Maximum Likelihood phylogenetic tree derived from 80 global samples of DENV-3 using 306 nucleotides of E/NS1 junction with a bootstrap analysis of 500 replicates.

<p>A DENV-1 strain (M87512) was used as outgroup. Horizontal branch lengths are drawn to scale. Aligned sequences were analyzed in the Modeltest 2.3 program and found that the best fit-model of nucleotide substitution for phylogenetic reconstruction was Tamura & Nei (TrN+I) with a proportion of invariable sites (I) of 0.5203 and gamma distribution with equal rates for all sites, using Akaike's Information Criterion (AIC). GenBank accession numbers: In_98901403_DSS_DV_3_98 (AB189125), In_98901437_DSS_DV_3_98 (AB189126), In_98901517_DHF_DV_3_98 (AB189127), In_FW01_04 (AY858040), In_FW06_04 (AY858041), In_KJ30i_04 (AY858042), In_KJ71_04 (AY858044), In_PH86_04 (AY858045), In_PI64_04 (AY858046), In_TB16_04 (AY858047), In_TB55i_04 (AY858048), In_BA51_04 (AY858037), In_den3_98 (AY858039), ET_D3_Hu_TL109NIID_2005 (AB214881), China_80_2_ (AF317645), BR_DEN3_RO1_02 (EF629370), BR_DEN3_RO2_02_ (EF629373), BDH02_1_02 (AY496871), BDH02_7_02 (AY496877), ThD3_0104_93_ (AY676350), ThD3_0055_93_ (AY676351), Thail_C0331_94_94 (AY876494), ThD3_0010_87_ (AY676352), VietN_BID_V1008_2006 (EU482452), VietN_BID_V1009_2006 (EU482453), VietN_BID_V1011_2006 (EU482455), VietN_BID_V1014_2006 (EU482458), VietN_BID_V1015_2006 (EU482459), VietN_BID_V1016_2006 (EU482460), VietN_BID_V1017_2006 (EU482461), VietN_BID_V1018_2006 (EU482462), VietN_BID_V1010_2006 (EU482454), VietN_BID_V1012_2006 (EU482456), VietN_BID_V1013_2006 (EU482457), Sing_8120_95 (AY766104), D3_H_IMTSSA_SRI_2000_1266 (AY099336), NC_001475 (NC_001475), Singapore (AY662691), D3_SG_SS710_2004 (EU081181), D3_SG_05K791DK1_2005 (EU081182), BR74886_02 (AY679147), BR_DEN3_95_04 (EF629366), BR_DEN3_97_04 (EF629367), BR_DEN3_98_04_ (EF629368), BR_DEN3_290_02 (EF629369), PtoR_BID_V1043_2006 (EU482555), PtoR_BID_V1078_2003 (EU482564), PtoR_BID_V1075_1998 (EU482563), PtoR_BID_V1088_1998 (EU482566), PtoR_BID_V859_1998 (EU482596), VEN_BID_V904_2001 (EU482612), PtoR_BID_V858_2003 (EU482595), D3/Hu/TL029NIID/2005 (AB214880), Indo_98_98901640 (AY912455), In KJ46 (AY858045), Philp56 H87 (L11423), 375 And03 (EU003494), 389 Guaj03 (EU003495), 395 NSan04 (EU003496), 400 Guaj04 (EU003497), 417 Guav04 (EU003498), 429 Huil04 (EU003499), 591 DV20 Ant05 (EU003513), DV06 Ant05 (EU003514), C0360 94 (AY923865), ThD3 1283 98 (AY676349), 98TW182 (DQ675520), Thail 98 KPS 4 0657 207 (AY912458), 99TW628 99 (DQ675533), D3 H IMTSSA MART 1999 1243 (AY099337), D3 H IMTSSA MART 2000 1567 (AY099338), D3 H IMTSSA MART 2000 1706 (AY099339), D3 H IMTSSA MART 2001 2012 (AY099340), D3 H IMTSSA MART 2001 2336 (AY099342), D3 H IMTSSA MART 2001 2023 (AY099341), BDH02_8_02 (AY496878), BDH02_6_02 (AY496876), ThD3_1687_98 (AY676348), DENV1 (M87512).</p

Bayesian phylogenetic trees derived from 79 global samples of DENV-3 NS1 gene sequences inferred with MrBayes program.

<p>The posterior probabilities expressed in percent are indicated at important nodes. DENV-1 (M87512) and DENV-4 (AY618992) strains were used as outgroup. Horizontal branch lengths are drawn to scale. Aligned sequences were analyzed in the MrModeltest 2.3 program to identify the best fit-model of nucleotide substitution for Bayesian phylogenetic reconstruction. The nucleotide substitution model used was under a General Time Reversible model of nucleotide substitution with gamma-distributed rate variation (G = 1.9241) and a proportion of invariable sites (I = 0.4401) (GTR+G+I), using Akaike's Information Criterion (AIC). Five runs of 4 chains each (one cold and tree heated, temperature = 0.20) were run for 1.5×10⁶ generations, with a burn-in of 6000 generations. GenBank accession numbers: D3BR PV7 03 (FJ481174), In_98901403_DSS_DV_3_98 (AB189125), In_98901437_DSS_DV_3_98 (AB189126), In_98901517_DHF_DV_3_98 (AB189127), In_98902890_DF_DV_3_98 (AB189128), ET_D3_Hu_TL018NIID_2005 (AB214879), ET_D3_Hu_TL109NIID_2005 (AB214881), ET_D3_Hu_TL029NIID_2005 (AB214880), ET_D3_Hu_TL129NIID_2005 (AB214882), China_80_2_ (AF317645), D3_H_IMTSSA_SRI_2000_1266 (AY099336), D3_H_IMTSSA_MART_1999_1243 (AY099337), BDH02_1_02 (AY496871), BDH02_3_02 (AY496873), BDH02_4_2 (AY496874), BDH02_7_02 (AY496877), In_Sleman_78 (AY648961), Singapore (AY662691), ThD3_0104_93_ (AY676350), ThD3_0055_93_ (AY676351), BR74886_02 (AY679147), PF89_320219_89 (AY744678), PF90_3056_90 (AY744680), PF92_4190_92 (AY744684), PF94_136116_94 (AY744685), In_BA51_04 (AY858037), In_den3_98 (AY858039), In_FW01_04 (AY858040), In_FW06_04 (AY858041), In_KJ30i_04 (AY858042), In_KJ71_04 (AY858044), In_PH86_04 (AY858045), In_PI64_04 (AY858046), In_TB16_04 (AY858047), In_TB55i_04 (AY858048), Thail_C0331_94_94 (AY876494), In_InJ_16_82 (DQ401690), PhMH_J1_97 (DQ401695), BR_DEN3_95_04 (EF629366), BR_DEN3_97_04 (EF629367), BR_DEN3_98_04_ (EF629368), BR_DEN3_290_02 (EF629369), BR_DEN3_RO1_02 (EF629370), BR_DEN3_RO2_02_ (EF629373), D3_SG_SS710_2004 (EU081181), D3_SG_05K791DK1_2005 (EU081182), D3_SG_05K843DK1_2005 (EU081187), D3_SG_05K4648DK1_2005 (EU081225), VietN_BID_V1008_2006 (EU482452), VietN_BID_V1009_2006 (EU482453), VietN_BID_V1010_2006 (EU482454), VietN_BID_V1011_2006 (EU482455), VietN_BID_V1012_2006 (EU482456), VietN_BID_V1013_2006 (EU482457), VietN_BID_V1014_2006 (EU482458), VietN_BID_V1015_2006 (EU482459), VietN_BID_V1016_2006 (EU482460), VietN_BID_V1017_2006 (EU482461), VietN_BID_V1018_2006 (EU482462), PtoR_BID_V1043_2006 (EU482555), PtoR_BID_V1049_1998 (EU482558), PtoR_BID_V1050_1998 (EU482559), PtoR_BID_V1075_1998 (EU482563), PtoR_BID_V1078_2003 (EU482564), PtoR_BID_V1088_1998 (EU482566), PtoR_BID_V858_2003 (EU482595), PtoR_BID_V859_1998 (EU482596), VEN_BID_V904_2001 (EU482612), VEN_BID_V906_2001 (EU482613), VEN_BID_V913_2001 (EU482614), Philip56_H87 (M93130), NC_001475 (NC_001475), ThD3_1687_98 (AY676348), 98TWmosq_98 (DQ675532), ThD3_1283_98 (AY676349), In_KJ46_04 (AY858043), Thail C0360 94 (AY923865), DENV1 (M87512), DENV4 (AY618992).</p

Estimated substitutions rates and dates for DENV-3 genotypes.

<p>Estimated substitutions rates and dates for DENV-3 genotypes.</p

Nucleotide and amino acid pairwise distances between DENV-3 strains used in this study for the envelope glycoprotein.

<p>In parenthesis are indicated the year of isolation and the genotype.</p

DNA Microarray Platform for Detection and Surveillance of Viruses Transmitted by Small Mammals and Arthropods

<div><p>Viruses transmitted by small mammals and arthropods serve as global threats to humans. Most emergent and re-emergent viral agents are transmitted by these groups; therefore, the development of high-throughput screening methods for the detection and surveillance of such viruses is of great interest. In this study, we describe a DNA microarray platform that can be used for screening all viruses transmitted by small mammals and arthropods (SMAvirusChip) with nucleotide sequences that have been deposited in the GenBank. SMAvirusChip was designed with more than 15,000 oligonucleotide probes (60-mers), including viral and control probes. Two SMAvirusChip versions were designed: SMAvirusChip v1 contains 4209 viral probes for the detection of 409 viruses, while SMAvirusChip v2 contains 4943 probes for the detection of 416 viruses. SMAvirusChip was evaluated with 20 laboratory reference-strain viruses. These viruses could be specifically detected when alone in a sample or when artificially mixed within a single sample. The sensitivity of SMAvirusChip was evaluated using 10-fold serial dilutions of dengue virus (DENV). The results showed a detection limit as low as 2.6E3 RNA copies/mL. Additionally, the sensitivity was one log₁₀ lower (2.6E2 RNA copies/mL) than quantitative real-time RT-PCR and sufficient to detect viral genomes in clinical samples. The detection of DENV in serum samples of DENV-infected patients (n = 6) and in a whole blood sample spiked with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples. In addition, in a pool of mosquito samples spiked with DENV, the virus was also detectable. SMAvirusChip was able to specifically detect viruses in cell cultures, serum samples, total blood samples and a pool of mosquitoes, confirming that cellular RNA/DNA did not interfere with the assay. Therefore, SMAvirusChip may represent an innovative surveillance method for the rapid identification of viruses transmitted by small mammals and arthropods.</p></div

Phospholipase A₂ Isolated from the Venom of <i>Crotalus durissus terrificus</i> Inactivates <i>Dengue virus</i> and Other Enveloped Viruses by Disrupting the Viral Envelope

<div><p>The <i>Flaviviridae</i> family includes several virus pathogens associated with human diseases worldwide. Within this family, <i>Dengue virus</i> is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against <i>Dengue virus</i> or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A₂ isolated from the venom of <i>Crotalus durissus terrificus</i> was able to inhibit <i>Dengue virus</i> and <i>Yellow fever virus</i> infection in Vero cells. Here, we present evidence that phospholipase A₂ has a direct effect on <i>Dengue virus</i> particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A₂ might gain access to the <i>Dengue virus</i> lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A₂ is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.</p></div

Results of statistical analyses of reference viruses and control samples tested with the SMAvirusChip platform.

<p>Results of statistical analyses of reference viruses and control samples tested with the SMAvirusChip platform.</p

SMAvirusChip v2 form (RAF) showing the results of statistical analysis of an array hybridized with ZIKV.

<p>SMAvirusChip v2 form (RAF) showing the results of statistical analysis of an array hybridized with ZIKV.</p

Detection of DENV-2 spiked in a whole blood sample and a pool of mosquitoes with SMAvirusChip v1.

<p>Detection of DENV-2 spiked in a whole blood sample and a pool of mosquitoes with SMAvirusChip v1.</p

Laboratory viruses used in this study.

<p>Laboratory viruses used in this study.</p