Comparison of the 5’UTR nucleotide sequences of Asian and African ZIKV isolates.

<p>The mean pairwise identity of all pairs at a given position is indicated by the identity bar; lilac is indicative of 100% pairwise identity, dark purple highlights positions possessing <100% pairwise identity. Positions and quantity of single nucleotide polymorphisms (SNPs) are represented as black bands within grey sequence bars. Sequences 1–32, highlighted orange, correspond to the outbreak originating in 2015 in Brazil. Microcephaly, adult mortality and ZIKV PE243 associated sequences are highlighted as previously described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005048#pntd.0005048.g001" target="_blank">Fig 1</a>.</p

Primers sequences used for 5’/3’ RACE of ZIKV viral termini.

<p>Primers sequences used for 5’/3’ RACE of ZIKV viral termini.</p

Activation of the IFN-β promoter by poly I:C in cells over expressing ZIKV sfRNA.

<p>A549 cells were co-transfected with either pDEST-DENV-3’UTR, pDEST-ZIKV PE243-3‘UTR or pDEST40-MBP (sfRNA over-expression plasmids and MBP-HDVr control, respectively) and p125Luc IFN-β promoter reporter (expressing Firefly luciferase) along with pRL-CMV (internal control, expressing <i>Renilla</i> luciferase). The IFN-β promoter was stimulated by transfecting poly I:C 24 h after the primary transfection. The relative luciferase activity (Firefly/<i>Renilla</i>) was analyzed at 24 h following the second transfection. The mean with standard error is shown for three independent experiments performed in triplicate; values of independent experiments were used for analysis. The data were normalized to cells transfected with pDEST40-MBP without any poly I:C treatment. Asterisk (*) indicates significance (2-way ANOVA, p<0.05).</p

Comparison of African and Asian lineage ZIKV protein coding regions.

<p>The mean pairwise identity of all pairs at a given position is indicated by the identity bar; light blue denotes 100% pairwise identity, dark blue highlights positions possessing less than 100% pairwise identity. Positions and quantity of amino acid substitutions are indicated by black bands within grey sequence bars. Sequences 1–37, highlighted yellow, correspond to the outbreak originating in 2015 in South America. Microcephaly, adult mortality and ZIKV PE243 associated sequences are highlighted as previously described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005048#pntd.0005048.g001" target="_blank">Fig 1</a>.</p

Primers used for cloning of ZIKV 3'UTR containing sfRNA.

<p>Primers used for cloning of ZIKV 3'UTR containing sfRNA.</p

The predicted structure of ZIKV PE243 3’UTR.

<p>5’-3’ of the ZIKV PE243 3’UTR sequence, left to right. The arrow indicates the predicted start of sfRNA. Nucleotides are indicated either counted from the 3’ (indicated as negative numbers) or from the start of the 3’UTR (positive number in brackets). SL, stem loop structure; DBL, dumbbell structure; 3’SL; 3’ end stem loop structure. The dotted line represents the predicted pseudoknot.</p

Bayesian maximum clade credibility tree generated from coding sequence data.

<p>Bayesian posterior probabilities are given at nodes of importance. Isolates which have been implicated in particular diseases are highlighted, as is the ZIKV PE243 isolate we have sequenced. GenBank accession numbers of all sequences used are given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005048#pntd.0005048.s001" target="_blank">S1 Table</a>. EC_2007 refers to the epidemic consensus sequence generated from the Yap Island outbreak in 2007 (EU545988).</p

sfRNA production in ZIKV PE243 infection.

<p>Top panel: Vero E6 cells were infected with ZIKV isolate PE243 and sfRNA detected by northern blot. Total RNA isolated from Vero E6 cells infected with ZIKV PE243 was separated on a denaturing agarose gel and transferred to a nylon membrane as described in Materials and methods. Radiolabeled DNA probe complementary to 3’UTR was used to detect genomic RNA and sfRNA. Bottom panel: assessed amounts of 18S ribosomal RNAs (fluorescently labelled with ethidium bromide) prior to transfer.</p