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Herpesviruses are well-known infectious agents with remarkably wide host ranges. Starting in 1975 (33), several reports have documented the presence of herpesvirus-like particles in land tortoises and freshwater and marine turtles (5, 7-9, 11, 15, 17, 19-23, 27-29, 30, 38). Recent investigations have revealed an association between the presence of herpesvirus and an upper respiratory tract disease in Mediterranean tortoises [spur-thighed tortoise (Testudo graeca) and Hermann's tortoise (T. hermanni)] (5,8,9,17,20,23,(27)(28)(29)30).In tortoises with herpesvirus infection, clinical signs range from a mild conjunctivitis to a severe stomatitis-glossitis and pharyngitis. Diphtheritic plaques can be observed on the dorsal surface of the tongue and on the hard palate of infected tortoises. Frequently, a clear serous to a mucopurulent nasal discharge is present. Signs of central nervous system disease have also been reported in Mediterranean tortoises with herpesvirus infection (17).Eosinophilic intranuclear inclusions, often seen in multiple tissues, are particularly prominent in tortoises with pharyngitis and glossitis. As seen with transmission electron microscopy, inclusions consist of numerous viral particles. The morphology and morphogenesis have been used to categorize the virus as herpesvirus.A diagnosis of herpesvirus infection is often made based solely upon light or electron microscopy findings. Antemortem diagnosis can be made using biopsy specimens of oral lesions. A serum neutralization (SN) test has been developed but is limited in its application since it is only available in a few research laboratories in Europe (10). In addition, time is a limiting factor with the SN test. Ten to 14 days are required to obtain the final reading and a laborious procedure is required. An easier and faster but equally reliable serodiagnostic test is needed. In this report, we describe the development of an enzyme-linked immunosorbent assay (ELISA) that can be used to monitor the exposure to herpesvirus of free-range, private, and zoo collections of tortoises. MATERIALS AND METHODSViruses. Herpesvirus isolates HV1976 and HV4295/7R/95 were used as antigens in the ELISAs and immunoblotting. HV1976 was isolated from a captive Hermann's tortoise from the United States (Washington), while HV4295/7R/95 was isolated from a captive Hermann's tortoise in Germany during a herpesvirus outbreak in a private collection (27).Antigen preparation for ELISA. The herpesvirus isolates were grown in terrapene heart cell monolayers (TH-1; ATCC-CCL 50 Sub-line B1; American Type Culture Collection, Rockville, Md.) in T-150 plastic flasks with ventilated caps (Corning, Rochester, N.Y.) for use as ELISA antigens. The TH-1 cells were grown in Dulbecco's modified Eagle's medium F12 (Gibco BRL, Grand Island, N.Y.) with 5% fetal bovine serum (Sigma, St. Louis, Mo.), gentamicin (60 mg/liter) (Sigma), penicillin G (120,000 U/liter), streptomycin (120,000 U/liter), and amphotericin B (300 g/liter) (ABAM; Sigma). Infected cell monolayers were scraped a...

Background: Pneumonia and stomatitis represent severe and often fatal diseases in different captive snakes. Apart from bacterial infections, paramyxo-, adeno-, reo-and arenaviruses cause these diseases. In 2014, new viruses emerged as the cause of pneumonia in pythons. In a few publications, nidoviruses have been reported in association with pneumonia in ball pythons and a tiger python. The viruses were found using new sequencing methods from the organ tissue of dead animals.Methods: Severe pneumonia and stomatitis resulted in a high mortality rate in a captive breeding collection of green tree pythons. Unbiased deep sequencing lead to the detection of nidoviral sequences. A developed RT-qPCR was used to confirm the metagenome results and to determine the importance of this virus. A total of 1554 different boid snakes, including animals suffering from respiratory diseases as well as healthy controls, were screened for nidoviruses. Furthermore, in addition to two full-length sequences, partial sequences were generated from different snake species. Results: The assembled full-length snake nidovirus genomes share only an overall genome sequence identity of less than 66.9% to other published snake nidoviruses and new partial sequences vary between 99.89 and 79.4%. Highest viral loads were detected in lung samples. The snake nidovirus was not only present in diseased animals, but also in snakes showing no typical clinical signs.Conclusions: Our findings further highlight the possible importance of snake nidoviruses in respiratory diseases and proof multiple circulating strains with varying disease potential. Nidovirus detection in clinical healthy individuals might represent testing during the incubation period or reconvalescence. Our investigations show new aspects of nidovirus infections in pythons. Nidoviruses should be included in routine diagnostic workup of diseased reptiles.

Ranaviruses in amphibians and fish are considered emerging pathogens and several isolates have been extensively characterized in different studies. Ranaviruses have also been detected in reptiles with increasing frequency, but the role of reptilian hosts is still unclear and only limited sequence data has been provided. In this study, we characterized a number of ranaviruses detected in wild and captive animals in Europe based on sequence data from six genomic regions (major capsid protein (MCP), DNA polymerase (DNApol), ribonucleoside diphosphate reductase alpha and beta subunit-like proteins (RNR-α and -β), viral homolog of the alpha subunit of eukaryotic initiation factor 2, eIF-2α (vIF-2α) genes and microsatellite region). A total of ten different isolates from reptiles (tortoises, lizards, and a snake) and four ranaviruses from amphibians (anurans, urodeles) were included in the study. Furthermore, the complete genome sequences of three reptilian isolates were determined and a new PCR for rapid classification of the different variants of the genomic arrangement was developed. All ranaviruses showed slight variations on the partial nucleotide sequences from the different genomic regions (92.6–100%). Some very similar isolates could be distinguished by the size of the band from the microsatellite region. Three of the lizard isolates had a truncated vIF-2α gene; the other ranaviruses had full-length genes. In the phylogenetic analyses of concatenated sequences from different genes (3223 nt/10287 aa), the reptilian ranaviruses were often more closely related to amphibian ranaviruses than to each other, and most clustered together with previously detected ranaviruses from the same geographic region of origin. Comparative analyses show that among the closely related amphibian-like ranaviruses (ALRVs) described to date, three recently split and independently evolving distinct genetic groups can be distinguished. These findings underline the wide host range of ranaviruses and the emergence of pathogen pollution via animal trade of ectothermic vertebrates.

Ranaviral disease in amphibians has been studied intensely during the last decade, as associated mass-mortality events are considered to be a global threat to wild animal populations. Several studies have also included other susceptible ectothermic vertebrates (fish and reptiles), but only very few cases of ranavirus infections in lizards have been previously detected. In this study, we focused on clinically suspicious lizards and tested these animals for the presence of ranaviruses. Virological screening of samples from lizards with increased mortality and skin lesions over a course of four years led to the detection of ranaviral infections in seven different groups. Affected species were: brown anoles (Anolis sagrei), Asian glass lizards (Dopasia gracilis), green anoles (Anolis carolinensis), green iguanas (Iguana iguana), and a central bearded dragon (Pogona vitticeps). Purulent to ulcerative-necrotizing dermatitis and hyperkeratosis were diagnosed in pathological examinations. All animals tested positive for the presence of ranavirus by PCR and a part of the major capsid protein (MCP) gene of each virus was sequenced. Three different ranaviruses were isolated in cell culture. The analyzed portions of the MCP gene from each of the five different viruses detected were distinct from one another and were 98.4-100% identical to the corresponding portion of the frog virus 3 (FV3) genome. This is the first description of ranavirus infections in these five lizard species. The similarity in the pathological lesions observed in these different cases indicates that ranaviral infection may be an important differential diagnosis for skin lesions in lizards.