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

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.

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.

[Dengue: epidemiological aspects and the first outbreak in the Middle Solimões Region of Coari in the State of Amazonas from 2008 to 2009]. / Dengue: aspectos epidemiológicos e o primeiro surto ocorrido na região do Médio Solimões, Coari, Estado do Amazonas, no período de 2008 a 2009.

INTRODUCTION: Dengue is one of the most prevalent infectious diseases in Brazil and is a major public health problem worldwide. It is most prevalent in tropical and subtropical regions with 2.5 to 3 billion people at risk of becoming infected. Thus, this study sought to demonstrate the epidemiological characteristics of individuals affected by dengue, its prevalence and its epidemic process in the Middle Solimões region of Coari in the state of Amazonas between 2008 and 2009. METHODS: Epidemiological data were obtained through epidemiological monitoring by the Municipal Health Facility of the city of Coari, AM. The variables analyzed were the month of notification, the number of confirmed cases, the cases' genders, the cases' ages and their neighborhoods of residence. RESULTS: In total, 1,003 cases were reported (635 in 2008 and 368 in 2009), and 639 cases were diagnosed as positive. Of these, +54% involved female subjects, and +46% were male. The majority of the affected individuals were between 10 and 49 years of age, and the spatial distribution was concentrated in neighborhoods near streams, lakes and areas in which housing had recently been disrupted. CONCLUSIONS: We concluded that, during the period studied, there was an outbreak of dengue in the City of Coari, AM. However, it is possible that a dengue epidemic may have occurred earlier in Coari without proper diagnosis or follow-up and that previously infected individuals may have traveled to the capital of Amazonas (Manaus), where the virus has circulated since 1998.

Notification of the first isolation of Cacipacore virus in a human in the State of Rondônia, Brazil.

Flavivirus is a genus of arthropod-transmitted viruses of the family Flaviviridae, and in Brazil, up to eleven different Flavivirus have been isolated. We collected blood from farmers in the municipality of Theobroma, which is located 320km from the City of Porto Velho, the former capital of the Brazilian State of Rondônia. For viral isolation, we used newborn mouse brain, followed by RT-PCR with specific universal Flavivirus primers. We obtained fragments 958bp and 800bp in length. Based on BLAST, these sequences were 91% similar to a sequence of Cacipacore virus.

Dengue: aspectos epidemiológicos e o primeiro surto ocorrido na região do Médio Solimões, Coari, Estado do Amazonas, no período de 2008 a 2009 / Dengue: epidemiological aspects and the first outbreak in the Middle Solimões Region of Coari in the State of Amazonas from 2008 to 2009

INTRODUÇÃO: A dengue é uma das doenças infecciosas mais frequentes no Brasil e um dos principais problemas de saúde pública no mundo, principalmente em regiões tropicais e subtropicais, com 2,5 a 3 bilhões de pessoas expostas ao risco de serem infectadas atualmente. Deste modo, o presente estudo teve como objetivo demonstrar as características epidemiológicas dos indivíduos acometidos por dengue, sua prevalência e seu processo epidêmico na região do Médio Solimões, Coari, Amazonas, no período de 2008 e 2009. MÉTODOS: Os dados epidemiológicos foram obtidos na Vigilância Epidemiológica da Secretaria Municipal de Saúde da Cidade de Coari-AM. As variáveis analisadas foram: mês da notificação, casos confirmados, gênero (sexo), faixa etária e bairro de residência. RESULTADOS: No total, foram notificados 1.003 casos (635 em 2008 e 368 em 2009), sendo diagnosticados 639 casos positivos. Destes, ± 54% acometerem indivíduos do sexo feminino e ± 46% do sexo masculino. As faixas etárias mais acometidas foram às observadas entre 10-49 anos; quanto à distribuição espacial, observamos o acometimento de indivíduos de bairros próximos a igarapés, lagos e com processo recente e desordenado de habitação. CONCLUSÕES: Deste modo, conclui-se que, durante o período estudado, houve um surto epidêmico de dengue na Cidade de Coari, AM. Entretanto, deve-se considerar que uma epidemia de dengue anterior pode ter ocorrido em Coari, sem que tenha tido o devido diagnóstico etiológico, ou que houveram pessoas com infecção passada que se deslocaram para a capital do Amazonas (Manaus), onde os vírus circulam desde 1998.

INTRODUCTION: Dengue is one of the most prevalent infectious diseases in Brazil and is a major public health problem worldwide. It is most prevalent in tropical and subtropical regions with 2.5 to 3 billion people at risk of becoming infected. Thus, this study sought to demonstrate the epidemiological characteristics of individuals affected by dengue, its prevalence and its epidemic process in the Middle Solimões region of Coari in the state of Amazonas between 2008 and 2009. METHODS: Epidemiological data were obtained through epidemiological monitoring by the Municipal Health Facility of the city of Coari, AM. The variables analyzed were the month of notification, the number of confirmed cases, the cases' genders, the cases' ages and their neighborhoods of residence. RESULTS: In total, 1,003 cases were reported (635 in 2008 and 368 in 2009), and 639 cases were diagnosed as positive. Of these, +54% involved female subjects, and +46% were male. The majority of the affected individuals were between 10 and 49 years of age, and the spatial distribution was concentrated in neighborhoods near streams, lakes and areas in which housing had recently been disrupted. Conclusions: We concluded that, during the period studied, there was an outbreak of dengue in the City of Coari, AM. However, it is possible that a dengue epidemic may have occurred earlier in Coari without proper diagnosis or follow-up and that previously infected individuals may have traveled to the capital of Amazonas (Manaus), where the virus has circulated since 1998.

Notification of the first isolation of Cacipacore virus in a human in the State of Rondônia, Brazil / Notificação do primeiro isolamento do vírus Cacipacoré em um ser humano, no Estado de Rondônia, Brasil

Flavivirus is a genus of arthropod-transmitted viruses of the family Flaviviridae, and in Brazil, up to eleven different Flavivirus have been isolated. We collected blood from farmers in the municipality of Theobroma, which is located 320km from the City of Porto Velho, the former capital of the Brazilian State of Rondônia. For viral isolation, we used newborn mouse brain, followed by RT-PCR with specific universal Flavivirus primers. We obtained fragments 958bp and 800bp in length. Based on BLAST, these sequences were 91% similar to a sequence of Cacipacore virus.

Flavivirus é um gênero dos vírus transmitidos por artrópode da família Flaviviridae e, no Brasil, são isolados onze Flavivirus diferentes. Foi coletado o sangue de um agricultor, no município de Theobroma situado a 320km de distância da Cidade de Porto Velho, capital do Estado Brasileiro, Rondônia. Para isolamento viral, foi usado cérebro de camundongos recém-nascido, seguido por RT-PCR com primers universais específicos de Flavivirus. Nós obtivemos fragmentos com 958bp e 800bp de comprimento. Ao Blast das sequências obtivemos 91% de similaridade com uma sequência do vírus de Cacipacoré.

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.

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics.

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.

Characterization of dengue virus serotype 1 in epidemics in Porto Velho, Rondônia, in 2001-2003.

The first dengue fever epidemic in the State of Rondônia (western region of Brazil) was recorded in 1997, without laboratory confirmation. Following this, there was an epidemic in Manaus, in the neighboring State of Amazon, in 1998, in which DENV-1 and DENV-2 viruses were isolated from patients. In the present paper, the serotype characterization of the dengue virus isolated from patients with clinically suspected dengue in Porto Velho, Rondônia, between 2001 and 2003 is described. One hundred and fifty blood samples were collected between the first and fifth days of symptoms. Seventy samples of virus isolates were subjected to dengue identification by means of RT-PCR using universal primers for the NS1 gene of DENV, which amplifies a 419 bp fragment. The amplicons obtained were subjected to enzymatic digestion to characterize the viral serotypes. All the samples analyzed were DENV-1. A nucleotide sequence randomly selected from one amplicon, which was also DENV-1, presented 98% similarity to sequences from Southeast Asia that were obtained from GenBank.

Characterization of dengue virus serotype 1 in epidemics in Porto Velho, Rondônia, in 2001-2003

The first dengue fever epidemic in the State of Rondônia (western region of Brazil) was recorded in 1997, without laboratory confirmation. Following this, there was an epidemic in Manaus, in the neighboring State of Amazon, in 1998, in which DENV-1 and DENV-2 viruses were isolated from patients. In the present paper, the serotype characterization of the dengue virus isolated from patients with clinically suspected dengue in Porto Velho, Rondônia, between 2001 and 2003 is described. One hundred and fifty blood samples were collected between the first and fifth days of symptoms. Seventy samples of virus isolates were subjected to dengue identification by means of RT-PCR using universal primers for the NS1 gene of DENV, which amplifies a 419 bp fragment. The amplicons obtained were subjected to enzymatic digestion to characterize the viral serotypes. All the samples analyzed were DENV-1. A nucleotide sequence randomly selected from one amplicon, which was also DENV-1, presented 98 percent similarity to sequences from Southeast Asia that were obtained from GenBank.

A primeira epidemia de febre do dengue no Estado de Rondônia, Região Ocidental do Brasil foi registrado em 1997, sem confirmação laboratorial. Em seguida, houve uma epidemia descrita em 1998, em Manaus, no vizinho Estado do Amazonas, onde os vírus DENV-1 e DENV-2 foram isolados de pacientes. No presente artigo, foi descrito a caracterização do sorotipo do vírus dengue isolado de pacientes com suspeitas clinicas de dengue em Porto Velho, Rondônia, entre 2001 a 2003. Foram coletadas 150 amostras de sangue, entre primeiro e quinto dia de sintomas. Setenta amostras de vírus isolados foram submetidas a identificação do dengue pela RT-PCR usando primers universais para gene da NS1 do DENV que amplifica um fragmento de 419pb. O amplicon obtidos foram submetidos a digestão enzimática para caracterização do sorotipo viral. Todas as amostras analisadas foram DENV-1. A seqüência nucleotídica de um dos amplicons aleatoriamente selecionada também DENV-1 demonstrou 98 por cento similaridade com as seqüências do Sudeste Asiático obtidas no GenBank.