A cell culture system and production of an infectious clone of Rhopalosiphum padi virus (Dicistroviridae)

Abstract

Rhopalosiphum padi virus (RhPV) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. RhPV, an icosahedral virus, belongs to the family Dicistroviridae. It has a 10 kb positive-sense RNA genome, with two viral open reading frames (ORFs). We have identified two cell lines, Z10-2 and DMII derived from the homopterans, Homalodisca coagulata and Dalbulus maidis that are permissive for RhPV. Infection, viral replication and production of virions was confirmed by northern blot hybridization, RT-PCR, western blot analysis and immunoelectron microscopy. A cell culture system that allows replication of the virus is crucial for further study of the biology of RhPV. The long term goal of this project is to produce aphid resistant transgenic plants that express RhPV. Acquisition of an infectious clone of RhPV is of primary importance for development of such an aphid resistance technology. A full-length cDNA clone of RhPV was constructed using overlapping reverse transcription PGR products. RNA transcribed from the full-length cDNA clone was infectious upon transfection into Z10-2 and DMII cells, resulting in production of progeny virus phenotypically indistinguishable from the parent virus. The in vitro transcript caused the same cytopathic effects as those caused by transfection of cells with the viral RNA. The virus-like particles (VLPs) purified from the cells transacted with the full-length transcript were infectious to Z10-2 cells and also to R. padi. The infectious cDNA clone of RhPV, together with the cell culture system, will provide valuable experimental tools for the study of replication and pathogenesis of RhPV. The recombinant baculovirus AcRhPV6, which contains the cDNA of RhPV under the control of the polyhedrin promoter, was constructed using the Bac-to-Bac baculovirus