The hidden diversity: Replication-strand asymmetry shapes short-term nucleotide and structural variation in an RNA virus

Orsay virus (OrV), a positive-sense RNA virus infecting Caenorhabditis elegans, establishes latent infections with minimal impact on host fitness. Using strand-specific RNA-seq across twelve time points spanning larval development, we characterized OrV replication dynamics and short-term evolutionary patterns. Viral accumulation followed four distinct phases and exhibited strong strand asymmetry, consistent with a predominance of the stamping-machine replication mechanism. Diversity analyses revealed that negative-strand genomes harbor more mutations than positive strands, with hotspots concentrated in the replicase C-terminal tail and structural protein regions. Many negative-strand variants failed to propagate to positive strands, suggesting within-cell constraints or purifying selection at the RNA level. We also report the first evidence of non-standard viral genomes in OrV, dominated by deletions in RNA2, including a recurrent large deletion that may function as a subgenomic RNA encoding a shorter version of the {delta} protein. These findings highlight replication asymmetry, strand-specific mutational filtering, and pervasive non-standard genomes as key features shaping OrV evolution during a primary infection.