Molecular Insights into Crimean-Congo Hemorrhagic Fever Virus

Zivcec, Marko; Scholte, Florine E. M.; Spiropoulou, Christina F.; Spengler, Jessica R.; Bergeron, Éric

The CCHFV infectious clone system allows the production of infectious recombinant CCHFV (rCCHFV) from DNA plasmids and contains the complete genome sequence under the control of a T7 promoter. Successful rescue of rCCHFV requires the addition of helper plasmids and transfection into cells. In the rCCHFV rescue process three plasmids, each containing the full sequence of one of the genome segments, are transfected into permissive cells (e.g., Huh7, BSR-T7/5). The genome is then transcribed by T7 into cRNA copies (Figure 7) that are used as a replication template for vRNA. Helper plasmids designed to produce NP and L proteins are provided in trans to synthesize and encapsidate vRNA, and finalize production of reconstituted genomic RNPs. After genomic RNPs are obtained, replication and transcription can be driven by NP and L protein produced from viral mRNA. The production of all viral proteins (NP, GPC and L protein) ultimately assembles into rCCHFV particles capable of infecting neighboring cells and performing multiple infection cycles. Since rCCHFV is infectious, all precautions and biosafety level restrictions associated with live CCHFV experimentation must be adhered to. A key to the success of this system is codon optimization of the DNA sequence of the L helper plasmid. Codon optimization of the L protein increases the activity and the amount of full-length L protein present in transfected cells [56] . While the infectious clone system is the most comprehensive model available of the virus replication cycle, the newly generated rCCHFV must functionally perform all the basic steps of the viral replication cycle for successful rescue of virus. Mutations that abolish a protein function that is critical for replication will prohibit virus rescue without insight into which specific aspects of the replication cycle have been disrupted. Minigenome and VLP systems can be used to more precisely dissect the effects of mutations or treatments on the individual steps in the viral replication cycle.