In this study, we developed new methods for differentiation of ranaviruses based on polymerase chain reaction and restriction enzyme analysis of DNA polymerase and neurofilament triplet H1-like (NF-H1) protein gene. Using these methods, we were able to differentiate the 6 known ranaviruses -Bohle iridovirus (BIV), European catfish virus (ECV), epizootic haematopoietic necrosis virus (EHNV), European sheatfish virus (ESV), frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV) -with 3 less characterised virus isolates : short-finned eel ranavirus (SERV), Rana esculenta virus Italy 282/I02 (REV 282/I02) and pike-perch iridovirus (PPIV). Doctor fish virus (DFV) and guppy virus 6 (GV6) were distinguished as a group from the other viruses. In addition, all 11 isolates were analysed and compared based on nucleotide sequences from 3 different genomic regions: major capsid protein (MCP), DNA polymerase and NF-H1. The partial DNA polymerase gene was sequenced from all analysed viruses. The complete sequence of the MCP and a fragment of the NF-H1 gene were obtained from BIV, ECV, EHNV, ESV, FV3, PPIV, REV 282/I02 and SERV. With the exception of GV6, DFV and SGIV, the sequence analyses showed only a few variations within the analysed viruses. The sequence data suggest that PPIV, REV 282/I02 and SERV are new members of the genus Ranavirus. The methods developed in this study provide tools to differentiate between closely related ranaviruses of different host and geographical origin.

The Santee-Cooper ranaviruses doctor fish virus (DFV), guppy virus 6 (GV6), and largemouth bass virus (LMBV) are members of the genus Ranavirus within the family Iridoviridae. The major capsid protein (MCP) is a main structural protein of iridoviruses and supports the differentiation and classification of ranaviruses. Presently the complete sequence of the MCP gene is known for most ranaviruses with the exception of the Santee-Cooper ranaviruses. In the present study, the complete nucleotide sequence of the MCP gene of DFV, GV6, and LMBV was determined. DFV and GV6 are identical within the MCP gene sequence. The identity compared to the corresponding sequence in LMBV amounts to 99.21%. The MCP gene of DFV, GV6, and LMBV exhibits only approximately 78% identity compared to the respective gene of other ranaviruses. Based on the sequence data obtained in the present study, a Rana MCP polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) analysis were developed to identify and differentiate ranaviruses, including DFV, GV6, and LMBV. KEY WORDS: Iridoviridae · Ranavirus · DFV · GV6 · LMBV · EHNV · MCP gene · Rana MCP PCR Resale or republication not permitted without written consent of the publisherDis Aquat Org 96: 195-207, 2011 196 bass Micropterus salmoides in the 1990s in the United States (Ahne et al. 1989, Pozet et al. 1992, Bovo et al. 1993, Hedrick & McDowell 1995, Jancovich et al. 1997, Plumb et al. 1996, Ariel & Owens 1997, Grizzle et al. 2002, Goldberg et al. 2003. Thus, ranaviruses have been isolated worldwide from fish, amphibians, and reptiles at an increasing frequency over the last few decades (Chinchar 2002, Duffus et al. 2008, Johnson et al. 2008, Chinchar et al. 2009, Gray et al. 2009, Mazzoni et al. 2009, Hoverman et al. 2010, Torrence et al. 2010, Whittington et al. 2010, Xu et al. 2010. Recent reports about ranavirus outbreaks in Pseudacris clarkii tadpoles in Texas underline the subject's topicality (Torrence et al. 2010), since the decline of amphibian populations is a severe problem on a global scale.While EHN and ranavirus infection of amphibians were listed as notifiable diseases in 2006 (Anonymous 2006), other ranavirus infections of fish, caused by ESV/ECV, PPIV, shortfin eel ranavirus (SERV), or Santee-Cooper ranaviruses, and the reptile ranavirus soft shelled turtle iridovirus (SSTIV) have thus far not been listed by the European Union nor by the World Organisation for Animal Health (Office International des Épi-zooties, OIE). In the light of the current knowledge on the significant genetic similarity and broad host range of ranaviruses, further discussion about the declaration and listing of ranavirus infections as exotic diseases in the EU is necessary.Ranaviruses are enveloped icosahedral virions with a diameter of 160 to 200 nm . The genome consists of a single linear double stranded DNA molecule of approximately 105 to 140 kbp, which is highly methylated, circularly permuted, and terminally redundant (Willis & Granoff 1980, G...

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