New Genotype of Dengue Type 3 Virus Circulating in Brazil and Colombia Showed a Close Relationship to Old Asian Viruses

Dengue type 3 genotype V viruses have been recently detected in Brazil and Colombia. In this study, we described another Brazilian isolate belonging to this genotype. Phylogenetic analysis including dengue type 3 viruses isolated worldwide showed that Brazilian and Colombian viruses were closely related to viruses isolated in Asia more than two decades ago. The characteristic evolutionary pattern of dengue type 3 virus cannot explain the close similarity of new circulating viruses with old viruses. Further studies are needed to confirm the origin of the new dengue type III genotype circulating in Brazil and Colombia

Phylogenetic relationship of dengue virus type 3 isolated in Brazil and Paraguay and global evolutionary divergence dynamics

Background: Dengue is the most important mosquito-borne viral disease worldwide. Dengue virus comprises four antigenically related viruses named dengue virus type 1 to 4 (DENV1-4). DENV-3 was re-introduced into the Americas in 1994 causing outbreaks in Nicaragua and Panama. DENV-3 was introduced in Brazil in 2000 and then spread to most of the Brazilian States, reaching the neighboring country, Paraguay in 2002. In this study, we have analyzed the phylogenetic relationship of DENV-3 isolated in Brazil and Paraguay with viruses isolated worldwide. We have also analyzed the evolutionary divergence dynamics of DENV-3 viruses. Results: The entire open reading frame (ORF) of thirteen DENV-3 isolated in Brazil (n = 9) and Paraguay (n = 4) were sequenced for phylogenetic analysis. DENV-3 grouped into three main genotypes (I, II and III). Several internal clades were found within each genotype that we called lineage and sub-lineage. Viruses included in this study belong to genotype III and grouped together with viruses isolated in the Americas within the lineage III. The Brazilian viruses were further segregated into two different sub-lineage, A and B, and the Paraguayan into the sub-lineage B. All three genotypes showed internal grouping. The nucleotide divergence was in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages. Phylogenetic trees constructed with any of the protein gene sequences showed the same segregation of the DENV-3 in three genotypes. Conclusion: Our results showed that two groups of DENV-3 genotypes III circulated in Brazil during 2002-2009, suggesting different events of introduction of the virus through different regions of the country. In Paraguay, only one group DENV-3 genotype III is circulating that is very closely related to the Brazilian viruses of sub-lineage B. Different degree of grouping can be observed for DENV-3 and each group showed a characteristic evolutionary divergence. Finally, we have observed that any protein gene sequence can be used to identify the virus genotype.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP PRONEX REDE DENGUE) [2010/50432-6]Instituto Nacional de Ciencia e Tecnologia (INCT) em Dengu

Molecular characterization of dengue type 3 isolated in Brazilian and Paraguay

RESUMO Alfonso Castro, H. L. Caracterização molecular de dengue tipo 3 isolados no Brasil e no Paraguai. 2010. 105f. Dissertação (Mestrado). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2010. O vírus da dengue (DENV), pertencente ao gênero Flavivirus da família Flaviviridae, é a arbovirose de maior impacto em saúde pública na atualidade. A infecção com qualquer do quatro sorotipos de dengue (DENV-1, -2, -3 e -4) pode ser assintomática ou causar doença febril (DF) que pode evoluir para uma forma mais grave, e algumas vezes fatais, caracterizada por derrame capilar, trombocitopenia. A introdução do DENV-3, genótipo III, nas Américas coincidiu com um aumento no número de casos graves da doença. Este vírus causou uma grande epidemia em 2002 no Rio de Janeiro e posteriormente se espalho em todas as regiões do pais, chegando inclusiva ao Paraguai. Diversos estudos filogenéticos e evolutivos foram realizados com o DENV-3 nas Américas, mas utilizando sequências genômicas parciais. Neste trabalho temos por objetivo analisar o relacionamento filogenético e evolutivo de DENV-3 isolados no Brasil e no Paraguai analisando a sequência genômica completa. A sequência de vírus isolados no Brasil (n=9) e no Paraguai (n=3) foram comparadas com 527 sequências depositadas no GenBank. As 12 cepas virais isoladas no Brasil e no Paraguai pertencem ao grupo americano do genótipo III. Analisando a árvore filogenética dos DENV-3 observamos três genótipos e diversas linhagens, sub-linhagens e clados dentro de cada genótipo. A distância genética entre os genótipos foi de 7,3 a 7,5%, entre as linhagens de 3,2 a 5,3%, entre as sub-linhagens 2,5 a 3,2% e entre os clados de 1,0 a 1,9%. A taxa evolutiva dos vírus variou entre 1,2x10-4 a 8,2x10-4 subs/sitio/ano. O ancestral comum do genótipo I teria surgido entre 1849-1945, do genótipo II entre 1916-1960, e do genótipo III entre 1876-1923. Os diferentes grupos genéticos apresentam motif de aminoácidos característicos. Estes dados serão de grande utilidade para uma melhor caracterização dos DENV-3 em futuras epidemias e, inclusive, poderão ser utilizados para seleção de candidatos a vacina.ALFONSO CASTRO, H. L. Molecular characterization of dengue type 3 isolated in Brazilian and Paraguay. 2010. 105f. Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2010. Infections of humans with dengue viruses (DENV), which belong to the genus Flavivirus(family, Flaviviridae), can be subclinical or cause illnesses ranging from a mild, flu-like syndrome with rash (dengue fever [DF]) to a severe and some times fatal disease, characterized by capillary leakage, thrombocytopenia, and sometimes hypovolemic shock (hemorrhagic dengue fever [DHF/DSS]). DENV are classified in four immunological distinct serotypes: DENV-1 to 4. Recently, a dramatically increase of DHF/DSS cases in the Americas have bee see, and this increase coincided with the introduction of the dengue virus type 3, genotype III. This virus causes a great epidemic in 2002 in the city of Rio de Janeiro and later, the virus spread in Paraguay. Phylogenetics and evolutionary studies have bee carried out with DENV-3 isolated worldwide, but using sequences partial genomic. In this work, we have analyzed the genetic diversity of DENV-3 of Brazilian and Paraguayan isolated, analyzing the complete sequences genomic. The Brazilian (n=9) and Paraguayan (n=3) isolated, were compared with 527 sequences deposited in the GeneBank. Theses isolated, belong to the American group of the genotype III. The phylogenetic analysis of complete genome of the DENV-3, confirmed the existence of three known genotypes and suggested the presence of other groups within each genotype named of the lineages, sub-lineages and clades. The genetic distance among the genotypes were of 7,3 to 7,5%, among the lineages of 3,2 to 5,3%, among the sub-lineages of 2,5 to 3,2% and among clades of 1,0 to 1,9%. The evolutionary rates of the viruses varied among 1,2x10-4 to 8,2x10-4 s/s/y. The age of the ancestral common more recent of the genotype I, possibly are among 1849-1945, the ancestral common of the genotype II, among 1916-1960 and the ancestral common more recent of the genotype III, among 1876-1923. The different genetic groups present motif of amino acids. These data could provide information for a better understanding of the evolution of theses viruses, and even for selection of candidate vaccin

Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and beta-thalassemia major and healthy blood donors

Human Parvovirus B19 (B19V) is a recognized cause of life-threatening conditions among patients with hemoglobinopathies. This study investigates B19V infection in patients with sickle cell disease and beta-thalassemia using different experimental approaches. A total of 183 individuals (144 with sickle cell disease and 39 with beta-thalassemia major) and 100 healthy blood donors were examined for B19V using anti-B19V IgG enzyme immunoassay, quantitative PCR, DNA sequencing, and phylogenetic analysis. Viremia was documented in 18.6% of patients and 1% of donors, and was generally characterized by low viral load (VL); however, acute infections were also observed. Anti-B19V IgG was detected in 65.9% of patients with sickle cell disease and in 60% of donors, whereas the patients with thalassemia exhibited relatively low seroreactivity. The seroprevalence varied among the different age groups. In patients, it progressively increased with age, whereas in donors it reached a plateau. Based on partial NS1 fragments, all isolates detected were classified as subgenotype 1A with a tendency to elicit genetically complex infections. Interestingly, quasispecies occurred in the plasma of not only patients but also donors with even higher heterogeneity. The partial NS1 sequence examined did not exhibit positive selection. Quantitation of B19V with a conservative probe is a technically and practically useful approach. The extensive spread of B19V subgenotype 1A in patients and donors and its recent introduction into the countryside of the Sao Paulo State, Brazil were demonstrated; however, it is difficult to establish a relationship between viral sequences and the clinical outcomes of the infection. J. Med. Virol. 84:16521665, 2012. (c) 2012 Wiley Periodicals, Inc.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Instituto Nacional de Ciencia e Tecnologia em Celulas Tronco e Terapia Celular (INCTC)Instituto Nacional de Ciencia e Tecnologia em Celulas Tronco e Terapia Celular (INCTC

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

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

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

Laboratory viruses used in this study.

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