Cloning of large positive-strand RNA viruses.

Full-length, biologically active cDNA clones of the positive-strand RNA plant viruses are indispensable for investigating the functions of viral genes and control elements as well as generating virus-derived gene expression and silencing vectors. Even though engineering of such clones for 4- to 10-kb viral RNAs has become routine, it remains a challenging task for 15- to 20-kb RNA genomes of the monopartite viruses in a family Closteroviridae. This unit describes strategic considerations and techniques used to generate an infectious cDNA clone of a closterovirus. The use of agroinfection to improve specific infectivity of the resulting clone is also explained.