Cloning of the fish cell line SSN-1 for piscine nodaviruses

Six cell clones were derived from the SSN-1 cell Line, which is composed of a mixed cell population and persistently infected with a C-type retrovirus (SnRV). These clones were susceptible to 4 piscine nodavirus strains belon ging to different genotypes (SJNNV, RGNNV, TPNNV and BFNNV [striped jack, r edspotted grouper, tiger puffer and barfin flounder nervous necrosis viruse s]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects a ppeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d po st-incubation. These observations were highly reproducible and formed the b asis for a successful virus titration system. Quantitative analysis using t he cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30 degreesC for strain SGWak97 (RGNNV), 20 to 25 degreesC for strain SJNag93 (SJNNV), 20 degreesC for strain TPKag93 (TPNNV), and 15 to 20 degreesC for strain JFIwa98 (BFNNV ). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 a nd E-9 cells, but PCR amplification for the pol gene and LTR region of the proviral DNA indicated the presence of the retrovirus in the other clones, including E-11. The cell clones obtained in the present study will be more useful for qualitative and quantitative analyses of piscine nodaviruses tha n the SSN-1 cell Line.