Production of abundant defective viral genomes by an insect-specific alphavirus revealed by nanopore sequencing

Defective viral genomes (DVGs) shape virus-host interactions and evolution, yet remain poorly characterized in insect-specific viruses. Using Nanopore direct RNA sequencing (DRS), we profile full-length RNA species of Eilat virus (EILV), a mosquito-restricted alphavirus. We found that EILV produces abundant deletion-type DVGs far exceeding those seen in related arboviruses under identical conditions. These DVGs retain intact terminal sequences with conserved junction motifs. A modified DRS approach reveals widespread premature termination during EILV genomic RNA synthesis, linking DVG formation to reduced replicase processivity. Notably, a subset of DVGs is selectively replicated and packaged, accumulating over serial passages and interfering with parental virus replication. Our findings uncover an intrinsic, high DVG-generating capacity in EILV, linking low-processivity RNA synthesis with increased genomic plasticity. This suggests that elevated DVG production may be an adaptive mechanism, providing important insights into alphavirus evolution and replication fidelity in insect-specific RNA viruses. Using Nanopore direct RNA sequencing, authors show Eilat virus has abundant deletion-type defective viral genomes; delineating it from other arthropod-borne alphaviruses. This is likely driven by low replicase processivity and premature termination.