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The members of the picornavirus family include various viruses which, due to their impressive oncolytic activity, have the potential to be used for the treatment of cancer. However, the replication of these oncolytic viruses (OV) is not limited to tumor cells but can also take place in various normal tissues. To increase the safety of these OV, target sites (miR-TS) of microRNAs, which are expressed in normal tissues but are absent or only expressed at low levels in cancer cells, can be inserted into the viral genome. Here we describe how miR-TS can easily be inserted into the complementary DNA (cDNA) of coxsackievirus B3 (CVB3) RNA genome using the In-Fusion cloning technology. Here we provide the step-by-step protocol, how miR-TS containing recombinant CVB3 can be generated from these viral cDNA constructs, how the virus is amplified, purified and concentrated, and how the functionality of the miR-TS within the viral genome can be confirmed.

Original publication




Journal article


Methods Mol Biol

Publication Date





259 - 282


Coxsackievirus B3, In-Fusion cloning, Oncolytic virus, Virus plaque purification, cDNA, microRNA target sites, microRNAs, DNA, Complementary, Enterovirus B, Human, Genome, Viral, HeLa Cells, Humans, MicroRNAs, Oncolytic Viruses, Virus Replication