Advanced vacuolation indicates propagation of various salmonid alphavirus type 2 isolates in Acholeplasma-infected BF-2 cells.

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During previous routine inspections of bluegill fry (BF-2) and rainbow trout gonad (RTG-2) cells incubated with organ samples from asymptomatic Arctic char Salvelinus alpinus, brook trout Salvelinus fontinalis, and rainbow trout Oncorhynchus mykiss, a distinctive, reproducible cytopathic effect (CPE) appeared. The striking CPE, involving progressive vacuolation turning into slowly proceeding pyknotic degeneration, was originally attributed exclusively to enhanced growth of Acholeplasma sp. However, at a recent re-examination of re-infected BF-2 cells using electron microscopy (EM), conventional PCR, and quantitative PCR (qPCR), a virus was also detected. Two days post inoculation (dpi), EM revealed characteristic virions inside cytoplasmic vacuoles and next to bacteria outside the cells. The nucleotide sequences of the viral nsP3 gene fragment obtained from supernatants of infected cells were 100% identical and representative for salmonid alphavirus type 2 (SAV 2). The 16S RNA gene (16S rDNA) fragment sequences of the Mollicutes-specific PCR product obtained from SAV-infected as well as virus-free BF-2 control cells were identical with Acholeplasma laidlawii. In addition, qPCR results indicated enhanced propagation of virus and bacteria increasing with vacuolation between 5 and 8 dpi. Advanced vacuolation can be regarded as a CPE of both SAV and A. laidlawii, suggesting a viral impact on the bacterial infection that turns a latent intracellular stage into an apparent degenerative condition.