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

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

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

Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

<div><p>Abstract INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.</p></div

Revalidation and genetic characterization of new members of Group C (<i>Orthobunyavirus</i> genus, <i>Peribunyaviridae</i> family) isolated in the Americas

<div><p>Group C serogroup includes members of the <i>Orthobunyavirus</i> genus (family <i>Peribunyaviridae</i>) and comprises 15 arboviruses that can be associated with febrile illness in humans. Although previous studies described the genome characterization of Group C orthobunyavirus, there is a gap in genomic information about the other viruses in this group. Therefore, in this study, complete genomes of members of Group C serogroup were sequenced or re-sequenced and used for genetic characterization, as well as to understand their phylogenetic and evolutionary aspects. Thus, our study reported the genomes of three new members in Group C virus (Apeu strain BeAn848, Itaqui strain BeAn12797 and Nepuyo strain BeAn10709), as well as re-sequencing of original strains of five members: Caraparu (strain BeAn3994), Madrid (strain BT4075), Murucutu (strain BeAn974), Oriboca (strain BeAn17), and Marituba (strain BeAn15). These viruses presented a typical genomic organization related to members of the <i>Orthobunyavirus</i> genus. Interestingly, all viruses of this serogroup showed an open reading frame (ORF) that encodes the putative nonstructural NSs protein that precedes the nucleoprotein ORF, an unprecedented fact in Group C virus. Also, we confirmed the presence of natural reassortment events. This study expands the genomic information of Group C viruses, as well as revalidates the genomic organization of viruses that were previously reported.</p></div

Group C viruses used in the present study.

<p>Group C viruses used in the present study.</p

ML phylogenetic trees based on alignments of nucleotide sequences of Group C viruses.

<p>(a) S segment (b) M segment and (c) L segment. Phylogenies are midpoint rooted for clarity of presentation. The scale bar indicates evolutionary distance in numbers of substitutions per nucleotides substitutions/site, and the principal bootstrap support levels were indicated. Branches are colour-coded according to group. Viruses strains sequenced in this study are highlighted with red color. The Bimiti, Guama, Catu and Mahogany hammock were used as outgroup. Pairwise distance based on alignments of nucleotide sequences of Group C viruses with (d) S segment (e) M segment and (f) L segment.</p

Reassortment events in Group C viruses.

<p>(a) Apeu, (b) Itaqui, (c) Oriboca and (d) Summary of RDP4 analysis to determine potential reassortants.</p

Genome organization of Group C viruses.

<p>(a) Apeu, (b) Caraparu, (c) Itaqui, (d) Madrid, (e) Marituba, (f) Murucutu, (g) Nepuyo and (h) Oriboca.</p