Reports of apparent mixed infections in Asia that included rickettsioses.

<p>Reports of apparent mixed infections in Asia that included rickettsioses.</p

Overview of PCR-based and DNA sequencing results.

*<p>positivity criteria in analogy to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002163#pntd.0002163-Bustin1" target="_blank">[19]</a>. GenBank accession numbers:</p>1<p>BankIt1587796 Seq2 KC283067.</p>2<p>BankIt1587796 Seq3 KC283068.</p>3<p>BankIt1587796 Seq1 KC283066.</p

Map of Lao PDR.

<p>Locations of the hometowns of the patients whose dengue viruses were sequenced are in grey (Luang Namtha, Latsavang (indicated by a star), Vientiane and Salavan).</p

Clinical details of the 11 patients with blood samples, all survived.

<p>N/A: data not available.</p

Genotype 1 subtree with genome sequences.

<p>Genotype 1 subtree, 843 sequences, from tree performed with the 39 Lao dengue 1 envelope sequences produced in this study aligned with the 1,318 DENV-1 almost complete sequences (>10,000 bases) downloaded from HFVdb. Evolutionary branches that do not include 2007–2010 Lao DENV-1 strains are not shown to increase the legibility. Origin, country (ISO 3166 code) and year of strains within these branches are indicated. The sequences from the Latsavang outbreak (XB) are indicated by red dots. The sequences from Luang Namtha (LNT) are indicated by squares, the one from Salavan (SV) by triangles and the ones from Vientiane (UI) by lozenges. Sequences from 2007 are in yellow, the ones from 2008 (except the ones from Latsavang) are in purple, the ones from 2009 in blue and the ones from 2010 in green. Groups of sequences supported by a high bootstrap value (>90) that contain at least one of Lao sequence are designated as cluster (1 to 7).</p

Dengue assay results.

<p>ELISA results: hl.IgG: High Level IgG; ‘+’: for NS1, IgM or IgG, positive according to the manufacturer's instruction; ‘−’ for negative result; ‘N/A’: no sample available; ‘Inter’: Interpretation, ‘Den’: for evidence of dengue infection. PCR results: ‘Den’: dengue All; ‘Den1’: dengue 1; ‘PF’: panflavivirus; ‘nPF’: heminested panflavivirus; ‘Seqce’: sequencing result of PF or nPF PCR product; ‘+’ in case of dengue genome amplification; ‘−’ in case of no detection of dengue or <i>Flavivirus</i> genome.</p

Temporal and geographical distribution of the strains from the ten DENV-1 clusters.

<p>3,108 DENV-1 envelope sequences, obtained from the European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database in April 2014, were aligned with the DENV-1 Lao sequences obtained in this study. A tree was built (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006203#pntd.0006203.g004" target="_blank">Fig 4</a>) and ten clusters (bootstrap >70) containing the strains from this study were identified. In order to visualize the circulation of all the strains from these different clusters, we plotted each strain according to time of collection and location. Each of the 10 clusters is displayed in a different color: yellow for the strains from cluster # 1, light green for cluster #2, red for cluster #3, blue for cluster #4, dark green for cluster #5, medium green for cluster # 6, pink for cluster #7, grey for cluster #8, light orange for cluster #9 and dark orange for cluster #10. The list of all the strains included in each cluster is provided as <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006203#pntd.0006203.s007" target="_blank">S6 Table</a>.</p

Neighbour-Joining tree of 2,199 dengue 1 envelope gene sequences.

<p>Tree produced using Mega 5.05 software with Kimura-2 model. Bootstrap values (in percentage), generated by using 500 replicates, are only indicated for the nodes that define the genotypes, and for clades inside genotype 1. The sequences from the Latsavang outbreak are indicated by red dots. The other Lao strains from this study are indicated by light blue dots while a dark blue dot is used for the 1996 Lao strain. The sequences from Vientiane are indicated by ‘VTE’, the ones from Luang Namtha by ‘LNT’, and the ones from Salavan by ‘SV’; all followed by year of collection.</p

Seasonality of recruited patients and confirmed dengue cases at the three sites.

<p>Total: recruited patients. Conf dengue: confirmed dengue cases, Average Temp: monthly mean temperature.</p

<i>Dengue virus</i> infection and serotypes detected per site and per year.

<p><i>Dengue virus</i> infection and serotypes detected per site and per year.</p