Evaluation of Cell Culture Methods for Detection of Largemouth Bass Virus

Largemouth bass virus (LMBV), a recently discovered iridovirus found in the eastern United States, is usually detected by isolation in cell culture. Although LMBV will replicate in several cell lines, optimal cell culture methods for the detection of this virus have not been determined. We tested inoculation method, adsorption time, incubation temperature, and various cell lines to determine the conditions that would provide the most sensitive cell culture assay for LMBV. The optimal inoculation procedure tested was to remove the culture medium from the culture well before the addition of the inoculum, and the optimal adsorption procedure tested was to allow the virus to adsorb for 40 min while the plates were on an orbital shaker. Following inoculation, incubation at 30°C resulted in a higher number of viral plaques than incubation at 25°C or 32°C. Four cell lines (bluegill fry (BF‐2), fathead minnow (FHM), epithelioma papulosum cyprini, and channel catfish ovary cells) inoculated with LMBV had similar susceptibility to infection. Similar percentages of LMBV‐positive samples were detected in BF‐2 and FHM cell cultures inoculated with homogenized organ samples from largemouth bass Micropterus salmoides; however, the use of two cell lines increased the number of infected samples discovered. A blind passage also increased the number of positive samples detected in cell culture. Subcultivation to confirm virus‐positive samples was useful for reducing false‐positive results.