Three Novel Herpesviruses Associated with Stomatitis in Sudan Plated Lizards (Gerrhosaurus Major) and a Black-Lined Plated Lizard (Gerrhosaurus Nigrolineatus)
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Journal of Zoo and Wildlife Medicine 35(1): 50±54, 2004 Copyright 2004 by American Association of Zoo Veterinarians THREE NOVEL HERPESVIRUSES ASSOCIATED WITH STOMATITIS IN SUDAN PLATED LIZARDS (GERRHOSAURUS MAJOR) AND A BLACK-LINED PLATED LIZARD (GERRHOSAURUS NIGROLINEATUS) James F. X. Wellehan, D.V.M., M.S., Donald K. Nichols, D.V.M., Dipl. A.C.V.P., Ling-ling Li, M.S., and Vivek Kapur, B.V.Sc., M.Sc., Ph.D. Abstract: Glossal stomatitis was observed in a Sudan plated lizard (Gerrhosaurus major) with severe dyspnea. On necropsy, intranuclear inclusion bodies were seen in the periglottal lingual epithelium. Labial stomatitis was seen in a second Sudan plated lizard and a black-lined plated lizard (G. nigrolineatus). Degenerate polymerase chain reaction (PCR) primers targeting a conserved region of herpesvirus DNA±dependent DNA polymerase gene were used to amplify products from lesions from each lizard. Nucleotide sequencing of the PCR products showed that the sequence from each lizard was unique. Phylogenetic and comparative sequence analyses suggest that these viruses are novel members of the subfamily Alphaherpesvirinae, and they are here termed gerrhosaurid herpesviruses 1±3. Results of our analyses suggest that the genus Gerrhosaurus can be infected by these novel herpesviruses. Key words: Gerrhosaurus major, Gerrhosaurus nigrolineatus, herpes, stomatitis, virus, plated lizard. INTRODUCTION reptiles. All reptile herpesvirus sequences available in the public databases (GenBank, National Center Herpesviridae is a diverse family of enveloped for Biotechnology Information, Bethesda, Mary- double-stranded DNA viruses found in many dif- land 20894, USA; EMBL, Cambridge, United ferent orders of vertebrates, including ®sh, amphib- Kingdom; and Data Bank of Japan, Mishima, Shi- ians, reptiles, birds, and mammals.15 Herpesviruses uoka, Japan) are from chelonians and appear to be have been further divided into the subfamilies Al- most closely related to members of the Alphaher- phaherpesvirinae, Betaherpesvirinae, and Gammah- 17,20 erpesvirinae on the basis of properties of infection, pesvirinae. host range, and behavior in culture. Evidence from The herpesviruses described in lizards are lac- DNA analysis has largely reinforced the extant ertid herpesvirus, iguana herpesvirus, and agamid classi®cation system and suggests the presence of herpesvirus. Lacertid herpesvirus has been seen by further subfamilies. All known sequences of ®sh electron microscopy associated with cutaneous pap- 18 and amphibian herpesviruses appear to be distinct illomatous lesions. Iguana herpesvirus was isolat- from the subfamilies listed above, and an oyster ed from iguana heart cell cultures. Cytopathic ef- herpesvirus may also be distinct.15 To date, pub- fects were seen in cell culture. Inoculation of 12 lished analyses of sequences of avian herpesviruses young iguanas produced no consistent pattern of all appear to belong to the subfamily Alphaherpes- lesions, and although there was a much higher mor- virinae.15,21 tality rate in the inoculated population, no causal 2 Herpesviruses of reptiles have been described in relationship was established. Agamid herpesvirus chelonians,3,5,6,10,11 snakes,7,12,16 and lizards,2,18,22 as- was seen by electron microscopy in the liver, lung, sociated with a variety of lesions, including sto- and spleen of two red-headed agamas (Agama aga- 22 matitis in tortoises.10 However, with a few excep- ma) that died. tions, little information is available regarding the This study describes stomatitis in two species of genetic classi®cation of the viruses recovered from gerrhosaurid lizards associated with the presence of three unique herpesviruses. These are the ®rst re- ported sequences for herpesviruses in lizards, and From the Departments of Microbiology and Veterinary the results of our analyses suggest that the genus Pathobiology, and Biomedical Genomics Center, Univer- Gerrhosaurus can be infected by members of the sity of Minnesota, St. Paul, Minnesota 55108, USA (Wel- Alphaherpesvirinae. lehan, Li, Kapur); and the Department of Pathology, Na- tional Zoological Park, Washington, D.C. 20008, USA MATERIALS AND METHODS (Nichols). Present address (Wellehan): Department of Small Animal Clinical Sciences, College of Veterinary Study animals Medicine, University of Florida, Gainesville, Florida 32610, USA. Correspondence should be directed to Dr. Case 1: A male Sudan plated lizard (G. major), Wellehan. at least 6 yr old, from a private collection in Min- 50 WELLEHAN ET AL.ÐTHREE NOVEL GERRHOSAURID HERPESVIRUSES 51 Figure 1. a. Sudan plated lizard (Gerrhosaurus major) with periglottal glossal stomatitis (case 1). b. Sudan plated lizard (G. major) with labial stomatitis (case 3). c. Black-lined plated lizard (G. nigrolineatus) with maxillary labial stomatitis (case 2). nesota was presented for necropsy (Fig. 1a) after a the methods described previously.21 The ®rst round 4-mo history of severe dyspnea and periglottal in- of ampli®cation used forward primers DFA (59- ¯ammation that was not responsive to enro¯oxacin GAYTTYGCNAGYYTNTAYCC-39,Y5 pyrimi- (Baytril, Bayer, Shawnee Mission, Kansas 66201, dine, N 5 nucleotide) and ILK (59-TCCTGGA- USA; 10 mg/kg, p.o., s.i.d.) or piperacillin (Pipra- CAAGCAGCARNYSGCNMTNAA-39,R5 pu- cil, Lederle, Carolina, Puerto Rico 00984, USA; rine, M 5 A or C) and reverse primer KG1 (59- 100 mg/kg, s.c., s.i.d.) treatments. No signi®cant GTCTTGCTCACCAGNTCNACNCCYTT-39), and radiographic lesions were identi®ed. No new lizards the second round of ampli®cation used forward had been added to the collection for more than 5 yr. primer TGV (59-TGTAACTCGGTGTAYGGNT- Case 2: A male black-lined plated lizard (G. ni- TYACNGGNGT-39) and reverse primer IYG (59- grolineatus) with labial stomatitis was purchased CACAGAGTCCGTRTCNCCRTADAT-39,D5 A, from a pet store in Minnesota (Fig. 1c), where it G, or T). For both Sudan plated lizard samples, the had been housed with a female black-lined plated ®rst round was modi®ed to use IYG instead of KG1 lizard without stomatitis. No further history was as a reverse primer; use of the original protocol did available. not result in a product for either Sudan plated lizard Case 3: A second male Sudan plated lizard (case herpesvirus. 3), at least 6 yr old, from the same private collec- The PCR products were puri®ed using the QIA- tion as case 1 had a history of a chronic labial pro- quick PCR puri®cation kit (Qiagen), and the prod- liferative growth that periodically became ulcerated ucts of the PCR reactions were sequenced either during at least the past 6 yr (Fig. 1b). The lizard directly or after cloning into pGEM-T plasmid vec- had not been observed traumatizing the rostrum, tors (Promega, Madison, Wisconsin 53711, USA) and the caging material was not abrasive. No prior using the Big-Dye Terminator kit (Perkin±Elmer, history was available. The two Sudan plated lizards Branchburg, New Jersey 08876, USA) and ana- had been housed separately. lyzed on ABI 3100 automated DNA sequencers at the University of Minnesota Advanced Genetic Polymerase chain reaction ampli®cation and Analysis Center. All products were sequenced in sequencing sense and antisense directions. DNA was extracted from paraf®nized tissue from The sequences were compared with known se- case 1, and very small amounts of fresh tissue were quences in the public genetic databases (GenBank, debrided from the lesions for therapeutic purposes EMBL, and Data Bank of Japan) using the in cases 2 and 3. DNA extraction was performed TBLASTX algorithm.1 Predicted protein sequences with the QIAamp DNA mini kit (Qiagen, Valencia, of homologous 55- to 61±amino acid segments of California 91355, USA). herpes DNA polymerases were aligned by the Jo- Nested PCR ampli®cation of herpesvirus DNA± tun±Hein algorithm9 using MegAlign (DNAstar, dependent DNA polymerase was performed using Madison, Wisconsin 53711, USA), and phyloge- 52 JOURNAL OF ZOO AND WILDLIFE MEDICINE Figure 2. Eosinophilic intranuclear inclusions in tongue of case 1. Inclusions are indicated with arrows. H&E. Bar 5 10 mm. netic analyses were performed with the PHYLIP ulocytic and lymphocytic in¯ammation with ero- 3.573c package.4 Trees were out-group rooted using sion of overlying epithelium. The periglottal lingual the corresponding region of the delta polymerase epithelium contained areas with eosinophilic intra- gene of Plasmodium falciparum (GenBank acces- nuclear inclusions with both granulocytic and lym- sion No. S17330). Gaps of all lengths were counted phocytic in¯ammation of the underlying tissue (Fig. as single events. A total of 100 bootstrapped rep- 2). Histology was not done on cases 2 and 3. licates were performed, each data set was shuf¯ed 10 times in random order of input to create most Polymerase chain reaction ampli®cation and probable trees, and a majority-rule consensus tree sequencing was then created. Ampli®cation of herpesvirus DNA polymerase Sequence data were submitted to GenBank and gene sequences from DNA extracted from cases 1 are available under accession Nos. AF416628± and 2 using the primers and ampli®cation condi- AF416630. tions described above resulted in a 231±base pair (bp) product. Ampli®cation of herpesvirus DNA RESULTS polymerase gene sequences from case 3 using the Necropsy of case 1, a Sudan plated lizard, re- same primers and conditions resulted in a 234-bp vealed very little body fat. The periglottal tongue product. TBLASTX searches of the public genetic was raised and tan (Fig. 1a). The lungs appeared databases for herpesvirus DNA polymerase se- grossly normal; no other gross lesions were ob- quence from cases 1 and 2 showed the highest served. The tongue, glottis, and proximal trachea score with gallid herpesvirus 2 DNA polymerase were ®xed in 10% formalin and paraf®nized. He- (GenBank accession No. AAA79862). In contrast, matoxylin and eosin staining of the ®xed tissue re- DNA polymerase sequence from case 3 showed the vealed areas inside the glottal trachea of both gran- highest score with bovine herpesvirus 2 DNA poly- WELLEHAN ET AL.ÐTHREE NOVEL GERRHOSAURID HERPESVIRUSES 53 Figure 3.