Ranavirus phylogeny and differentiation based on major capsid protein, DNA polymerase and neurofilament triplet H1-like protein genes

Abstract

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.