Plasmid-Based Reverse Genetics System to Generate Colorado Tick Fever Virus

Colorado tick fever virus (CTFV) is a non-enveloped virus with a multi-segmented double-stranded RNA genome.

To generate CTFV, take a mixture of genome and expression plasmids.

The genome plasmids encode all viral genome segments under a T7 promoter, while the expression plasmids encode all viral proteins under a mammalian promoter.

Introduce a transfection reagent to form complexes.

Apply these complexes to mammalian cells that express T7 RNA polymerase and incubate to allow plasmid entry. The expression plasmids enter the nucleus for transcription and subsequent protein production.

Remove the medium and co-culture these cells with virus-permissive host cells to support virus amplification.

The T7 polymerase transcribes the genome plasmids into single-stranded genomic RNAs, while the expression plasmids express viral proteins.

The viral polymerase generates complementary strands, producing double-stranded RNA genomes.

Complete virions assemble and are released into the medium, which infect the permissive host cells.

Their replication causes a cytopathic effect in the host cells, confirming the successful generation of infectious CTFV.