Análise molecular dos vírus dengue tipo 1 e 2 no Brasil, baseada nas seqüências da região da junção dos genes do envelope e da proteína não estrutural 1

The genomic sequences of the Envelope-Non-Structural protein 1 junction region (E/NS1) of 84 DEN-1 and 22 DEN-2 isolates from Brazil were determined. Most of these strains were isolated in the period from 1995 to 2001 in endemic and regions of recent dengue transmission in São Paulo State. Sequence data for DEN-1 and DEN-2 utilized in phylogenetic and split decomposition analyses also include sequences deposited in GenBank from different regions of Brazil and of the world. Phylogenetic analyses were done using both maximum likelihood and Bayesian approaches. Results for both DEN-1 and DEN-2 data are ambiguous, and support for most tree bipartitions are generally poor, suggesting that E/NS1 region does not contain enough information for recovering phylogenetic relationships among DEN-1 and DEN-2 sequences used in this study. The network graph generated in the split decomposition analysis of DEN-1 does not show evidence of grouping sequences according to country, region and clades. While the network for DEN-2 also shows ambiguities among DEN-2 sequences, it suggests that Brazilian sequences may belong to distinct subtypes of genotype III.Foram determinadas as seqüências nucleotídicas da junção dos genes do envelope e da proteína não estrutural 1 (E/NS1) de 84 cepas de DEN-1 e 22 cepas de DEN-2 do Brasil. A maioria dessas cepas foi isolada no período de 1995-2001, em regiões endêmicas e de transmissão recente no Estado de São Paulo. Seqüências da junção E/NS1 de DEN-1 e DEN-2 de outras regiões geográficas brasileiras e mundiais, obtidas do GenBank, foram também utilizadas neste estudo. As análises foram efetuadas utilizando-se as técnicas de Verossimilhança Máxima e Bayesiana de inferência filogenética. Os resultados das análises das seqüências de DEN-1 e DEN-2 são ambíguos e o suporte para a maioria dos grupos é baixo, sugerindo que a região E/NS1 não é filogeneticamente informativa. O gráfico gerado na análise de decomposição dos grupos de DEN-1 não mostrou evidências de agrupamento das seqüências de acordo com os países, as regiões ou clados. No entanto, para DEN-2 evidenciou a existência de ambigüidades entre as seqüências, sugerindo que as brasileiras pertencem a subtipos distintos do genotipo III

Vírus da encefalite São Luis: primeiro isolamento de humano no Estado de São Paulo, Brasil

O presente estudo relata o isolamento do vírus da encefalite São Luis (SLEV) de um caso febril humano suspeito de dengue, em São Pedro, Estado de São Paulo. MAC-ELISA realizado com soros das fases aguda e convalescente foi inconclusivo e anticorpos IgG foram detectados por inibição da hemaglutinação para flavivirus. Imunofluorescência indireta com cultura de células C6/36 inoculadas com soro da fase aguda foi positivo para flavivirus mas negativo quando testado com anticorpos monoclonais para dengue. O RNA extraído de cultura de células infectadas foi amplificado na presença de primers universais para o gênero Flavivirus, deduzidos de uma região da proteína não estrutural 5 e diretamente sequenciado. Os resultados da pesquisa no BLAST indicaram que a seqüência apresenta 93% de similaridade de nucleotídeos com a seqüência de SLEV (cepa MS1.7), confirmado por RT-PCR, realizado com primers específicos para SLEV. O fato de SLEV ter sido identificado como a causa de doença humana indica a necessidade de aprimorar a vigilância a fim de detectar precocemente esse agente no Estado de São Paulo e no Brasil. Esse caso é também um alerta para os profissionais de saúde sobre a necessidade de investigações clínicas e epidemiológicas mais completas sobre doenças febris como no caso relatado. Infecções por SLEV podem não ser reconhecidas ou confundidas com outras causadas por arbovírus como a dengue.This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue

Dengue Virus Type 4 Phylogenetics in Brazil 2011: Looking beyond the Veil

Dengue Fever and Dengue Hemorrhagic Fever are diseases affecting approximately 100 million people/year and are a major concern in developing countries. In the present study, the phylogenetic relationship of six strains of the first autochthonous cases of DENV-4 infection occurred in Sao Paulo State, Parana State and Rio Grande do Sul State, Brazil, 2011 were studied. Nucleotide sequences of the envelope gene were determined and compared with sequences representative of the genotypes I, II, III and Sylvatic for DEN4 retrieved from GenBank. We employed a Bayesian phylogenetic approach to reconstruct the phylogenetic relationships of Brazilian DENV-4 and we estimated evolutionary rates and dates of divergence for DENV-4 found in Brazil in 2011. All samples sequenced in this study were located in Genotype II. The studied strains are monophyletic and our data suggest that they have been evolving separately for at least 4 to 6 years. Our data suggest that the virus might have been present in the region for some time, without being noticed by Health Surveillance Services due to a low level of circulation and a higher prevalence of DENV-1 and DENV- 2

Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 – 2018

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species

Surveillance of arbovirus infections in the atlantic forest region, State of São Paulo, Brazil: I. detection of hemagglutination-inhibition antibodies in wild birds between 1978 and 1990

We report data related to arbovirus antibodies detected in wild birds periodically captured from January 1978 to December 1990 in the counties of Salesópolis (Casa Grande Station), Itapetininga and Ribeira Valley, considering the different capture environments. Plasmas were examined using hemagglutination-inhibition (HI) tests. Only monotypic reactions were considered, except for two heterotypic reactions in which a significant difference in titer was observed for a determined virus of the same antigenic group. Among a total of 39,911 birds, 269 birds (0.7%) belonging to 66 species and 22 families were found to have a monotypic reaction for Eastern equine encephalitis (EEE), Venezuelan equine encephalitis (VEE), Western equine encephalitis (WEE), Ilheus (ILH), Rocio (ROC), St. Louis encephalitis (SLE), SP An 71686, or Caraparu (CAR) viruses. Analysis of the data provided information of epidemiologic interest with respect to these agents. Birds with positive serology were distributed among different habitats, with a predominance of unforested habitats. The greatest diversity of positive reactions was observed among species which concentrate in culture fields.Apresentam-se os resultados referentes a anticorpos para arbovírus em aves silvestres capturadas, periodicamente, de janeiro de 1978 a dezembro de 1990, nos municípios de Salesópolis (Estação de Casa Grande); Itapetininga e municípios do Vale do Ribeira, considerando-se os diferentes ambientes de captura. Plasmas foram examinados, por testes de Inibição de Hemaglutinação (IH). Considerou-se apenas as reações monotípicas, com exceção de duas reações heterotípicas, onde ocorreu uma diferença de título significativa para um determinado vírus de um mesmo grupo antigênico. Em um total de 39.911 aves, foram encontradas 269 aves pertencentes a 66 espécies e 22 famílias, com reação monotípica para os vírus Encefalite Equina do Leste (EEE), Encefalite Equina Venezuelana (VEE), Encefalite Equina do Oeste (WEE), Ilhéus (ILH), Rocio (ROC), Encefalite São Luis (SLE), SP AN 71686, ou Caraparu (CAR). A análise dos resultados contribue com informações de interesse epidemiológico em relação àqueles agentes. Observou-se distribuição das aves com sorologia positiva em diferentes habitats, predominando os que excluem mata e a presença de maior diversidade de reações positivas nas espécies que se concentram em campos de cultura

Vírus Mayaro: casos importados de infecção humana no Estado de São Paulo, Brasil

Mayaro virus (MAYV) is an arbovirus (Togaviridae: Alphavirus) enzootic in tropical South America and maintained in a sylvan cycle involving wild vertebrates and Haemagogus mosquitoes. MAYV cases occur sporadically in persons with a history of recent activities inside or around forests. This paper reports three cases of MAYV fever detected in men infected in Camapuã, MS, Brazil. Serum samples collected at four days and two months after the onset of the symptoms and examined by hemagglutination inhibition test, revealed monotypic seroconversion to MAYV. Isolation of the virus was obtained from one of the samples by inoculation of the first blood samples into newborn mice. A suspension of the infected mouse brain was inoculated into C6/36 cells culture and the virus was identified by indirect immunofluorescent assay with alphavirus polyclonal antibodies. RT-PCR, performed with RNA extracted from the supernatant of C6/36 infected cells in the presence of alphavirus generic primers as well as specific MAYV primers, confirmed these results. The reported cases illustrate the importance of laboratory confirmation in establishing a correct diagnosis. Clinical symptoms are not always indicative of a disease caused by an arbovirus. Also MAYV causes febrile illness, which may be mistaken for dengue.O vírus Mayaro (MAYV) é um arbovírus do gênero Alphavirus, família Togaviridae, enzoótico na América do Sul, sendo mantido em ciclo silvestre envolvendo vertebrados e mosquitos Haemagogus. Casos de MAYV são esporádicos e ocorrem em pessoas com história de recentes atividades dentro ou próximo a florestas. Este artigo relata infecção por MAYV detectada em três pacientes, infectados em Camapuã, MS, Brasil. Amostras de sangue, coletadas no 4º dia e no 2º mês após o início dos sintomas, foram usadas para teste de inibição da hemaglutinação, que revelou soroconversão monotípica para MAYV. O isolamento do vírus foi obtido somente de uma das amostras, por inoculação em camundongos lactentes. Suspensão de cérebro de camundongo infectado foi inoculada em cultura de células C6/36 e o vírus foi identificado por imunofluorescência indireta com anticorpos policlonais para alphavirus. RT-PCR realizado com RNA extraído do sobrenadante de células C6/36 infectadas, na presença de "primers" genéricos para alphavirus assim como "primers" para MAYV, confirmou os resultados. Os casos relatados ilustram a importância da confirmação laboratorial em estabelecer um diagnóstico correto. Os sintomas clínicos não são sempre indicativos de uma doença causada por arbovírus. MAYV causa doença febril, que pode ser confundida com dengue

Febre amarela silvestre: estudo clínico e laboratorial, enfatizando a viremia, de um caso humano

The authors report the clinical, laboratorial and epidemiological aspects of a human case of jungle yellow fever. The patient suffered from fever, chills, sweating, headaches, backaches, myalgia, epigastric pains, nausea, vomiting, diarrhea and prostration. He was unvaccinated and had been working in areas where cases of jungle yellow fever had been confirmed. Investigations concerning the yellow fever virus were performed. Blood samples were collected on several days in the course of the illness. Three of these samples (those obtained on days 5,7 and 10) were inoculated into suckling mice in attempt to isolate virus and to titrate the viremia level. Serological surveys were carried out by using the IgM Antibodies Capture Enzyme Linked Immunosorbent Assay (MAC-ELISA), Complement Fixation (CF), Hemagglulinalion Inhibition (HI) and Neutralization (N) tests. The yellow fever virus, recovered from the two first samples and the virus titration, showed high level of viremia. After that, specific antibodies appeared in all samples. The interval between the end of the viremia and the appearance of the antibodies was associated with the worsening of clinical symptoms, including bleeding of the mucous membrane. One must be aware of the risk of having a urban epidemics in areas where Aedes aegypti is found in high infestation indexes.Os autores estudaram um caso humano de febre amarela silvestre, sob os aspectos clínico, laboratorial e epidemiológico. O paciente apresentava febre (39ºC), calafrios, sudorese, cefaléia, dor lombar, mialgia, dor abdominal em epigástrio, náuseas, vômitos, diarréia e prostração. Relatava permanência em área onde foram constatados casos de febre amarela silvestre e não havia histórico de vacinação anterior. Frente às suspeitas que levaram à investigação do vírus da febre amarela, foram colhidas várias amostras de sangue no curso da doença. As amostras do 5º, 7º e 10º dias foram submetidas a provas de isolamento e quantificação do vírus, o que possibilitou o estudo da viremia. Empregando-se os testes de MAC-ELISA (detecção de IgM), Fixação de Complemento (FC), Inibição de Hemaglutinação (IH) e teste de Neutralização (N), foi observada a resposta imune para anticorpos específicos nas amostras do 7º ao 26º dias. Os resultados mostraram que no 5º e 7º dias havia persistência da fase virêmica, com títulos elevados. Ao término desta fase, com o aparecimento de anticorpos específicos, foi observado um agravamento do quadro clínico, com sangramento de mucosas. Os autores alertam para a possibilidade de ocorrerem epidemias urbanas em áreas com alta infestação de Aedes aegypti

New arenavirus isolated in Brazil

Adolfo Lutz Institute. Virus Service. São Paulo, SP, Brazil.Adolfo Lutz Institute. Virus Service. São Paulo, SP, Brazil.Paulista Medical School. Infectious Diseases Clinics. São Paulo, SP, Brazil.Adolfo Lutz Institute. Virus Service. São Paulo, SP, Brazil.Adolfo Lutz Institute. Virus Service. São Paulo, SP, Brazil.Adolfo Lutz Institute. Virus Service. São Paulo, SP, Brazil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Pan American Health Organization. Communicable Diseases Program. Washington, DC, USA.World Health Organization. Microbiology and Immunology Support Section. Geneva, Switzerland.Yale University. School of Medicine. Department of Epidemiology and Public Health. Yale Arbovirus Research Unit. New Haven, CT.Yale University. School of Medicine. Department of Epidemiology and Public Health. Yale Arbovirus Research Unit. New Haven, CT.US Army Medical Research Institute of Infectious Diseases. Virology Division. Frederick, Maryland, USA.Yale University. School of Medicine. Department of Epidemiology and Public Health. Yale Arbovirus Research Unit. New Haven, CT.A new arenavirus, called Sabiá, was isolated in Brazil from a fatal case of haemorrhagic fever initially thought to be yellow fever. Antigenic and molecular characterisation indicated that Sabiá virus is a new member of the Tacaribe complex. A laboratory technician working with the agent was also infected and developed a prolonged, non-fatal influenza-like illness. Sabiá virus is yet another arenavirus causing human disease in South America

Detection of a New Yellow Fever Virus Lineage Within the South American Genotype I in Brazil

Nucleotide sequences of two regions of the genomes of 11 yellow fever virus (YFV) samples isolated from monkeys or humans with symptomatic yellow fever (YF) in Brazil in 2000,2004, and 2008 were determined with the objective of establishing the genotypes and studying the genetic variation. Results of the Bayesian phylogenetic analysis showed that sequences generated from strains from 2004 and 2008 formed a new subclade within the clade 1 of the South American genotype I. The new subgroup is here designated as 1E. Sequences of YFV strains recovered in 2000 belong to the subclade 1D, which comprises previously characterized YFV strains from Brazil. Molecular dating analyses suggested that the new subclade 1E started diversifying from 1D about 1975 and that the most recent 2004-2008 isolates arose about 1985. J. Med. Virol. 82:175-185, 2010. (C) 2009 Wiley-Liss, Inc.Secretaria de Estado da Saude de Sao Paulo/SPFAPESP[05/53973-0