Robust profiling of plus-stranded RNA virus replication dynamics using RT-qPCR with viral genomic RNA as an internal...

Plus-stranded RNA (+RNA) viruses replicate through negative-strand intermediates and frequently generate subgenomic RNAs (sgRNAs) to regulate viral gene expression. Accurately profiling viral replication dynamics is essential for studying host-virus interactions, yet conventional methods such as Northern blot are labor-intensive and require large amounts of RNA. Here, we present a robust and efficient RT-qPCR-based strategy to profile +RNA virus replication dynamics by targeting sgRNAs while using genomic RNA-specific gene as an internal control. Using a Flock House virus-derived replicon named FR1gfp in Caenorhabditis elegans, we demonstrate that RT-qPCR measurements of sgRNA accumulation closely mirror replication dynamics obtained by Northern blot analysis. This approach reliably captures the temporal onset and progression of viral replication while substantially reducing time, cost, and RNA input requirements. Our results establish RT-qPCR targeting sgRNAs as a sensitive and scalable alternative for monitoring +RNA virus replication in vivo, providing a practical framework applicable to viruses that produce single or multiple sgRNAs.