Isolation and Characterization of two different Isolation and Characterization of two different Ranaviruses from Edible Frogs Ranaviruses from Edible Frogs ( Pelophylax Pelophylax kl. kl. esculentus esculentus ) in Switzerland ) in Switzerland Anke C. St Anke C. Stöhr hr 1 , Alexandra Hoffmann , Alexandra Hoffmann 2 , , Tibor Tibor Papp Papp 1,3 1,3 , Nicolas B. M. Pruvost , Nicolas B. M. Pruvost 2 , Heinz Heinz-Ulrich Reyer Ulrich Reyer 2 , , Rachel E. Marschang Rachel E. Marschang 1 1 Fachgebiet Fachgebiet für Umwelt r Umwelt- und Tierhygiene, Universit und Tierhygiene, Universität t Hohenheim, Stuttgart, Germany Hohenheim, Stuttgart, Germany 2 2 Institut f Institut für Evolutionsbiologie und Umweltwissenschaften, Universit r Evolutionsbiologie und Umweltwissenschaften, Universität Z t Zürich, Z rich, Zürich, Switzerland rich, Switzerland 3 Institute for Veterinary Medical Research, Centre for Agricultur Institute for Veterinary Medical Research, Centre for Agricultur al Research of the Hungarian Academy of Science, al Research of the Hungarian Academy of Science, Budapest, Hungary Budapest, Hungary Ranaviruses are large dsDNA viruses belonging to the family Iridoviridae. They are important pathogens of amphibians, fish and reptiles. In amphibians, systemic clinical infection is often associated with fatal mass-mortality events and is suspected to play an important role in the global amphibian decline (1, 2). Ranaviral disease is therefore considered an emerging infectious disease in amphibians and is notifiable to the Word Organisation for Animal Health (OIE). An increasing number of infections have been reported during the last decade on different continents. In Europe, ranaviruses have been detected in the United Kingdom (3, 4, 5, 6), Spain (7, 8), Denmark (9), Italy (10), Croatia (11, 12), former Yugoslavia (13) and the Netherlands (14) in different free-living amphibian species. In 2008, edible frogs (Pelophylax kl. esculentus) were collected from ponds in several European countries (Fig.1) for crossing experiments and behavioural studies and transported to a research facility in Switzerland. Several days after their arrival in Switzerland, frogs began dying with reddening of the skin (legs, abdomen) and haemorrhages in the gastrointestinal tract. A ranavirus was identified as a causative agent. New animals were added to the surviving group yearly. In the following three years, another two outbreaks with low to high mortality between asymptomatic periods took place (Fig.2). Animals were tested repeatedly for the presence of ranaviruses. Virus isolates were characterized based on partial nucleotide sequences from four genomic regions and compared to each other and frog virus 3 (FV3), the type species of the genus ranavirus. Introduction Tissue samples from edible frogs which died during disease outbreaks, skin/cloacal swabs from apparently healthy animals and euthanized individuals were tested over three years for the presence of ranaviruses by PCR (14, 15) and virus isolation methods on iguana heart cells (IgH-2, ATCC: CCL-108) (Fig.2). The obtained isolates were further characterized based on partial nucleotide sequences from four genomic regions using additional PCR’s (5, 10, 16) targeting major capsid protein (MCP, 1402bp), DNA polymerase (DNApol, 560bp), and ribonucleoside diphosphate reductase alpha (RNR-α, 806bp) and beta (RNR-β, 646bp) subunit genes. In a retrospective study, samples which were taken each year before transport to Zurich and one ethanol-fixed edible frog which died shortly after collection in 2008 were also tested. Materials and Methods During the first two years, the same ranavirus (RV1) was detected repeatedly during two outbreaks with high to low mortality. Consequently, a new ranavirus (RV2) was isolated in association with the second mass-mortality event in 2010 (Fig.2). The two ranaviruses showed high similarity to each other and to FV3 in the analyzed genomic regions. Slight variations were found in the nucleotide sequences of the partial sequences from the MCP, DNApol and RNR-α subunit genes; the partial sequences from the RNR-β subunit gene were 100% identical to one another. Comparison of the amino acid sequences showed that all differences except those on the RNR-α subunit were silent mutations (Fig.3). Furthermore, a quiescent infection was demonstrated in two animals of eight tested frogs. These apparently healthy frogs were euthanized in autumn 2010 and the previously detected ranavirus (RV1) was isolated from kidney and spleen (Fig.2). In the retrospective study, the ethanol-fixed frog - which was collected from a pond in Germany - tested positive for ranavirus (RV1). Results and Discussion RNR-β Typ1 Typ2 FV3 Typ1 100% 98,68% Typ2 100% 98,68% FV3 98,51% 98,51% RNR - β RV1 RV2 FV3 RV1 100% 98,68% RV2 100% 98,68% FV3 98,51% 98,51% MCP Typ1 Typ2 FV3 Typ1 99,79% 98,07% Typ2 100% 98,15% FV3 97,74% 97,74% RNR-α Typ1 Typ2 FV3 Typ1 99,74% 98,82% Typ2 99,21% 98,82% FV3 98,82% 97,82% MCP RV1 RV2 FV3 RV1 99,79% 98,07% RV2 100% 98,15% FV3 97,74% 97,74% RNR - α RV1 RV2 FV3 RV1 99,74% 98,82% RV2 99,21% 98,82% FV3 98,82% 97,82% DNApol Typ1 Typ2 FV3 Typ1 99,81% 99,84% Typ2 100% 98,15% FV3 98,27% 98,27% DNApol RV1 RV2 FV3 RV1 99,81% 99,84% RV2 100% 98,15% FV3 98,27% 98,27% Fig.3: Ranavirus sequence identity of the four analyzed parts of the genomes. The two different ranaviruses (RV1 / RV2) detected in this study are presented in comparison to FV3. For each gene sequence, the upper diagonal shows the values for the nucleotide sequence identity, the amino acid identity values are listed in the lower diagonal. • Two different ranaviruses have been identified as causative agents for recurring disease outbreaks with low to high mortality in a group of edible frogs. • It has been shown, that animals can be sublethally infected and harbour quiescent virus over a period of at least one year. • During this study a ranavirus was detected in a wild amphibian from Germany for the first time. Conclusions Rachel.marschang @googlemail.com Fig.1: Map of Europe. The countries from which frogs were collected 2008 -2010 are marked in green. Locations of ponds of origin are indicated in red. Edible frog infected with ranavirus: left: ventral haemorrhages („red leg“) right: haemorrhagic ulceration of digitis Ethanol-fixed edible frog Taking skin swab from an apparently healthy animal Fig.2: Schematic timeline of outbreaks and testing of frogs. Box sizes are proportional to numbers of animals for the categories collection of animals, hibernation and disease outbreaks. RV1: Ranavirus 1; RV2: Ranavirus 2; : no virus detected at sampling time point (1) Daszak P., Berger L., Cunningham A.A., Hyatt A.D., Green E., Speare R., 1999. Emerging infectious diseases and amphibian population declines. Emerg Infect Dis 1999;5:735–48. (2) Gray M.J., Miller D.L., Hoverman J.T., 2009. Ecology and pathology of amphibian ranaviruses. Dis Aquat Org 87:243– 266. (3) Drury, S.E.N., Gough, R.E., Cunningham, A.A., 1995. Isolation of an iridovirus-like agent from common frogs (Rana temporaria). Vet Rec 137, 72–73. (4) Cunningham A.A., Langton T.E.S., Bennett P.M., Lewin J.F., Drury S.E.V., Gough R.E., et al., 1996. Pathological and microbiological findings from incidents of unusual mortality of the common frog (Rana temporaria). Philos Trans R Soc London B 1996;351: 1539–57. (5) Hyatt A., Gould A., Zupanovic Z., Cunningham A., Hengstberger S., Whittington R., et al., 2000. Comparative studies of piscine and amphibian iridoviruses. Arch Virol 145:301–31. (6) Duffus, A.L.J., Cunningham, A.A., 2010. Major disease threats to European amphibians. Herpetol J 20, 117–127. (7) Balseiro, A., Dalton, K.P., del Cerro, A., Marquez, I., Cunningham, A.A., Parra, F., Prieto, J.M., Casais, R., 2009. Pathology, isolation and characterization of a ranavirus from the common midwife toad, Alytes obstetricans, on the Iberian Peninsula. Dis Aquat Org 84, 95–104. (8) Balseiro, A., Dalton, K.P., del Cerro, A., Marquez, I., Parra, F., Prieto, J.M., Casais. R., 2010. Outbreak of common midwife toad virus in alpine newts (Mesotriton alpestris cyreni) and common midwife toad (Alytes obstetricans) in northern Spain: a comparative pathological study of an emerging ranavirus. Vet J 186 (2): 256-258 (9) Ariel, E., Kielgast, J., Svart, H.E., Larsen, K., Tapiovaara, H., Jensen, B.B., Holopainen, R., 2009. Ranavirus in wild edible frogs, Pelophylax kl. esculentus in Denmark. Dis Aquat Org 85, 7– 14. (10) Ariel E, Holopainen R, Olesen NJ, Tapiovaara H., 2010. Comparative study of ranavirus isolates from cod (Gadus morhua) and turbot (Psetta maxima) with reference to other ranaviruses. Arch Virol 155:1261–1271. (11) Fijan, N., Matašin, Z. Petrinec, Z., Valpotić, I., Zwillenberg, L.O.,1991. Isolation of an iridovirus-like agent from the green frog (Rana esculenta L.). Vet Arhiv 61, 151–158. (12) The World Organisation for Animal Health. Available online: http://www.Oie.Int/ (13) Kunst L., Valpotic I., 1968. Nova zarazna bolest zaba uzrokovana virusom. Vet Arhiv 38, 108–113. (14) Kik M., Martel A., Spitzen- van der Sluijs A., Pasmans F., Wohlsein P., Gröne A., Rijks J.M., 2011. Ranavirus-associated mass mortality in wild amphibians, The Netherlands, 2010: A first report. Vet J 190: 284–286. (14) Mao J., Hedrick R.P., Chinchar V.G., 1997. Molecular characterization, sequence analysis, and taxonomic position of newly isolated fish iridoviruses. Virology 229: 212–20. (15) Marschang R. E., Becher P., Posthaus H., Wild P., Thiel H. J., Müller-Doblies U., Kaleta E. F., Bacciarini L. N., 1999. Isolation and characterization of an iridovirus from Hermann´s tortoises (Testudo hermanni). Arch. Virol. 144, S.1909-1922. (16) Holopainen R., Ohlemeyer S., Schütze H., Bergmann S.M., Tapiovaara H., 2009. Ranavirus phylogeny and differentiation based on major capsid protein, DNA polymerase and neurofilament triplet H1-like protein genes. Dis Aquat Org 85: 81-91. References This study was partially financed by grants from the Swiss National Science Foundation through a grant to HUR (No. 3100A0-1200225/1) and AAZV through a grant to REM. Acknowledgements