A small RNA virus isolated from apparently healthy wild sandbar shiners, Notropisscepticus (Jordan & Gilbert)

As part of the National Wild Fish Health Survey, the US Fish and Wildlife Service initiated a survey of pathogens present in wild fish populations. During that survey, a filterable agent was isolated from a pooled sample of sandbar shiners, Notropis scepticus (Jordan & Gilbert), at the Warm Springs Fisheries Center, Fish Health Laboratory, Warm Springs, Georgia. The shiners were collected from North Carolina’s Deep River at the SR 1456 bridge on October 22 1998. The liver, kidney, spleen and intestine of five apparently healthy sandbar shiners were pooled and homogenized in Hank’s balanced salt solution (HBSS, Sigma) at a dilution of 1:10 (w/v). The homogenate was further diluted in HBSS to 1:100 and centrifuged at 2500 g for 20 min at 4 °C. The resulting supernatant was stored overnight at 4 °C and inoculated onto epithelioma papulosum cyprini (EPC) and fathead minnow (FHM) cell lines cultured in minimum essential medium (Gibco) supplemented with 10% foetal bovine serum (FBS) (Fryer & Lannan 1994). Cell cultures were incubated at 20 °C; when a cytopathic effect (CPE) was observed, the supernatant was removed, passed through a 0.45-mm syringe filter, and transferred to fresh EPC and FHM cells. A CPE of focal cell rounding occurred in the FHM cell line 72-h post-inoculation. Focal aggregates of refractile, rounded cells became numerous and at 13 days post-inoculation the monolayer was destroyed. The EPC cell line was also susceptible to the viral agent; however, the CPE was more diffuse and exhibited cellular hypotrophy in addition to cell rounding. Monolayers of this cell line were completely destroyed following a 13-day incubation at 25 °C. Inoculation of the rainbow trout gonad cell line (RTG-2) with the isolate resulted in a focal CPE similar to that observed in the FHM cells (Fig. 1). Determination of particle size and morphology was achieved using electron microscopy of negatively stained particles. Briefly, the monolayers of EPC cells were inoculated with the virus suspension and incubated at 25 °C until the CPE was extensive. Monolayers were scraped from the flask surface and suspended in the culture supernatant. Cell-associated virions were released from the cells with two freeze–thaw cycles and the supernatant was clarified using centrifugation (Marathon 6K, Fisher Scientific, Pittsburgh, PA, USA) at 6000 g for 30 min. The supernatant was removed and centrifuged at 106000 g for 60 min in SSC buffer (0.15 M sodium chloride, 0.15 M sodium citrate, pH 7.4). The resulting pellet was suspended in 20 L SSC buffer. Negatively stained preparations examined by electron microscopy revealed spherical virions measuring c. 20–25 nm in diameter (Fig. 2). The thymidine analogue, 5-iodo-2-deoxyuridine (IDU), was used to determine the nucleic acid composition of the viral genome. Epithelioma papulosum cyprini cells were seeded at c. 90% confluency on a 48-well plate and incubated overnight in a 50-mg mL IDU solution at 25 °C (Rovozzo & Burke 1973). Media was removed from the cell monolayers; the cells were then inoculated with tenfold dilutions of the virus suspension in IDU solution and incubated at room temperature for 1 h. Fresh IDU solution supplemented with 10% FBS was then added to the Correspondence A E Goodwin, Aquaculture/Fisheries Center, University of Arkansas at Pine Bluff, 1200 N. University Drive, P.O. Box 4912, Pine Bluff, AR 71611, USA (e-mail: agoodwin@uaex.edu)