Major capsid protein gene sequence analysis of the Santee-Cooper ranaviruses DFV, GV6, and LMBV.

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.

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

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.