De Novo Generation and Characterization of New Zika Virus Isolate Using Sequence Data from a Microcephaly Case

Zika virus (ZIKV) has recently emerged and is the etiological agent of congenital Zika syndrome (CZS), a spectrum of congenital abnormalities arising from neural tissue infections in utero. Herein, we describe the de novo generation of a new ZIKV isolate, ZIKV_Natal, using a modified circular polymerase extension reaction protocol and sequence data obtained from a ZIKV-infected fetus with microcephaly. ZIKV_Natal thus has no laboratory passage history and is unequivocally associated with CZS. ZIKV_Natal could be used to establish a fetal brain infection model in IFNAR^-/- mice (including intrauterine growth restriction) without causing symptomatic infections in dams. ZIKV_Natal was also able to be transmitted by Aedes aegypti mosquitoes. ZIKV_Natal thus retains key aspects of circulating pathogenic ZIKVs and illustrates a novel methodology for obtaining an authentic functional viral isolate by using data from deep sequencing of infected tissues. IMPORTANCE The major complications of an ongoing Zika virus outbreak in the Americas and Asia are congenital defects caused by the virus's ability to cross the placenta and infect the fetal brain. The ability to generate molecular tools to analyze viral isolates from the current outbreak is essential for furthering our understanding of how these viruses cause congenital defects. The majority of existing viral isolates and infectious cDNA clones generated from them have undergone various numbers of passages in cell culture and/or suckling mice, which is likely to result in the accumulation of adaptive mutations that may affect viral properties. The approach described herein allows rapid generation of new, fully functional Zika virus isolates directly from deep sequencing data from virus-infected tissues without the need for prior virus passaging and for the generation and propagation of full-length cDNA clones. The approach should be applicable to other medically important flaviviruses and perhaps other positive-strand RNA viruses.