Oumar Faye; Caio C. M. Freire; Atila Iamarino; Ousmane Faye; Juliana Velasco C. de Oliveira; Mawlouth Diallo; Paolo M. A. Zanotto; Amadou Alpha Sall

“ The association of the animal sources with viral lineages (Figure S2B) suggested that ZIKV dispersed widely among distinct animal species without a clear pattern of preference, maybe associated to the enzootic cycle of ZIKV in Africa, whose natural cycle allows a broad range of hosts [68] . Nevertheless, we found significant MC (p-value≈1.00 E-4, Dataset S2) for ZIKV strains isolated from A. dalzieli, suggesting a possible important role of this zoophilic vector [69] in West Africa. This association was found to be robust to the exclusion of vertebrate host from the analysis. ”

“ Furthermore, using the concatenated sequences of E and NS5 genes, we employed a discrete model attributing state characters representing isolation locality, animal source, recombination and N- linked glycosylation on E protein of each of the strains with the Bayesian Stochastic Search Variable (BSSVS) algorithm [55] , implemented in BEAST. This method estimates the most probable state at each node in the MCC trees, allowing us to reconstruct plausible ancestral states on these nodes. ”

“ Virus isolates
ZIKV strains were provided by CRORA at the Institute Pasteur of Dakar. The strains were obtained from mosquitoes, humans and other mammals isolated in Burkina Faso, Central African Republic, Côte d'Ivoire and Senegal in West Africa (Table S1) . Viral stocks were prepared by inoculating viral strains into Aedes pseudoscutellaris clone 61 monolayer in Leibovitz 15 (L-15) growth medium (GibcoBRL, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) (GibcoBRL, Grand Island, NY, USA) , 10% Tryptose Phosphate and antibiotics (Sigma, Gmbh, Germany) . ”

“ Maximum likelihood phylogenetic tree inferred for concatenated of sequences from Envelope and NS5 genes of Zika virus.Consensus tree summarized after 1000 non-parametric bootstrap replicates, with support values greater than 60% shown in the nodes. The cluster the Ugandan MR766 prototype strain was highlighted by the yellow sector and the Nigerian cluster was highlighted by the green sector. The strains from Senegal and Côte d'Ivoire are shown in green and orange, respectively. ”

“ In West Africa, surveillance programs have reported periodic circulation of the virus since 1968. To help fill the gap in understanding ZIKV evolution, 43 ZIKV samples were analyzed. We focused on: (i) adaptive genetic changes including protein glycosylation patterns, (ii) phylogenetic relationship among isolates and their spatiotemporal patterns of spread across Africa and Asia and, (iii) dispersion among vertebrate reservoirs and invertebrate vector species. ”

“ Those studies report three main ZIKV lineages, one from Asia and two from Africa. Aiming to fill this gap and gain better insights ZIKV molecular evolution in the 20th century, we investigated 43 ZIKV strains, sampled from 1947 to 2007 in Africa and Asia, to describe phylogenetic relationships, selective influences, recombination events, phylodynamics, phylogeography, host correlations with viral lineages and glycosylation patterns. ”

“ We found a single event in E sequences with estimated breakpoints near to the 414th and 1065th site of E gene reaching nine viral strains: ArA986, HD78788, ArA27101, ArA27290, ArA27096, ArA27443, ArA27407, ArA27106 and ArA982. These results were found by Bootscan, Maxchi, Chimaera, SiSscan and 3Seq methods and supported by significant p-values of 1.31E-5, 2.85E-3, 1.59E-3, 1.79E-29 and 6.85E-19, respectively. Likewise, only one recombination event was detected in NS5 sequences with estimated breakpoints near sites 1581 and 2152 of the NS5 gene from strains ArD158084, ArB1362 and ArD157995. ”

“ This suggests frequent purging of deleterious polymorphisms in functionally important genes. Likewise, the lack of positively selected sites, indicated by ω>0, is typical of highly adapted phenotypes and shows no detectable directional change on the available data. Our findings were expected, as the infection and transmission modes of ZIKV allow the accumulation of synonymous mutations and negatively selected sites [65] . The alternation between arthropod vector and mammal hosts may impose several barriers to non-synonymous mutations in important genes [66] . ”

“ Moreover, we represented the viral migration in Google Earth (http://www.google.com/earth/) , using the SPREAD v1.0.3 program [56] . We evaluated the correlation among viral states and inferred phylogenies from PST by the parsimony score (PS) , association index (AI) and monophyletic clade size (MC) , with BaTS v1.0 [57] after 10000 null replications. ”

“ Uganda (UG) , Central African Republic (CF) , Dezidougou in Côte d'Ivoire (DE) , Sokala-Sobara in Côte d'Ivoire (SS) , Kedougou in Senegal (KE) , Saboya in Senegal (SA) , Bandia in Senegal (BA) , Dakar in Senegal (DA) , Burkina Faso (BF) , Nigeria (NG) , Malaysia (MY) and Yap Island in the Federated States of Micronesia (FM) ; (B) most probable animal source; (C) recombination events per region; and (D) glycosylation polymorphisms. (TIF)
Click here for additional data file. Table S1 Source, country and year of isolation from ZIKV strains used in this study. ”

“ Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proc Natl Acad Sci U S A 94: 6815–6819 pmid:9192648↑ Schmidt H a, Strimmer K, Vingron M, von Haeseler A (2002) TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. ”

“ Predicting functional gene links from phylogenetic-statistical analyses of whole genomes. PLoS Comput Biol 1: e3. doi:10.1371/journal.pcbi.0010003 pmid:16103904↑ Suchard MA, Weiss RE, Sinsheimer JS (2001) Bayesian Selection of Continuous-Time Markov Chain Evolutionary Models. Mol Biol Evol 18: 1001–1013 doi:10.1093/oxfordjournals.molbev.a003872 pmid:11371589↑ Posada D, Crandall K a (2002) The effect of recombination on the accuracy of phylogeny estimation. ”