Zika virus (ZIKV) is an emerging flavivirus primarily transmitted through the bite of Aedesmosquitoes. It spread explosively around the tropics in the 21st century, and still presents a threat to human health. There is a need to better understand the drivers of emergence of ZIKV and likely consequences of its future evolution. RNA recombination is a process of genetic exchange which occurs in positive-sense RNA viruses and contributes to virus evolution. We have developed a recombinant-specific PCR assay for detection of ZIKV recombinants in mammalian and insect cell co-infections. Using this system, we demonstrated the ability of distinct geographic and genetic isolates to recombine and tested their viability in tissue culture. Initial ZIKV recombinants produce plaques with a significantly smaller diameter than either parental strain. Recombinant large-plaque variants were isolated through serial passage and plaque purification, and the genome analysed for adaptive mutations. A single nucleotide coding mutation in NS1 was detected in several independent large-plaque variants. The role of this mutation in virus replication was investigated through reverse genetics to assess whether it gave the recombinant a replicative advantage. Understanding the genetic changes that arise during, and following recombination is important in the context of virus evolution. It is particularly relevant for ZIKV, as co-circulation of the African and Asian strains of ZIKV in nature has already occurred in parts of Latin America and Africa. Current and future studies will focus on the phenotype of recombinant ZIKV, their replication in mammalian and insect cell lines, and in mosquitoes.

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