Dengue 4 in Ceará, Brazil: characterisation of epidemiological and laboratorial aspects and causes of death during the first epidemic in the state.

BACKGROUND The first dengue cases in Brazil with laboratory confirmation occurred in the northern region of the country, with the isolation of two serotypes, dengue virus 1 (DENV-1) and DENV-4. In Ceará, the introduction of DENV-4 was reported during a DENV-1 epidemic in 2011, with only two isolations. OBJECTIVES The aim of this study was to characterise the first DENV-4 epidemic in the state of Ceará, Brazil. METHODS The study population was composed of patients with suspected dengue that were reported to health care units from January to December 2012. The laboratory confirmation of infection was made by viral isolation, reverse transcription polymerase chain reaction (RT-PCR), AgNS1, immunohistochemistry and IgM enzyme-linked immunosorbent assay (ELISA). MAIN CONCLUSIONS In the study year, 72,211 suspected dengue cases were reported and 51,865 of these cases (71.8%) were confirmed to be positive. Co-circulation of three serotypes, DENV-1, DENV-3 and DENV-4, was detected with a predominance of DENV-4 (95.3%). Most cases were not severe, but there were 44 fatal outcomes. DENV-4 Genotype II was identified for the first time in Ceará.

First detection of dengue virus in the saliva of immunocompetent murine model.

The lack of an experimental animal model for the study of dengue pathogenesis is a limiting factor for the development of vaccines and drugs. In previous studies, our group demonstrated the susceptibility of BALB/c mice to infection by dengue virus (DENV) 1 and 2, and the virus was successfully isolated in several organs. In this study, BALB/c mice were experimentally infected intravenously with DENV-4, and samples of their saliva were collected. Viral RNA extracted from the saliva samples was subjected to qRT-PCR, with a detection limit of 0.002 PFU/mL. The presence of DENV-4 viral RNA was detected in the saliva of two mice, presenting viral titers of 109 RNA/mL. The detection of DENV RNA via saliva sampling is not a common practice in dengue diagnosis, due to the lower detection rates in human patients. However, the results observed in this study seem to indicate that, as in humans, detection rates of DENV RNA in mouse saliva are also low, correlating the infection in both cases. This study reports the first DENV detection in the saliva of BALB/c immunocompetent mice experimentally infected with non-neuroadapted DENV-4.

Zika virus infection: epidemiology, clinical manifestations and diagnosis.

PURPOSE OF REVIEW: Zika virus (ZIKV) is an arbovirus previously believed to cause only a mild and self-limiting illness. Recently, it has emerged as a new public health threat that caused a large outbreak in French Polynesia in 2013-2014 and since 2015 an explosive outbreak in Brazil, with an increase in severe congenital malformations (microcephaly) and neurological complications, mainly Guillain-Barré syndrome (GBS). Since then, it has spread through the Americas. On 1 February 2016, the WHO declared the ZIKV epidemic in Brazil a Public Health Emergency of International Concern. We reviewed the epidemiology of ZIKV infection, clinical presentations and diagnosis. We highlighted the clinical features and nonvector borne transmission of the virus. RECENT FINDINGS: Association between ZIKV infection and severe foetal outcomes, including microcephaly and other birth defects; increased rate of GBS and other neurological complications due to the ongoing ZIKV outbreak; increased evidence to date of ZIKV being the only arbovirus linked to sexual transmission; the challenge of ZIKV diagnosis; and the need for a specific point-of care test in epidemic scenarios. SUMMARY: The findings illustrate the emergence of a viral disease with the identification of new associated disorders, new modes of transmission, including maternal-foetal and sexual transmission.

Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance.

In Niterói, state of Rio de Janeiro, dengue virus type 4 (DENV-4) was isolated for the first time in March 2011. We analysed the laboratory findings of the first cases and evaluated the use of molecular techniques for the detection of DENV-4 in Aedes aegypti that were field-caught. Conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and Simplexa™ Dengue real-time RT-PCR confirmed DENV-4 infection in all cases. Additionally, DENV-4 was confirmed in a female Ae. aegypti with 1.08 x 10(3) copies/mL of virus, as determined by quantitative real-time RT-PCR. This is the first time the Simplexa™ Dengue real-time assay has been used for the classification of cases of infection and for entomological investigations. The use of these molecular techniques was shown to be important for the surveillance of dengue in humans and vectors.

Dengue virus type 3 in Brazil: a phylogenetic perspective.

Circulation of a new dengue virus (DENV)-3 genotype was recently described in Brazil and Colombia, but the precise classification of this genotype has been controversial. Here we perform phylogenetic and nucleotide-distance analyses of the envelope gene, which support the subdivision of DENV-3 strains into five distinct genotypes (GI to GV) and confirm the classification of the new South American genotype as GV. The extremely low genetic distances between Brazilian GV strains and the prototype Philippines/L11423 GV strain isolated in 1956 raise important questions regarding the origin of GV in South America.

Potential risk of re-emergence of urban transmission of Yellow Fever virus in Brazil facilitated by competent Aedes populations.

Yellow fever virus (YFV) causing a deadly viral disease is transmitted by the bite of infected mosquitoes. In Brazil, YFV is restricted to a forest cycle maintained between non-human primates and forest-canopy mosquitoes, where humans can be tangentially infected. Since late 2016, a growing number of human cases have been reported in Southeastern Brazil at the gates of the most populated areas of South America, the Atlantic coast, with Rio de Janeiro state hosting nearly 16 million people. We showed that the anthropophilic mosquitoes Aedes aegypti and Aedes albopictus as well as the YFV-enzootic mosquitoes Haemagogus leucocelaenus and Sabethes albiprivus from the YFV-free region of the Atlantic coast were highly susceptible to American and African YFV strains. Therefore, the risk of reemergence of urban YFV epidemics in South America is major with a virus introduced either from a forest cycle or by a traveler returning from the YFV-endemic region of Africa.

A simple heat dissociation method increases significantly the ELISA detection sensitivity of the nonstructural-1 glycoprotein in patients infected with DENV type-4.

The secreted form of the dengue virus (DENV) nonstructural-1 (NS1) glycoprotein has been shown to be useful for the diagnosis of DENV infections in patients' serum samples. In a number of studies, the sensitivity of the commercially available DENV NS1 glycoprotein detection assays was higher against some DENV serotypes (DENV-1>DENV-3>DENV-2=DENV-4) than others and were also lower using patients' serum samples with secondary versus primary DENV infections. In this study, 471 DENV-4 positive acute phase patients' serum samples were selected from a large panel collected in Brazil from March 2011 to October 2012 by RT-PCR and/or virus isolation followed by serotype determination. The sera from primary (n=228) and secondary (n=238) DENV-4 infections were identified using IgM and IgG capture ELISAs. The sensitivity of a commercial DENV NS1 glycoprotein detection ELISA was then assessed when these serum samples were not pre-treated or pre-treated by acid or heat dissociation prior to being tested. Acid and heat dissociation of patients' serum samples with primary and secondary DENV-4 infections increased significantly the sensitivity of the DENV NS1 glycoprotein detection ELISA from 54.4% to 77.2% (p<0.05) and 82% (p<0.05) and from 39.1% to 63.9% (p<0.05) and 73.1% (p<0.05), respectively. Treatment of DENV infected patients' serum samples using simple and rapid heat dissociation step (100°C for 5min) was, therefore, shown to be very useful for increasing the sensitivity of the DENV NS1 glycoprotein detection ELISA using serum samples from either primary or secondary DENV infected patients.

Virological surveillance for early warning of dengue epidemics in the State of Rio de Janeiro, Brazil.

BACKGROUND: The state of Rio de Janeiro has been important since 1986 as a portal for the introduction of dengue virus (DENV) into Brazil and dissemination of the virus throughout the country. This study describes an active surveillance of DENV in the state of Rio de Janeiro from 2004 to 2008. METHOD: A total of 14 408 samples from patients suspected to be infected with DENV were tested by virus isolation, and nested reverse transcriptase (RT)-PCR assay or anti-DENV dengue IgM antibody capture ELISA (MAC-ELISA), or both. RESULTS: By the use of these different methods, a total of 2324 (16.1%) cases were confirmed as dengue infection. The study covers an inter-epidemic period (2004-2005), the DENV-3 circulation in 2006, the re-emergence of DENV-2 in 2007 and the severe epidemic caused by DENV-2 in the summer of 2008. During the period, 69 dengue fatal cases were reported, 14 (20.2%) deaths being attributable to DENV-3 and 55 (79.7%) to DENV-2. CONCLUSION: Our results emphasize the role of the laboratory in the early detection of dengue virus transmission and provide information on the dynamics of DENV introduction and spread, important for the assessment of intervention strategies.

Dengue 4 in Ceará, Brazil: characterisation of epidemiological and laboratorial aspects and causes of death during the first epidemic in the state

BACKGROUND The first dengue cases in Brazil with laboratory confirmation occurred in the northern region of the country, with the isolation of two serotypes, dengue virus 1 (DENV-1) and DENV-4. In Ceará, the introduction of DENV-4 was reported during a DENV-1 epidemic in 2011, with only two isolations. OBJECTIVES The aim of this study was to characterise the first DENV-4 epidemic in the state of Ceará, Brazil. METHODS The study population was composed of patients with suspected dengue that were reported to health care units from January to December 2012. The laboratory confirmation of infection was made by viral isolation, reverse transcription polymerase chain reaction (RT-PCR), AgNS1, immunohistochemistry and IgM enzyme-linked immunosorbent assay (ELISA). MAIN CONCLUSIONS In the study year, 72,211 suspected dengue cases were reported and 51,865 of these cases (71.8%) were confirmed to be positive. Co-circulation of three serotypes, DENV-1, DENV-3 and DENV-4, was detected with a predominance of DENV-4 (95.3%). Most cases were not severe, but there were 44 fatal outcomes. DENV-4 Genotype II was identified for the first time in Ceará.

Dengue Virus Serotype 2 Established in Northern Mozambique (2015­2016)

After the report of an outbreak of dengue virus serotype 2 in 2014 in Nampula and Pemba cities, northernMozambique, a surveillance system was established by the National Institute of Health. A study was performed during2015­2016 to monitor the trend of the outbreak and confirm the circulating serotype of dengue virus (DENV). After theinclusion of consenting patients who met the case definition, samples from 192 patients were tested for the presence ofnonstructural protein1antigen,and60/192(31%)sampleswerepositive.FurtheranalysisincludedDENVIgMantibodies,with 39 (20%) IgM positive cases. Reverse transcriptase (RT) PCR was performed for identification of the prevailing DENVserotype; 21/23 tested samples were DENV-2 positive, with DENV-2 present in both affected cities. When sequencingDENV, phenotype Cosmopolitan was identified. The surveillance indicates ongoing spread of DENV-2 in northernMozambique 2 years after thefirst report of the outbreak.

Polyclonal antibodies against properly folded Dengue virus NS1 protein expressed in E. coli enable sensitive and early dengue diagnosis.

The non-structural 1 (NS1) protein plays an important role in dengue diagnosis because it has been detected as a soluble serum antigen in both primary and secondary infections. The NS1 protein was expressed in Escherichia coli cells, and the efficiency of four different refolding protocols was tested. All of the protocols generated dimeric NS1 in a conformation similar to that of the protein expressed by eukaryotic cells. A polyclonal antibody produced from the properly folded E. coli recombinant NS1 (rNS1) protein proved to be a useful tool for the diagnosis of Dengue virus because it detected 100% of the Dengue virus 2 (DENV2) in infected patients' sera and 60% of the DENV IgM-positive sera not detected by commercial NS1-based diagnostic kits. These data suggest a high-efficiency method for correctly folding rNS1 that maintains its structural and immunogenic properties. In addition, a detection method using the polyclonal antibody against correctly folded rNS1 seemed to be more sensitive and efficient for NS1 detection in serum, highlighting its usefulness for developing a high-sensitivity diagnostic kit.

Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance

In Niterói, state of Rio de Janeiro, dengue virus type 4 (DENV-4) was isolated for the first time in March 2011. We analysed the laboratory findings of the first cases and evaluated the use of molecular techniques for the detection of DENV-4 in Aedes aegypti that were field-caught. Conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and SimplexaTM Dengue real-time RT-PCR confirmed DENV-4 infection in all cases. Additionally, DENV-4 was confirmed in a female Ae. aegypti with 1.08 x 10³ copies/mL of virus, as determined by quantitative real-time RT-PCR. This is the first time the SimplexaTM Dengue real-time assay has been used for the classification of cases of infection and for entomological investigations. The use of these molecular techniques was shown to be important for the surveillance of dengue in humans and vectors.

Dengue virus type 3 in Brazil: a phylogenetic perspective

Circulation of a new dengue virus (DENV)-3 genotype was recently described in Brazil and Colombia, but the precise classification of this genotype has been controversial. Here we perform phylogenetic and nucleotide-distance analyses of the envelope gene, which support the subdivision of DENV-3 strains into five distinct genotypes (GI to GV) and confirm the classification of the new South American genotype as GV. The extremely low genetic distances between Brazilian GV strains and the prototype Philippines/L11423 GV strain isolated in 1956 raise important questions regarding the origin of GV in South America.

Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome

We have determined the complete nucleotide and the deduced amino acid sequences of Brazilian dengue virus type 3 (DENV-3) from a dengue case with fatal outcome, which occurred during an epidemic in the state of Rio de Janeiro, Brazil, in 2002. This constitutes the first complete genetic characterization of a Brazilian DENV-3 strain since its introduction into the country in 2001. DENV-3 was responsible for the most severe dengue epidemic in the state, based on the highest number of reported cases and on the severity of clinical manifestations and deaths reported.

Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain

Diagnosis of dengue by using reverse transcriptase-polymerase chain reaction

A rapid identification of dengue viruses from clinical samples by using a nested reverse transcriptase-polymerase chain reaction (RT-PCR) procedure was carried out for diagnostic and epidemiological purposes. RT-PCR identified DEN-1 and DEN-2 viruses in 41 per cent (41/100) of previously confirmed cases and provided an accurate confirmation of DHF in four fatal cases. RT-PCR was also useful for detecting and typing dengue viruses in suspected cases, allowing a rapid identification of new serotypes in endemic areas.

Dengue in the State of Rio de Janeiro, Brazil, 1986-1998

This paper presents epidemiological, laboratory, and clinical data on 12 years of dengue virus activity in the State of Rio de Janeiro from the time the disease was first confirmed virologically in April 1986 through April 1998. DEN-1 and DEN-2 viruses are the serotypes circulating in the state and were responsible for the epidemics reported during the last 12 years. The results published here show both the impact of dengue virus infections on the population and laboratory advances that have improved dengue diagnosis.

Obtenção, caracterização e utilização de polipeptídeos recombinantes de vírus dengue tipo 2 no diagnóstico das infecções por dengue / Attainment, characterization and use of recombinant polypeptides of dengue viruses type 2 in the diagnosis of dengue infections

Com o objetivo de se produzir antígenos recombinantes para o diagnóstico sorológico das infecções por vírus dengue (DENV), foi realizado o sequenciamento completo de uma cepa Brasileira (BR64022/98) de DENV-2 pela amplificação de segmentos contínuos, de aproximadamente 500pb, ao longo de todo o genoma viral. Vinte fragmentos amplificados por RT-PCR foram clonados e as sequências de nucleotídeos e de aminoácidos determinadas, resultando na primeira caracterização genética completa de uma cepa de DENV-2 do Brasil. Em uma segunda etapa, os fragmentos clonados foram expressos em Escherichia coli e purificados por cromatografia de afinidade. A reatividade dos polipeptídeos expressos e purificados aos anticorpos IgG anti-DENV presentes em soros humanos foi analisada por Western Blot e Enzyme Linked Immunosorbent Assay (ELISA). O polipeptídeo pD2-3 (E), localizado na porção N-terminal da proteína de envelope (E), apresentou maior reatividade quando utilizado na sua forma nativa, sendo reconhecido por 88 por cento dos soros testados e, consequentemente identificado como um antígeno candidato para utilização no diagnóstico das infecções pelos DENV. Soros de pacientes com febre amarela apresentaram ausência ou baixa reatividade contra pD2-3 (E). O pD2-3 (E) não foi reconhecido como antígeno recombinante sorotipo-específico, sendo reativo com soros de pacientes infectados por DENV-1, DENV-2 e DENV-3, com sensibilidade variando de 76,2 por cento a 93,3 por cento, nestes grupos específicos. O teste imunoenzimático indireto para a detecção de anticorpos IgG utilizando o pD2-3 (E) (REC IgG-ELISA) apresentou 89,1 por cento de sensibilidade e 96,4 por cento de especificidade, com maior sensibilidade para a confirmação de casos secundário s(98,1 por cento) quando comparados com casos primários de infecção por dengue (77,5 por cento). A não detecção de anticorpos IgG anti-DENV residuais pelo pD2-3(E) foi significante e demonstrou que um resultado reativo por REC IgG-ELISA é indicativo de infecção ativa por DENV. O REC IgG-ELISA tende a ser formatado como um teste qualitativo, dispensando a abordagem convencional de análises de títulos de IgG para a confirmação de infecções por DENV.