Clinical significance of intra-host variability of Dengue-1 virus in venous and capillary blood  E. Descloux, C. La Fuentez, Y. Roca, X. De Lamballerie  Clinical Microbiology and Infection  Volume 20, Issue 3, Pages O167-O175 (March 2014) DOI: 10.1111/1469-0691.12368 Copyright © 2014 European Society of Clinical Infectious Diseases Terms and Conditions

Fig. 1 Phylogenetic tree based on 68 nucleotide sequences of partial E-gene of dengue virus (DENV) (neighbour-joining method, Kimura 2 algorithm). The consensus tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Taxon names of the 2009 Bolivian strains correspond to D1 Bolivia 2009 with the patient number in brackets. Taxon names of Bolivian strains previously characterized [23] correspond to D1 Bolivia patient number/last two digits of year of isolation, and the epidemic year (E) in brackets. Taxon names of GenBank sequences correspond to D1.country/last two digits of year of isolation. Their GenBank accession numbers are indicated in brackets. All patients included in this study were infected by DENV-1 strains belonging to the genotype V ‘America-Africa’. These strains isolated during the 2009 Bolivian outbreak constitute a homogeneous group of sequences sharing a common ancestor with Bolivian strains involved in the 2007 outbreak that share themselves a common ancestor with strains isolated in Puerto Rico (2006) and Venezuela (2007). The 2007 and 2009 Bolivian strains are more distantly related to Bolivian strains from the 1998, 2000 and 2001 outbreaks characterized by E-gene sequences highly similar to those of viruses that circulated in Brazil and Argentina during the same period [23]. The 2009 Bolivian DENV-1 viruses may have evolved in situ in Bolivia after the introduction of new variants in 2007 from neighbouring countries (Puerto Rico, Venezuela). Clinical Microbiology and Infection 2014 20, O167-O175DOI: (10.1111/1469-0691.12368) Copyright © 2014 European Society of Clinical Infectious Diseases Terms and Conditions

Fig. 2 Dengue virus type 1 (DENV-1) genetic variability in patients with and without haemorrhagic symptoms. The extent of intra-host genetic variability of DENV-1 based on the global analysis of clone sequences (average 56 clones/patient) from both venous and capillary sectors was compared between five patients with haemorrhagic symptoms and five patients without haemorrhagic symptoms using a Mann-Witney test. The percentage of mutant clones (number of clones with nucleotide (nt) and amino acid (aa) mutation/total number of clones), the percentage of mutations among nt and aa sequences (number of nt and aa mutations/number of nt and aa sequenced), the pairwise distance (p-distance) among nt sequences (π nt) and among aa sequences (π aa) presented as boxplots (with central line indicating the median value) on the left axes, respectively, were calculated using the MEGA 4.0.2 package [22]. All parameters of genetic variability were significantly lower in patients with haemorrhagic symptoms (p <0.05 using a Mann-Witney test), except mean π nt. Detailed results of the analysis restricted to the venous sector and the capillary sector are presented in the Supplementary material, Table S1. Clinical Microbiology and Infection 2014 20, O167-O175DOI: (10.1111/1469-0691.12368) Copyright © 2014 European Society of Clinical Infectious Diseases Terms and Conditions