Simultaneous detection of positive-sense RNA1 and RNA2 of nervous necrosis virus in viral factories of infected brain cells using RNA-fluorescence in situ hybridization.

Nervous necrosis virus (NNV) is a bipartite positive-sense RNA virus that causes viral nervous necrosis in marine larval fish worldwide. Understanding the intracellular trafficking and spatial organization of viral RNAs is essential for elucidating the NNV replication cycle; however, methods that allow strand-specific and simultaneous visualization of multiple NNV RNAs at the subcellular level are limited. In the present study, we developed a strand-specific RNA-fluorescence in situ hybridization (RNA-FISH) protocol for visualizing the subcellular localization of NNV RNAs during infection. Detection specificity was first validated by Northern blot analysis, which distinguished positive-strand RNA1 and RNA2 from their corresponding negative-strands. The designed probes selectively detected each viral RNA without cross-reactivity, enabling simultaneous visualization of two viral RNA species within a single infected cell. This technique can be combined with immunocytochemistry or organelle trackers staining to examine the spatial relationship between viral RNAs and viral or host proteins. Incorporation of 5-bromouridine (BrU) allowed the detection of newly synthesized viral RNAs and the tracking of their intracellular trafficking. Using this approach, nascent viral RNAs were observed to move out of mitochondrial replication/transcription sites toward vesicles associated with coat protein translation and subsequently to the remodeled microtubule-organizing center (MTOC) for viral assembly. This protocol provides a robust tool for studying the intracellular dynamics of viral RNAs and their coordination with viral and host factors and can be adapted for the investigation of other RNA viruses.