Publication Abstract

Emerging vesiculo-type virus infections of freshwater fishes in Europe

A. Betts, D. Stone, K Way, C. Torhy, S.Chilmonczyk, A. Benmansour, P. de Kinkelin

Rhabdoviruses were isolated from perch (Perca fluviatilis), pike perch (Stizostedion stizostedion), and grayling (Thymallus.thymallus), undergoing clinical signs of infection and initial experiments suggested these viruses were distinct from spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV). More recently, characterisation of putative SVCV and PFRV isolates has suggested that there are four distinct genogroups of fish vesiculoviruses (Stone et al, submitted). Hence biochemical and genetic characterisation studies were carried out to further classify these viruses.

The virus isolates were passaged on BF-2 cells and purified using ultracentrifugation through a 25% glycerol pad. Purified virus isolated were lysed in SDS buffer and the relative mobility patterns of viral proteins were analysed using polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. Genetic analysis was initially attempted by PCR of viral G gene regions, however due to significant sequence differences within the G genes of the fish vesiculoviruses this approach was unsuccessful. Therefore, primers were designed to conserved domains within the L gene of the Mononegavirales using previously published sequences. RNA was extracted from clarified virus preparations and RT-PCR carried out in duplicate to amplify L gene fragments. Amplified DNA was cloned and multiple clones sequenced in both directions. DNA sequences were analysed using the Megalign software package.

PAGE analysis of the perch, pike perch and grayling viral proteins showed an identical banding pattern to those of a previously characterised perch rhabdovirus (Dorson et al 1984). However, the M protein from these viruses was shown to be smaller than that of the spring viraemia virus group. Sequence data from the virus isolates was compared to that of SVCV, PFRV and other fish vesiculoviruses available in our laboratory. Phylogenetic analysis of sequence data identified the four tentative fish vesiculovirus genogroups. The pike, pike perch and grayling isolates showed a >80% homology at the amino acid level and a >75% nucleic acid homology when compared to PFRV and were sufficiently distinct to be placed in a new group of tentative species within genus Vesiculovirus.

In summary, biochemical and genetic data suggest evidence for a fifth group of tentative species of fish vesiculoviruses containing the perch, pike perch and grayling rhabdoviruses.

Reference:

A. Betts, D. Stone, K Way, C. Torhy, S.Chilmonczyk, A. Benmansour, P. de Kinkelin. 2004. Emerging rhabdovirus infections of fresh water fish. Diseases of Aquatic Organisms, 57: 201-212