Isolation of Flaviviruses and Alphaviruses with Encephalitogenic Potential Diagnosed by Evandro Chagas Institute (Pará, Brazil) in the Period of 1954-2022: Six Decades of Discoveries.

Viruses with encephalitogenic potential can cause neurological conditions of clinical and epidemiological importance, such as Saint Louis encephalitis virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Dengue virus, Zika virus, Chikungunya virus, Mayaro virus and West Nile virus. The objective of the present study was to determine the number of arboviruses with neuroinvasive potential isolated in Brazil that corresponds to the collection of viral samples belonging to the Department of Arbovirology and Hemorrhagic Fevers, Evandro Chagas Institute (SAARB/IEC) of the Laboratory Network of National Reference for Arbovirus Diagnosis from 1954 to 2022. In the analyzed period, a total of 1,347 arbovirus samples with encephalitogenic potential were isolated from mice; 5,065 human samples were isolated exclusively by cell culture; and 676 viruses were isolated from mosquitoes. The emergence of new arboviruses may be responsible for diseases still unknown to humans, making the Amazon region a hotspot for infectious diseases due to its fauna and flora species characteristics. The detection of circulating arboviruses with the potential to cause neuroinvasive diseases is constant, which justifies the continuation of active epidemiological surveillance work that offers adequate support to the public health system regarding the virological diagnosis of circulating arboviruses in Brazil.

Phylogeography of dengue virus serotype 4, Brazil, 2010-2011.

Dengue virus serotype 4 (DENV-4) reemerged in Roraima State, Brazil, 28 years after it was last detected in the country in 1982. To study the origin and evolution of this reemergence, full-length sequences were obtained for 16 DENV-4 isolates from northern (Roraima, Amazonas, Pará States) and northeastern (Bahia State) Brazil during the 2010 and 2011 dengue virus seasons and for an isolate from the 1982 epidemic in Roraima. Spatiotemporal dynamics of DENV-4 introductions in Brazil were applied to envelope genes and full genomes by using Bayesian phylogeographic analyses. An introduction of genotype I into Brazil from Southeast Asia was confirmed, and full genome phylogeographic analyses revealed multiple introductions of DENV-4 genotype II in Brazil, providing evidence for >3 introductions of this genotype within the last decade: 2 from Venezuela to Roraima and 1 from Colombia to Amazonas. The phylogeographic analysis of full genome data has demonstrated the origins of DENV-4 throughout Brazil.

Molecular epidemiology of Oropouche virus, Brazil.

Oropouche virus (OROV) is the causative agent of Oropouche fever, an urban febrile arboviral disease widespread in South America, with >30 epidemics reported in Brazil and other Latin American countries during 1960-2009. To describe the molecular epidemiology of OROV, we analyzed the entire N gene sequences (small RNA) of 66 strains and 35 partial Gn (medium RNA) and large RNA gene sequences. Distinct patterns of OROV strain clustered according to N, Gn, and large gene sequences, which suggests that each RNA segment had a different evolutionary history and that the classification in genotypes must consider the genetic information for all genetic segments. Finally, time-scale analysis based on the N gene showed that OROV emerged in Brazil ≈223 years ago and that genotype I (based on N gene data) was responsible for the emergence of all other genotypes and for virus dispersal.

Oropouche orthobunyavirus: Genetic characterization of full-length genomes and development of molecular methods to discriminate natural reassortments.

Oropouche orthobunyavirus (OROV) has significant impact in public health in Amazon region. This arbovirus is one of the most common causes of febrile illness in Brazil, and is responsible for several epidemics since 1960's. In this study, we sequenced and characterized the complete coding sequences (S-, M- and L-RNA) of 35 OROV isolates from Brazil. Here, we classified 20 strains in genotype I from Pará and Maranhão states, nine as genotype II from Pará and Rondônia states confirmed, four classified into genotype III from Acre, Maranhão, Minas Gerais and Rondônia states and two genotype IV from Amazonas State. Also, we did not observe reassortment events involving the OROV isolates. In addition, we developed novel RT-PCR tools to identify reassortment events among OROV strains. These data will be useful to better understand the molecular epidemiology and diagnostic of OROV infections.

Zika virus in the Americas: Early epidemiological and genetic findings.

Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

Full-length sequencing and genetic characterization of Breu Branco virus (Reoviridae, Orbivirus) and two related strains isolated from Anopheles mosquitoes.

Breu Branco virus (BE AR 492347) was isolated from Anopheles (Nyssorhynchus) triannulatus mosquitoes captured in Tucuruí, Pará State, northern Brazil, in 1988. No cross-reactivity by complement-fixation tests was observed between Breu Branco virus and other known arboviruses. Results of electron microscopy and physicochemical tests suggested that Breu Branco virus may be a member of the family Reoviridae. In order to elucidate its taxonomic status, a comprehensive genetic characterization was conducted for Breu Branco virus and related strains (BE AR 494475 and BE AR 486204) that were also isolated from Anopheles mosquitoes in the same area. This included full-length genome sequencing, determination of genetic traits and phylogenetic analysis. Breu Branco virus showed a similar genome organization to members of the genus Orbivirus, family Reoviridae. Genetically, Breu Branco virus was indistinguishable from strains BE AR 494475 and BE AR 486204. Phylogenetic analysis suggested that Breu Branco virus BE AR 492347 and its related strains constitute a novel species of the genus Orbivirus. Breu Branco virus is the first Brazilian orbivirus and the fifth orbivirus in the world to be sequenced fully.

Reemergence of Oropouche fever, northern Brazil.

Oropouche fever has reemerged in Parauapebas and Porto de Moz municipalities, Pará State, Brazil. Serologic analysis (immunoglobulin M-ELISA) and virus isolation confirmed Oropouche virus (OROV) in both municipalities. Nucleotide sequencing of 2 OROV isolates from each location indicated genotypes I (Parauapebas) and II (Porto de Moz) in Brazil.

Molecular epidemiology of dengue virus serotypes 2 and 3 isolated in Brazil from 1991 to 2008 / Epidemiologia molecular dos sorotipos 2 e 3 do vírus dengue, isolados no Brasil de 1991 a 2008

The dengue virus (DENV1-4) causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in tropical and subtropical areas. The aim of this study was to evaluate the circulating genotypes of DENV. This was accomplished by sequencing the PrM and E genes of Brazilian isolates of DENV2 and DENV3 that were obtained between 1991 and 2008 from various geographic regions. Phylogenetic analyses of DENV2 demonstrated that the genotype III (Southeast Asian/American), in spite of several nucleotide and amino acid changes, was the only one that circulated over the past 19 years. Since its introduction in 2000, the DENV3 isolates that have been analyzed have all grouped intogenotype III (Indian subcontinent) and there has been no evidence of DENV3 belonging to other genotypes in this study.

O vírus dengue (DENV1-4) causa a dengue clássica e a febre hemorrágica da dengue / síndrome de choque da dengue (FHD/SCD) em regiões tropicais e subtropicais. O objetivo deste estudo foi avaliar os genótipos circulantes de DENV2 e DENV3 obtidos em distintas regiões geográficas no período de 1991 a 2008. As análises filogenéticas de DENV2 demonstraram que o genótipo III (Sudeste da Ásia/América), apesar das diversas alterações nucleotídicas e de aminoácidos, foi o único a circular durante os últimos 19 anos. Desde a sua introdução no estudo, em 2000, todas as amostras isoladas de DENV3 analisadas foram agrupadas no genótipo III (subcontinente indiano). Não foram encontradas evidências de que o DENV3 pertença a outros genótipos investigados.

A febre do Oropouche: uma revisão dos aspectos epidemiológicos e moleculares na Amazônia brasileira / Oropouche fever: an overview of the epidemiological and molecular aspects in the brazilian Amazon region

O vírus Oropouche (VORO Bunyaviridae, Orthobunyavirus) é um dos mais importantes arbovírus que infectam humanos na Amazônia Brasileira, sendo o agente causador da febre do Oropouche. Entre os anos de 1961 e 2006, um grande número de epidemias foi registrado em diferentes centros urbanos do estado do Pará (Belém, Santa Isabel, Castanhal, Santarém, Oriximiná, Serra Pelada, zona Bragantina - Igarapé Açu, Maracanã e Magalhães Barata), do Amazonas (Manaus e Barcelos), Acre (Xapuri), Amapá (Mazagão), Maranhão (Porto Franco), Tocantins (Tocantinópolis) e Rondônia (Ariquemes e Oro Preto D'Oeste). Estudos moleculares têm demonstrado a presença de pelo menos três linhagens distintas do VORO na Amazônia Brasileira (genótipos I, II e III), sendo os genótipos I e II os mais frequentemente encontrados em regiões da Amazônia ocidental e oriental, respectivamente. O genótipo III do VORO, previamente encontrado somente no Paraná, foi recentemente descrito na região Sudeste do Brasil. A associação de dados epidemiológicos e moleculares vêm contribuindo substancialmente para a caracterização das cepas do VORO isoladas durante epidemias, no período de pelo menos quatro décadas, bem como permitindo um melhor entendimento a respeito da epidemiologia molecular do VORO no que tange à emergência de novas linhagens genéticas e à dinâmica evolutiva desse arbovírus nas Américas e principalmente na Amazônia Brasileira. Este trabalho tem por objetivo apresentar uma revisão dos aspectos epidemiológicos e moleculares do VORO enfatizando sua distribuição, a dinâmica das epidemias ocorridas entre 1961 e 2006, bem como a dispersão de diferentes genótipos no Brasil.

Dengue virus type 3 in Boa Vista, Northern Brazil

Casos de doença febril aguda ocorridos em Boa Vista, estado de Roraima, Brasil foram investigados em julho de 2005. Amostras de sangue (n igual a 142) foram obtidas de pacientes clinicamente suspeitos de febre do dengue (FD). A tentativa de isolamento viral foi realizada em células C6/36 para pacientes com menos de cinco dias de doença (n igual a 96). As cepas isoladas foram identificadas pelo teste de imunofluorescência indireta (IFI) utilizando anticorpos monoclonais, bem como pela técnica de RT-PCR. Amostras de soro foram testadas pelo método de inibição da hemaglutinação (IH) e confirmadas pelo IgM-ELISA. O vírus dengue 3 (VDEN-3) foi isolado e detectado po RT-PCR em 41 pacientes. A região E de sete cepas foi sequenciada, sendo as mesmas identificadas como pertencentes ao genótipo III. Pelo teste de IH, 98 amostras de soros foram positivas para anticorpos IH para o vírus da Dengue, sendo os resultados confirmados pela detecção de anticorpos IgM para dengue. Clinicamente, todos os pacientes mostraram quadro de FD, sendo todas as faixas etárias afetadas independentes do sexo. A epidemia de dengue ocorrida em Roraima foi causada pelo VDEN-3, genótipo III, genótipo este que circula nas Américas desde 1994.