Deep Sequencing Analysis of Virome Components, Viral Gene Expression and Antiviral RNAi Responses in Aphids.

The green peach aphid () is a generalist pest damaging crops and transmitting viral pathogens. Using Illumina sequencing of small (s)RNAs and poly(A)-enriched long RNAs, we analyzed aphid virome components, viral gene expression and antiviral RNA interference (RNAi) responses. Myzus persicae densovirus (family ), a single-stranded (ss)DNA virus persisting in the aphid population, produced 22 nucleotide sRNAs from both strands of the entire genome, including 5'- and 3'-inverted terminal repeats. These sRNAs likely represent Dicer-dependent small interfering (si)RNAs, whose double-stranded RNA precursors are produced by readthrough transcription beyond poly(A) signals of the converging leftward and rightward transcription units, mapped here with Illumina reads. Additionally, the densovirus produced 26-28 nucleotide sRNAs, comprising those enriched in 5'-terminal uridine and mostly derived from readthrough transcripts and those enriched in adenosine at position 10 from their 5'-end and mostly derived from viral mRNAs. These sRNAs likely represent PIWI-interacting RNAs generated by a ping-pong mechanism. A novel ssRNA virus, reconstructed from sRNAs and classified into the family , co-persisted with the densovirus and produced 22 nucleotide siRNAs from the entire genome. Aphids fed on plants versus artificial diets exhibited distinct RNAi responses affecting densovirus transcription and flavivirus subgenomic RNA production. In aphids vectoring turnip yellows virus (family ), a complete virus genome was reconstituted from 21, 22 and 24 nucleotide viral siRNAs likely acquired with plant phloem sap. Collectively, deep-sequencing analysis allowed for the identification and de novo reconstruction of virome components and uncovered RNAi mechanisms regulating viral gene expression and replication.