Reverse Genetics of Zika Virus Using a Bacterial Artificial Chromosome.

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has become a global threat to human health. Although ZIKV has been known to circulate for decades causing mild febrile illness, the more recent ZIKV outbreaks in the Americas and the Caribbean have been associated with severe neurological disorders and congenital abnormalities. The development of ZIKV reverse genetics approaches have allowed researchers to address key questions on the biology of ZIKV by genetically engineering infectious recombinant (r)ZIKV. This has resulted in a better understanding of the biology of ZIKV infections, including viral pathogenesis, molecular mechanisms of viral replication and transcription, or the interaction of viral and host factors, among others aspects. In addition, reverse genetics systems have facilitated the identification of anti-ZIKV compounds and the development of new prophylactic approaches to combat ZIKV infections. Different reverse genetics strategies have been implemented for the recovery of rZIKV. All these reverse genetics systems have faced and overcome multiple challenges, including the viral genome size, the toxicity of viral sequences in bacteria, etc. In this chapter we describe the generation of a ZIKV full-length complementary (c)DNA infectious clone based on the use of a bacterial artificial chromosome (BAC) and the experimental procedures for the successful recovery of rZIKV. Importantly, the protocol described in this chapter provides a powerful method for the generation of infectious clones of other flaviviruses with genomes that have stability problems during bacterial propagation.