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However, the percentage of sense vsiRNAs was significantly higher in the 25-26 nt long vsiRNAs. Distinct vsiRNA hotspots, identifying highly abundant reads of different unique vsiRNA sequences, were observed in both viral genomes. Previous studies found an A or U bias at the 5' terminal nucleotide position of 21 nt vsiRNAs; in contrast, our analysis revealed a C and U nucleotide bias. This study provides insights that will help further elucidate differential processing of vsiRNAs in plant antiviral defense.Capsule summary word count Non