Zika virus (ZIKV) has evolved into a global health threat because of its unexpected causal link to microcephaly. Phylogenetic analysis reveals that contemporary epidemic strains have accumulated multiple substitutions from their Asian ancestor. Here we show that a single serine-to-asparagine substitution [Ser139→Asn139 (S139N)] in the viral polyprotein substantially increased ZIKV infectivity in both human and mouse neural progenitor cells (NPCs) and led to more severe microcephaly in the mouse fetus, as well as higher mortality rates in neonatal mice. Evolutionary analysis indicates that the S139N substitution arose before the 2013 outbreak in French Polynesia and has been stably maintained during subsequent spread to the Americas. This functional adaption makes ZIKV more virulent to human NPCs, thus contributing to the increased incidence of microcephaly in recent ZIKV epidemics.

Animal models are critical to understand disease and to develop countermeasures for the ongoing epidemics of Zika virus (ZIKV). Here we report a non-human primate model using a 2016 contemporary clinical isolate of ZIKV. Upon subcutaneous inoculation, rhesus macaques developed fever and viremia, with robust excretion of ZIKV RNA in urine, saliva, and lacrimal fluid. Necropsy of two infected animals revealed that systematic infections involving central nervous system and visceral organs were established at the acute phrase. ZIKV initially targeted the intestinal tracts, spleen, and parotid glands, and retained in spleen and lymph nodes till 10 days post infection. ZIKV-specific immune responses were readily induced in all inoculated animals. The non-human primate model described here provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccine and therapeutics.

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Viral replicase recruitment and long-range RNA interactions are essential for RNA virus replication, yet the mechanism of their interplay remains elusive. Flaviviruses include numerous important human pathogens, e.g., dengue virus (DENV) and Zika virus (ZIKV). Here, we revealed a highly conserved, conformation-tunable cis-acting element named 5′-UAR-flanking stem (UFS) in the flavivirus genomic 5′ terminus. We demonstrated that the UFS was critical for efficient NS5 recruitment and viral RNA synthesis in different flaviviruses. Interestingly, stabilization of the DENV UFS impaired both genome cyclization and vRNA replication. Moreover, the UFS unwound in response to genome cyclization, leading to the decreased affinity of NS5 for the viral 5′ end. Thus, we propose that the UFS is switched by genome cyclization to regulate dynamic RdRp binding for vRNA replication. This study demonstrates that the UFS enables communication between flavivirus genome cyclization and RdRp recruitment, highlighting the presence of switch-like mechanisms among RNA viruses.DOI: http://dx.doi.org/10.7554/eLife.17636.001

c cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5= cyclization sequence (5=CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5=CS, and the presence of DCS-PK facilitates the formation of 5=-3= RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution. T he flavivirus genus contains numerous important agents of human infectious diseases, including dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV). Human infection by flaviviruses can result in symptoms ranging from mild fever to severe encephalitis and hemorrhagic fever. Due to lack of effective vaccines and specific medicines against many flaviviruses, they pose a significant threat to human health around the world.Flaviviruses are enveloped RNA viruses with single-stranded, positive-sense genomes. Their 5= capped viral genome RNA (vRNA) is approximately 10 to 11 kb and contains a single open reading frame (ORF) flanked by 5= and 3= untranslated regions (UTRs). The ORF encodes a polyprotein with more than 3,000 residues, which is cotranslationally and/or posttranslationally proteolytically processed into three structural proteins (capsid, pre-membrane/membrane, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by viral and cellular proteases. NS3 and NS5 perform essential enzyme activities required for vRNA replication. The C-terminal domain of NS3 has helicase/NTPase (1) and RNA triphosphatase activities (2, 3). The N-terminal domain of NS5 encodes guanylyltransferase (4) and methyltransferase activities (5, 6), whereas the C-terminal domain of NS5 has RNA-dependent RNA polymerase (RdRp) activity. These enzyme activities are involved in vRNA synthesis, 5= capping, and internal adenosine methylation (7).The 5=UTR, 3=UTR, and capsid-coding sequence are involved in vRNA replication, translation, and perhaps encapsidation, and many cis-acting elements have ...