Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention1,2, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests.

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital syndromes1,2. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States and since then, hundreds of locally-acquired infections have been reported in Florida3,4. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least four introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission likely started in the spring of 2016 - several months before initial detection. By analyzing surveillance and genetic data, we discovered that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions are linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions.

No abstract

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital syndromes 1,2 . In July 2016, mosquito-borne ZIKV transmission was first reported in the continental United States and since then, hundreds of locally-acquired infections have been reported in Florida 3 . To gain insights into the timing, source, and likely route(s) of introduction of ZIKV into the continental United States, we tracked the virus from its first detection in Miami, Florida by direct sequencing of ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least four distinct ZIKV introductions contributed to the outbreak in Florida and that local transmission likely started in the spring of 2016 -several months before its initial detection. By analyzing surveillance and genetic data, we discovered that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions are phylogenetically linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. By comparing mosquito abundance and travel flows, we describe the areas of southern Florida that are especially vulnerable to ZIKV introductions. Our study provides a deeper understanding of how ZIKV initiates and sustains transmission in new regions.

2Despite great attention given to the recent Zika virus (ZIKV) epidemic in the Americas, much remains unknown about its epidemiology and evolution, in part due to a lack of genomic data. We applied multiple sequencing approaches to generate 100 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analyzed the timing and patterns of introductions into distinct geographic regions, confirming phylogenetic evidence for the origin and rapid expansion of the outbreak in Brazil 1 , and for multiple introductions from Brazil into Honduras, Colombia, Puerto Rico, other Caribbean islands, and the continental US. We find that ZIKV circulated undetected in many regions of the Americas for up to a year before the first locally transmitted cases were confirmed, highlighting the challenge of effective surveillance for this virus. We further characterize genetic variation across the outbreak to identify mutations with possible functional implications for ZIKV biology and pathogenesis.Since its introduction into Brazil in 2013 1 , mosquito-borne ZIKV (Family: Flaviviridae) has spread rapidly throughout the Americas, causing hundreds of thousands of cases of ZIKV disease, as well as ZIKV congenital syndrome and likely other neurological complications 2-4 . Comparative phylogenomic analysis of ZIKV can reveal the trajectory of the outbreak and detect mutations that may be associated with new disease phenotypes or affect molecular diagnostics. Despite the nearly 60 years since its discovery and the scale of the recent outbreak, however, fewer than 100 ZIKV genomes have been sequenced directly from clinical samples. This is due in part to technical challenges posed by low viral loads (often orders of magnitude lower than in Ebola or dengue virus infection 5-7 ), as well as issues of RNA preservation in samples collected without the unique requirements of sequencing in mind. Culturing the virus can greatly increase the material available for sequencing, but it can introduce artefacts and is time-consuming and difficult.We sought to gain a deeper understanding of the viral populations underpinning the ZIKV epidemic by extensive genome sequencing of the virus directly from samples collected as part of ongoing surveillance. We initially pursued metagenomic RNA sequencing in order to capture both ZIKV and other potential co-infections in an unbiased way. In most of the 37 samples examined by this approach, however, the amount of ZIKV material was not sufficient for genome assembly. Unbiased RNA sequencing still proved valuable because it provided ZIKV data to verify results from other methods. We also observed 3 other viruses in 7 samples (Extended Data Table 1) in our metagenomic data; notably, these did not include chikungunya or dengue, viruses known to be cocirculating with ZIKV in many affected regions 8,9 .In order to capture sufficient ZIKV content for genome assembly, we turned to two targeted enrichment approaches: hybrid c...