Characterization of Two Novel Insect-Specific Viruses Discovered in the Green Leafhopper, Cicadella viridis

Simple Summary Insect-specific viruses (ISVs) have gained increasing attention for their potential use as biological agents. In this study, the full genomes of two ISVs (Cicadella viridis iflavirus 1, CvIfV1; Cicadella viridis nido-like virus 1, CvNiLV1) were revealed in green leafhoppers, using metatranscriptome, RT-PCR, and RACE approaches, respectively. CvIfV1 is a member of iflavirus and has a typical iflavirus genome organization. An antiviral RNA interference (RNAi) was triggered when host insects were challenged with CvIfV1, which resulted in an abundant accumulation of 21-nt virus-derived siRNAs (vsiRNAs). CvNiLV1 clusters are within a distinct unclassified clade of viruses in the order Nidovirales, and evolutionary related with viruses that infect vertebrate hosts. CvNiLV1 was also targeted by the host antiviral RNAi pathway, and generated the 21-nt vsiRNAs with a strong A/U bias in the 5 '-terminal. Our study provided valuable information on ISVs in leafhoppers, and might prove useful in pest management. Insect-specific viruses (ISV) are one of the most promising agents for the biological control of insects. The green leafhopper, Cicadella viridis (Linnaeus), is an significant pest in agriculture, and causes economic losses to fruit trees, willows, and field crops. As a representative species of the large family Cicadellidae, ISVs in C. viridis have never been studied, to date. In this study, the full genome sequences of two ISVs, named Cicadella viridis iflavirus 1 (CvIfV1), and Cicadella viridis nido-like virus 1 (CvNiLV1), were revealed using a metatranscriptomic approach. A homology search and phylogenetic analysis indicated that CvIfV1 is a new member in the family Iflaviridae (genus Iflavirus) with a typical iflavirus genome organization, whereas CvNiLV1 belongs to the unclassified clade/family of the order Nidovirales. In addition, analysis of virus-derived small interfering RNAs (vsiRNAs) was performed to investigate the antiviral RNA interference (RNAi) response of C. viridis. The vsiRNAs exhibit typical patterns produced by host siRNA-mediated antiviral immunity, including a preference of 21-nt vsiRNAs derived equally from the sense and antisense genomic strands, and a strong A/U bias in the 5 '-terminus of the viral genomes. Our study provides valuable information for ISVs in leafhoppers for the first time, which might prove useful in the control of C. viridis in future.