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

Abstract

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 belonging to different genotypes (SJNNV, RGNNV, TPNNV and BFNNV [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects appeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d post-incubation. These observations were highly reproducible and formed the basis for a successful virus titration system. Quantitative analysis using the cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30°C for strain SGWak97 (RGNNV), 20 to 25°C for strain SJNag93 (SJNNV), 20°C for strain TPKag93 (TPNNV), and 15 to 20°C for strain JFIwa98 (BFNNV). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 and 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 than the SSN-1 cell line. KEY WORDS: Nodavirus · Viral nervous necrosis, VNN · SSN-1 cell line · Cell cloning · C-type retrovirus · Snakehead retrovirus Resale or republication not permitted without written consent of the publisherDis Aquat Org 43: [81][82][83][84][85][86][87][88][89] 2000 that a new cell line (GF-1) derived from grouper Epinephelus coioides was useful for the isolation and proliferation of a piscine nodavirus (GNNV, grouper nervous necrosis virus).Piscine nodaviruses can be divided into 4 genotypic groups based on partial sequences of the coat protein gene (Nishizawa et al. 1997): SJNNV (striped jack nervous necrosis virus), RGNNV (redspotted grouper nervous necrosis virus), TPNNV (tiger puffer nervous necrosis virus), and BFNNV (barfin flounder nervous necrosis virus). In a previous study, we demonstrated that the SSN-1 cell line was useful for propagating and differentiating 17 isolates of piscine nodavirus collected from 13 host fish species in 5 countries (Iwamoto et al. 1999). However, one problem with the practical use of the SSN-1 cell line was that this cell line was composed of a mixed population of cells, causing inconsistencies in the cytopathic effects (CPE) observed during virus infection. For this reason, FAT was used to titrate the virus instead of CPE as described in our previous study (Iwamoto et al. 1999). FAT was laborious and costly due to the requirement of special test chambers, and it has proved to be inadequate for the quantitative analysis of a large number of samples. In addition, the fact that the SSN-1 cell line is spontaneously infected by a C-type retrovirus designated as SnRV (Frerich...

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“…According to the IFAT results, all the IHC examinations, performed on 170 slides from infected brain and eye tissues revealed positive results compared to the 10 control slides. These results were similar to previous results reported; Iwamoto et al (2000).…”

Betanodavirus of Marine and Freshwater Fish: Distribution, Genomic Organization, Diagnosis and Control Measures

“…SSN-1 cell line is useful for propagating and differentiating genotypic variants of piscine nodavirus [59]. The cell line has been cloned and since it shows stable cytopathic effect expression, it can be used for qualitative and quantitative analyses of piscine nodaviruses rather than the SSN-1 cell line [60]. Yet another cell line derived from the brain tissue of Barramundi L. calcarifer was developed which was used to study the mechanisms of NNV-persistent infection in vitro and in vivo [24].…”

Cloning of a new fibroblast cell line from an early primary culture from mandarin fish (Siniperca chuatsi) fry for efficient proliferation of megalocytiviruses

“…After removing the supernatant, 5 ml of growth DMEM was added to re-suspend the cells, which were seeded into a 25 cm 2 flask, and incubated at 25°C. After three rounds of subculturing, primary cells in the eighth passage were subjected to single cell cloning using a standard limitation dilution in a 96-well plate (Imajoh et al 2007;Iwamoto et al 2000). The colonized cells were kept in a 25°C incubator with 5 % CO 2 and monitored under an inverted microscope (Nikon, Tokyo, Japan).…”