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 To cite this version: 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 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Manuscript 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Manuscript 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: 16 [email protected] 17 18 Abstract 19 A consensus Acceptednested 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.