TriLock-FISH enables in situ discrimination of influenza cRNA and mRNA for single-cell analysis of viral replication and transcription

ABSTRACT Influenza A virus produces three RNA species—viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA)—whose synthesis and trafficking are spatially compartmentalized. However, the high sequence similarity between cRNA and mRNA hinders their distinction in situ , limiting understanding of the spatial organization of the viral replication-transcription dynamics. Here, triple-lock fluorescence in situ hybridization (TriLock-FISH) was developed to distinguish vRNA, cRNA, and mRNA in situ with high specificity. TriLock-FISH enabled a genome-wide, time-resolved single-cell atlas of RNA abundance and localization, revealing marked cell-to-cell variation in RNA abundance but relatively conserved spatial organization. cRNA was found to be predominantly nuclear, with a small but reproducible cytoplasmic fraction. Application to infected mouse lung tissue demonstrated simultaneous spatial detection of all viral RNA species. This approach provides a highly specific framework for single-cell analysis of RNA virus replication and transcription. IMPORTANCE Influenza virus relies on three closely related RNA molecules for replication and gene expression, yet distinguishing them inside infected cells has remained technically challenging. This limitation has hindered understanding of viral replication in space and time. We developed triple-lock fluorescence in situ hybridization, which enables simultaneous visualization of all three viral RNA species across all eight influenza genome segments in infected cells and tissues. Single-cell analysis revealed striking heterogeneity in viral RNA abundance across infected cells, whereas intracellular RNA localization patterns remained relatively conserved. Complementary RNA, a replication intermediate long thought to be strictly nuclear, was also detected at low levels in the cytoplasm. These findings provide new insight into influenza virus replication and establish a broadly applicable tool for studying RNA virus biology.