Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro from a full-length cDNA clone

Department of Entomology, Iowa State University, Ames, IA 50011, USA.
Virology (Impact Factor: 3.32). 07/2008; 375(2):401-11. DOI: 10.1016/j.virol.2008.02.008
Source: PubMed


Availability of a cloned genome from which infectious RNA can be transcribed is essential for investigating RNA virus molecular mechanisms. To date, no such clones have been reported for the Dicistroviridae, an emerging family of invertebrate viruses. Previously we demonstrated baculovirus-driven expression of a cloned Rhopalosiphum padi virus (RhPV; Dicistroviridae) genome that was infectious to aphids, and we identified a cell line (GWSS-Z10) from the glassy-winged sharpshooter, that supports RhPV replication. Here we report that RNA transcribed from a full-length cDNA clone is infectious. Transfection of GWSS-Z10 cells with the RhPV transcript resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions, consistent with virus infection. Virions from transcript-infected cells were infectious in aphids. This infectious transcript of a cloned RhPV genome provides a valuable tool, and a more tractable system without interference from baculovirus infection, for investigating replication and pathogenesis of dicistroviruses.

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    • "Most information about cellular infection and the replication cycle of dicistroviruses comes from studies on DCV, and by comparison with mammalian picornaviruses [1]. Many members of the Dicistroviridae family are considered novel candidates to be used as biopesticides [1,13-17]. "
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    • "The blocks were sectioned at the Lisa Muma Weitz Advanced Microscopy & Cell Imaging Core Laboratory (University of South Florida) and the sections were laid on nickel grids. Gold labeling of the nickel grids was carried out as described previously [28]. Grids were treated with 25 µl of TBS-supplemented buffer (0.05 M Tris, 0.85% NaCl, pH 8.3–8.5, 0.5% normal goat serum, 0.5% normal pig serum and 0.5% BSA) with 3% nonfat dry milk for 2 h at room temperature. "
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    • "Recently, successful in vitro expression of a positive-strand RNA virus was reported (Pal et al., 2007; Boyapalle et al., 2008). A clone of the Rhopalosiphum padi virus genome was expressed in a baculovirus expression system and found to be infectious and pathogenic to its aphid host (Pal et al., 2007; Boyapalle et al., 2008). This research permits further studies of viruses in which a cell line is unavailable (e.g., for S. invicta). "
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