Infectious full-length clones of calibrachoa mottle virus (CbMV)

Abstract

Full-length cDNA clones derived from genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) of Calibrachoa mottle virus (CbMV) were constructed under the control of the T7 RNA promoter and ligated into plasmid pUC-18. The capped and uncapped in vitro transcripts, synthesized from full length genomic cDNA clone pUC-CbMV-FL generated chlorotic local lesions on mechanical inoculated Chenopodium quinoa within 10 to 15 days post-inoculation. The progeny virions recovered from inoculated symptomatic leaves had the same morphological properties as the parental virions. These virions are also reacted positively with CbMV antiserum in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and the viral coat protein was detected by Western blotting. The presence of five open reading frames (ORFs) on the CbMV genome and synthesis of corresponding proteins by gRNA and sgRNAs were confirmed by Northern blotting and in vitro translation. The transcription start site of three sgRNAs of CbMV was also determined. Successful construction of full-length infectious cDNA clone of CbMV makes it possible to develop molecular tools that can be used to understand the gene functions of this virus. © 2011 Gulati-Sakhuja A, et al.