A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Zika virus (ZIKV) infection is linked to severe neurological issues, including congenital abnormalities and Guillain-Barré syndrome.
• Reverse genetic systems are crucial for understanding ZIKV's molecular mechanisms but are hindered by challenges in constructing full-length cDNA clones.
• Flaviviral cDNA toxicity in bacteria, caused by cryptic promoters, impedes the creation of stable plasmids.

Purpose of the Study:

• To develop an efficient method for generating stable, full-length Zika virus (ZIKV) cDNA clones.
• To overcome the toxicity issues associated with flaviviral cDNA amplification in *Escherichia coli*.
• To create novel ZIKV research tools, including reporter viruses and replicons.

Main Methods:

• In silico prediction of cryptic prokaryotic promoters within ZIKV cDNA sequences.
• Mutational silencing of identified putative promoters to reduce toxicity.
• Construction and amplification of full-length ZIKV cDNA clones on single bacterial plasmids.
• Generation of infectious ZIKV, reporter viruses, and sub-genomic replicons.

Main Results:

• Successfully generated stable, full-length cDNA clones for both historical (MR766) and contemporary (H/PF/2013) ZIKV strains.
• The developed method overcomes previous difficulties in ZIKV cDNA cloning and amplification.
• Generated functional luciferase and fluorescent reporter viruses, and sub-genomic replicons.
• Infectious viruses produced exhibit properties comparable to those from more complex methods.

Conclusions:

• In silico promoter prediction and silencing is an effective strategy for creating stable flaviviral cDNA clones.
• The generated ZIKV tools facilitate research into viral biology and the development of antiviral therapies.
• This approach provides a simplified and efficient route for manipulating ZIKV genomes.