Susceptibility of the fish cell line SAF-1 to betanodavirus.

Betanodavirus infections of fish, generally known as viral nervous necrosis or viral encephalopathy and retinopathy, are a serious threat to marine fish farming throughout the world (Munday, Kwang & Moody 2002). The culture of betanodavirus has proved complex because most commonly available fish cell lines do not allow the growth of these viruses, as reviewed by Munday & Nakai (1997). The difficulties in finding cell lines that supported betanodavirus replication delayed isolation of the virus (Frerichs, Rodger & Peric 1996) and limited knowledge concerning viral infectious mechanisms. The first successful isolation of a nodavirus was made from sea bass, Dicentrarchus labrax (L.), using the SSN-1 cell line derived from striped snakehead, Ophicephalus striatus (Bloch) (Frerichs et al. 1996). It has been demonstrated that this cell line shows a high permissivity for other fish nodaviruses although some isolates do not produce cytopathic effect (CPE) (Iwamoto, Mori, Arimoto & Nakai 1999). To overcome these and other problems shown by SSN-1 cells, Iwamoto, Nakai, Mori, Arimoto & Furusawa (2000) cloned this cell line and reported that the clones obtained were more useful than the SSN-1 cells for qualitative and quantitative analysis of betanodaviruses. However, these cloned cell lines, like SSN-1, are persistently infected by a C-type retrovirus. Other authors have reported that another fish cell line (GF-1), derived from grouper, Epinephelus coioide (Hamilton), was useful for the isolation and replication of a nodavirus obtained from the same fish species, but did not show susceptibility of this cell line to other nodaviruses (Chi, Hu & Lo 1999). Recently, a new fish cell line, SAF-1 (ECACC no. 00122301), derived from gilthead seabream, Sparus auratus L. (Bejar, Borrego & Alvarez 1997), has proven to be suitable to detect and quantify lymphocystis viruses that do not replicate in standard fish cell lines, as well as other fish viruses (Perez-Prieto, Rodriguez-Saint Jean, Garcı́a-Rosado, Castro, Alvarez & Borrego 1999). In the present study, we report the suitability of SAF-1 cells to support replication and quantification assays of nodavirus strains and compare the results obtained using this cell line with those obtained using SSN-1 cells. The SAF-1 cell line is not infected with retrovirus (J. J. Borrego, unpublished data). Eleven samples (Table 1) obtained from three fish species farmed in the Iberian Peninsula were used in this study. These samples had previously been demonstrated to be positive for nodavirus by reverse transcriptase-polymerase chain reaction (RT-PCR) in a routine analysis performed in the Instituto de Acuicultura facilities. Briefly, the samples (whole individuals when using larvae, the head in the case of small fish <1 g, and a pool of eyes and brain from larger fish) were mixed (1:10) with Earle’s balanced salt solution (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with antibiotics Journal of Fish Diseases 2006, 29, 633–636