, 210-212.
339 Jacobson, E.R., Gardiner, C.H., Foggin, C.M., 1984. Adenovirus-like infection in two Nile
340 crocodiles. J. Am.Vet. Med. Assoc. 185, 1421-1422.
341 Jacobson, E.R., Gaskin, J.M., Gardiner, C.H., 1985. Adenovirus-like infection in a boa
342 constrictor. J. Am. Vet. Med. Assoc. 187, 1226-1227.
343 Juhász, A., Ahne, W., 1992. Physico-chemical properties and cytopathogenicity of an
344 adenovirus-like agent isolated from corn snake ( Elaphe guttata ). Arch. Virol. 130,
345 429-439.
346 Marschang, R.E., Mischling, M., Benk ı, M., Papp, T., Harrach, B., Böhm, R., 2003.
347 Evidence for wide-spread atadenovirus infection among snakes. In: Jestin, A.,
348 Clement, G. (Eds.), Virus persistence and evolution. Proceedings of the 6 th
349 International Congress of Veterinary Virology. ZOO-POLE développement –ISPAIA,
350 Ploufragan, France. p. 152.
351 Ogawa, M., Ahne,Accepted W., Essbauer, S., 1992. Reptilian Manuscript viruses: adenovirus-like agent isolated
352 from royal python ( Python regius ). J. Vet. Med.. B, Infectious Diseases and
353 Veterinary Public Health 39, 732-736.
15 Page 15 of 27 354 Perkins, L.E, Campagnoli, R.P., Harmon, B.G., Gregory, C.R., Steffens, W.L., Latimer, K.,
355 Clubb, S., Crane, M., 2001. Detection and confirmation of reptilian adenovirus
356 infection by in situ hybridization. J. Vet. Diagn. Invest. 13, 365-368.
357 Ramis, A., Fernandez-Bellon, H., Majo, N., Martinez-Silvestre, A., Latimer, K., Campagnoli,
358 R., Harmon, B.G., Gregory, C.R., Steffens, W.L., Clubb, S., Crane, M., 2000.
359 Adenovirus hepatitis in a boa constrictor ( Boa constrictor ). J. Vet. Diagn. Invest. 12,
360 573-576.
361 Russell, W.C., Benk ı, M., 1999. Animal adenoviruses. In: Granoff, A., Webster, R.G. (Eds.),
362 Encyclopedia of Virology. Academic Press, New York, pp. 14-21.
363 Schumacher, J., Jacobson, E.R., Burns, R., Tramontin, R.R., 1994. Adenovirus-like infection
364 in two rosy boas ( Lichanura trivirgata ). J. Zoo and Wildlife Med. 25, 461-465.
365 Wellehan, J.F.X., Johnson, A.J., Harrach, B., Benk ı, M., Pessier, A.P., Johnson, C.M.,
366 Garner, M.M., Childress. A., Jacobson, E.R., 2004. Detection and analysis of six
367 lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian
368 origin for the atadenoviruses. J Virol. 78, 13366-9.
369 Zsivanovits, P., Monks, D.J., Forbes, N.A., Ursu, K., Raue, R., Benk ı, M., 2006.
370 Presumptive identification of a novel adenovirus in a Harris hawk ( Parabuteo
371 unicinctus ), a Bengal eagle owl ( Bubo bengalensis ), and a Verreaux’s eagle owl
372 (Bubo lacteus ). J. Avian Med. Surgery 20, 105-112. 373 Accepted Manuscript
16 Page 16 of 27 374 Table 1. Adenovirus positive samples with short case histories, the results of virus isolation
375 on cell-culture and GenBank accession numbers of the obtained partial DNA polymerase
376 gene sequences. Only PCR positive animals are listed.
377 Species Ident. Age Case history Sample Virus AdV Investigation GenBank No. type isolation PCR for other Acc. No. agents Imported from the EU914202 continuous USA, CNS o./cl. problems with 1 ? symptoms, __ all pos. swabs coccidiosis, IIV- opisthotonus, PCR negativ spasms o./cl. __ all __ EU914203 2 ? No report swabs pos. organ, __ EU914203 3 ? No report all pos. IIV-PCR negativ feaces Abnormal EU914204 blinking; inactivity, Lung of this apathy, hiding, animal, as well anorexia, dark liver, Bearded as swab of 4 adult colouration, kidney, __ all pos. dragon partner animal rounded back with lung Pogona was positive in fallen tail, vitticeps IIV-PCR tachycardia, dyspnoea, exitus . EU914205 Chronic blood, swab __ 5 adult gastrointestinal __ cl. swab pos. problems
Paralysis of the EU914206 hind limbs and blood, tail, trembling, swabs 6 adult o./cl. __ IIV-PCR negativ hyperesthesia: pos. swabs root of the tail and the back Kept together with EU914207 Gila monsters and leopard tortoises. intestine, Rana- and 7* juv. Outbreak with liver, all + all pos. Herpesvirus- Mexican several deaths heart (IgH2 cells) PCR negativ beaded among the lizard Heloderma spp . Heloderma Swab taken 2 EU914207 horridum weeks later from o./cl. __ 8* juv. the two survivors __ all pos. swabs of the above group (both pos.) Swab taken 2 AY576680 † weeks later from __ 9* juv. a survivor Gila in oral swab __ all pos. the same enclosure AcceptedSwabs taken half ManuscriptAY576680 † a year p.ob., from 2 new animals Gila oral __ 10* juv. which had no 1 swab + all pos. monster swabs contact with the (IgH2 cells) Heloderma others (both suspectum positive) Swab taken a AY576680 † year p. ob., from 21 Gila and 7 oral 12 Gila __ 11* juv. beaded lizard, all 1 swab + swabs pos. new animals, no (IgH2 cells) contact with the others Emerald 12 ? Died, hepatitis liver, __ spleen, __ EU914208
17 Page 17 of 27 monitor spleen, kidney Varanus kidney pos. prasinus 3 of the survivors EU914209 Asp viper Group of nine internal positive for PMV Vipera a. 13 ? snakes, this one __ pos. organs in RT-PCR, this aspis died one not Common Isolate from an DQ106414 † boa earlier study, VH2 cell __ 14 ? + pos. Boa served as a passage constrictor positive control 378 379 cl.= cloacal, CNS= central nervous system, Ident. No.= identification number, IgH2= iguana
380 heart cell line (ATCC: CCL-108), IIV = invertebrate iridovirus, juv.= juvenile, o.= oral,
381 PMV= paramyxovirus, p.ob.= post outbreak, RT-PCR= reverse trancriptase polymerase
382 chain reaction, USA= United States of America, * = from the same owner, †Corresponding
383 sequence identical to that of the existing GenBank entry
384
Accepted Manuscript
18 Page 18 of 27 385 Figure captions:
386
387 Fig. 1. Adenovirus isolate from a Gila monster ( Heloderma suspectum ). Cytopathic effect
388 (CPE) five days after inoculation. A- Negative control IgH-2. B - 3 rd passage of an isolate
389 from an oral swab from case No.11 on IgH2 cells 5 days post inoculation. (Magnification:
390 400 x)
391 Fig. 2. Electron micrographs of icosahedral non-enveloped virus particles in cell culture
392 supernatant from two Helodermatid isolates. (Both 3 rd passage on IgH2 cells 5 days post
393 inoculation). A- Mexican beaded lizard isolate with identification No.7. B- Isolate from an
394 oral swab from Gila monster with identification No.10
395 Fig. 3. Identity matrix of the nucleotide (above diagonal, with white letters) and the deduced
396 amino acid (below diagonal, with black letters) sequences of the amplified DNA-polymerase
397 gene region. Accepted and proposed members of the Atadenovirus genus from GenBank are
398 marked with lighter background colouring compared to the sequences newly reported here
399 with darker grey background. Highest values for the new sequences compared to GenBank
400 sequences are shown in bold. For accession numbers see legend of Fig. 5; Agamid AdV,
401 Austria (AAY83284), Agamid AdV, USA (AY576678).
402 Fig. 4. Alignment of predicted partial DNA-polymerase sequences of the amplified region
403 from the atadenoviruses and the sturgeon adenovirus. (Identical sequences are represented
404 by one selected virus. Start of alignment corresponds to the codon at position 6458 of
405 HAdV-1 wholeAccepted genomic nucleotide sequence, Accession Manuscript No.: AF534906.) Sequences of
406 reptilian viruses are printed with capital letters, sequences new to science are highlighted
407 with bold printed names. OAdV-7 type species was taken as a null-sequence for the
408 comparison, identical amino acids are marked with dots in the rest of the alignment. Virus
409 species with more than one serotype are separated with lines and indicated with grey
19 Page 19 of 27 410 background shading of the names. Vertical background shading of sequences in black and
411 grey refers to 80% identity and similarity of conserved amino acid positions respectively,
412 regarding the alignment of all available sequences for this region. (helodermatid and
413 agamid isolates show a maximum of 3 amino acid variations between one another, and are
414 therefore also grouped together as they most probably belong to one species.) For
415 abbreviations and GenBank accession numbers see legend of Fig. 5.
416 Fig 5. Phylogenetic distance tree of partial adenovirus DNA-polymerase amino acid
417 sequences. The tree was generated using the Protdist program with Dayhoff matrix, followed
418 by Fitch program with global rearrangements. Bootstrap values above 50 (for 100
419 replications) are shown beside the branches, lower value branchings are shown with dotted
420 lines. Accepted and presumed virus species are indicated with grey circles, genera with half
421 circles. Names of reptilian viruses are printed in italics, and those representing sequences
422 from our study (too), are in bold italics.
423 Abbreviations and GenBank accession numbers (in brackets): B=Bovine, C=Canine,
424 D=Duck, F=Fowl, H=Human, M=Murine, O=Ovine, P=Porcine, Rus=BadV-4 strain Rus,
425 S=Simian, T=Turkey, TS=Tree shrew
426 Agamid AdV (AY576678 and AAY83284), BAdV-1 (YP_094032), BAdV-2 (AP_000006),
427 BAdV-3 (AP_000026), BAdV-4 (AAK13183), BAdV-4 Rus (not yet released), Bat AdV
428 (AB303301), CAdV-1 (AAB05434), CAdV-2 (AAB38716), Chameleonid AdV
429 (AY576679), DAdV-1 (AP_000080), Eublepharid AdV (AY576677), FAdV-1 (AP_000410),
430 FAdV-5 (DQ159938),Accepted FAdV-9 (AC_000013), Manuscript Frog (AAF86924), Gekkonid AdV
431 (AY576681), HAdV-9 (CAI05960), HAdV-12 (CAA51882), HAdV-17 (AP_000143),
432 HAdV-40 (AAC13953), HAdV-52 (ABK35035), HAdV-B=HAdV-3 (ABB17778) + HAdV-
433 7 (AP_000539) + HAdV-35 (AAN17476), HAdV-E=HAdV-4 (AAS66917) + SAdV-25
434 (AP_000304), Helodermatid AdV (AAS89696), MAdV-1 (AP_000342), OAdV-7
20 Page 20 of 27 435 (AAD45950), PAdV-3 (AB026117), PAdV-5 (AAK26504), Parakeet AdV (EU056825),
436 SAdV-1 (AAX19399), SAdV-3 (AAT84618), Scincid AdV (AY576682), Snake AdV
437 (AAL89790), Sturgeon AdV (not yet released), TAdV-3 (AAC64523), Tortoise AdV
438 (EU056826), TSAdV (YP_068060),
439
440
Accepted Manuscript
21 Page 21 of 27 Figure 1A
Accepted Manuscript
Page 22 of 27 Figure 1B
Accepted Manuscript
Page 23 of 27 Figure 2
Accepted Manuscript
Page 24 of 27 Figure 3
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 0,597 OAV-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 0,579 BAdV-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 0,601 SnAdV-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 1,000 Chameleonid 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 0,627 Gekkonid 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 0,565 Scincid 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 0,645 Helodermatid 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 0,697 Eublepharid 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 0,671 Agamid-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 0,649 Agamid-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 0,653 No.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 0,649 No.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 0,645 No.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 0,645 No.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 0,645 No.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 0,649 No.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 0,701 No.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 0,697 No.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 0,604 No.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 0,649 No.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
Accepted Manuscript
Page 25 of 27 Figure 4
HAdV-E
10 20 30 40 50 60 70 80 90 | | | | | | | | | | | | | | | | | | Ovine 7 salthplpygktlnafeanaqidyfqellqr-kekidyfdnsikpmivvadceppsldyldvlpplcskksgklcwsnetlinevltsidl Duck 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.BAdV-D 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.I GEKKONID ...... 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.I HELOD.No8HeAdV ...S..M...L..SPLD.SVAMAR..DK.ES-TG.LSF..KD.L....K...F..P.YH...... R...T..P.LG....TV.I AGAMID ...S..M.C.R..PPLD.SIE.RR..DK.DK-PH..S...PNL.....A...I..P.NE...... A..R...T..P.VG...... AGAMAAgAdV 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.I VARAN.No12 ...?..M.S.NPESP.D.AIS.EN.RR..EQ-I.P.....PR...... TV.AD...D.Q...... RH..R...T..P.RK....TV.I
Sturgeon ...... m...yp.epl..sih.kl.....d.-.e..s..ndtv....lsi.ah..nin...t...... rq..r...t..a..s.iv..l.c
Accepted Manuscript
Page 26 of 27 Figure 5
B3 S3 H40 TS H52 M1 C1 C2 P5 S1 P3 100 100 H12 Bat 75 89 H17 100 H9 F9 F5 57 B1 71 B2 F1
99 Parrot 100 No.12 Varanus 100 Frog 52 100 66 54 Sturgeon Tortoise
Parakeet 97
100 No.13 Vipera 73 Gekkonid B4 100 T3 Rus 100 Scincid
Agamid O7 D1 Snake-1 Euble- Chameleonid Helo- pharid dermatid (Gila) No. 7+8 0.1 Accepted ManuscriptHeloderma
Page 27 of 27