Iris yellow spot virus (IYSV) is one of the most devastating viral pathogens, which causes high economic losses in theonion yield. Physiological and genetic changes are associated with the appearance of chlorotic symptom in the infectedplants. IYSV-N gene sequence analysis revealed that it shared sequence identity of 99% with other Egyptian isolates, atboth genomic and proteomic levels. In addition, N protein sequence with computational examination indicated many motifsinvolved and played different roles in the virus activity and its regulation and stability were detected. In the DifferentialDisplay-Polymerase Chain Reaction (DD-PCR) study, a highly up-regulated gene at 15 days post-biological IYSV inoculation(dpi) was selected for sequencing. Based on the sequencing results that showed the identified gene was coding for achloroplast-related gene, degenerate specific primers were designed for Real-Time PCR analysis. A significant change inthe transcription level of the chloroplast-related gene after 15 dpi suggested that some IYSV proteins interact and/orregulate with chloroplast proteins and this finding supports the DD-PCR results. At 20 dpi, the ultrathin sections showedthat IYSV infection caused many dramatic chloroplasts malformations. The malformation appeared as chloroplast brokenenvelope with the presence of numerous spherical particles inside it and chloroplasts with long stromule. Our findingsindicated that IYSV interrupts normal chloroplast functions, as a part of the onion defence response, however many crucialfactors remain to be elucidated and further studies are needed at both biological and molecular levels.