Exploratory study of humoral and cellular immunity to 17DD Yellow Fever vaccination in children and adults residents of areas without circulation of Yellow Fever Virus.

The present investigation comprised two independent observational arms to evaluate the influence of pre-existing flavivirus humoral immunity and the age-impact on 17DD-YF vaccination immunity. Flavivirus (YFV; DENV; ZIKV) serology and YF-specific cellular immunity was evaluated in 288 children/9Mths-4Yrs and 288 adults/18-49Yrs residents of areas without YFV circulation. Data demonstrated that flavivirus seropositivity at baseline was higher in Adults as compared to Children (26%;87%;67% vs 6%;13%;15%, respectively). The heterologous flavivirus seropositivity (DENV; ZIKV) did not impact the YF-specific cellular immune response at baseline. However, higher levels of NCD4, EMCD8, IFN-MCD8, NCD19 and nCMCD19 were observed in subjects with pre-existing YFV seropositivity. Primary vaccination of YFV-seronegative volunteers led to higher levels of YF-neutralizing antibodies in Adults as compared to Younger Children (9Mths-2Yrs). Although similar seropositivity rates observed amongst Children and Adults at D30-45, lower rates were observed in Younger Children (9Mths-2Yrs) at D365 (94%;95%;100% vs 87%;96%;99%, respectively). A progressive decline in antibody levels were reported at D365, being more expressive in Children as compared to Adults. All age-subgroups exhibited at D30-45 increased levels of eEfCD4, EMCD4, IFN-MCD8 and nCMCD19 together with a decrease of eEfCD8 and CMCD8. While an increase of EMCD8 were observed in all subgroups at D30-45, a declined duration at D365 was reported only in Younger Children (9Mths-2Yrs). Biomarker signatures further support that only Younger Children (9Mths-2Yrs) presented a progressive decline of EMCD8 at D365. Together, these findings demonstrated that regardless the similarities observed in YF-neutralizing antibodies, the age impacts the duration of cellular immune response to primary 17DD-YF vaccination.

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 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.

Twenty years of DENV-2 activity in Brazil: molecular characterization and phylogeny of strains isolated from 1990 to 2010.

In Brazil, dengue has been a major public health problem since its introduction in the 1980s. Phylogenetic studies constitute a valuable tool to monitor the introduction and spread of viruses as well as to predict the potential epidemiological consequences of such events. Aiming to perform the molecular characterization and phylogenetic analysis of DENV-2 during twenty years of viral activity in the country, viral strains isolated from patients presenting different disease manifestations (n = 34), representing six states of the country, from 1990 to 2010, were sequenced. Partial genome sequencing (genes C/prM/M/E) was performed in 25 DENV-2 strains and full-length genome sequencing (coding region) was performed in 9 strains. The percentage of similarity among the DENV-2 strains in this study and reference strains available in Genbank identified two groups epidemiologically distinct: one represented by strains isolated from 1990 to 2003 and one from strains isolated from 2007 to 2010. No consistent differences were observed on the E gene from strains isolated from cases with different clinical manifestations analyzed, suggesting that if the disease severity has a genetic origin, it is not only due to the differences observed on the E gene. The results obtained by the DENV-2 full-length genome sequencing did not point out consistent differences related to a more severe disease either. The analysis based on the partial and/or complete genome sequencing has characterized the Brazilian DENV-2 strains as belonging to the Southeast Asian genotype, however a distinction of two Lineages within this genotype has been identified. It was established that strains circulating prior DENV-2 emergence (1990-2003) belong to Southeast Asian genotype, Lineage I and strains isolated after DENV-2 emergence in 2007 belong to Southeast Asian genotype, Lineage II. Furthermore, all DENV-2 strains analyzed presented an asparagine (N) in E390, previously identified as a probable genetic marker of virulence observed in DHF strains from Asian origin. The percentage of identity of the latter with the Dominican Republic strain isolated in 2001 combined to the percentage of divergence with the strains first introduced in the country in the 1990s suggests that those viruses did not evolve locally but were due to a new viral Lineage introduction in the country from the Caribbean.

Vinte e cinco anos de DENV-1 no Brasil: epidemiologia Molecular de Cepas Isoladas entre 1986 e 2011 / Twenty-five years of denv-1 in brazil: molecular epidemiology of strains isolated from 1986 to 2011

Este estudo apresenta a fiogenia e caracterização molecular baseados na análise do gene do envelope (E) (1.485 nucleotídeos) dos DENV-1 (n=48) e na região codificante completa (10.176 nucleotídeos) de seis representantes dentre as 48 amostras analisadas, isolados durante as epidemias ocorridas desde a introdução do sorotipo em 1986 até 2011. Possíveis eventos de recombinação genômica também foram analisados. Os resultados baseados na análise do gene E demonstraram que os DENV-1 isolados dos estados do Rio de Janeiro, Espírito Santo, Minas Gerais, Mato Grosso do Sul, Alagoas, Ceará, Piauí e Rio Grande do Norte pertencem ao genótipo V (América/África), mas agrupam-se em clades distintos. Três grupos foram identificados, um datando entre 1986-2002 (linhagem 1a), um segundo grupo representado por vírus isolados entre 2009 e 2011 e uma cepa isolada representativa do ano de 2002 (linhagem 2) e um grupo de cepas isoladas em 2010 e 2011 (linhagem 1b). A linhagem 2 apresentou um alto suporte de bootstrap garantindo a sua separação das outras linhagens. As linhagens 1a e 1b, apesar de se agruparem em ramos distintos, foram caracterizadas no mesmo grupo, com bootstrap acima de 75 porcento. Além disso, as linhagens 1a e 1b foram mais relacionadas com as cepas americanas e a linhagem 2 com as cepas asiáticas. As substituições de aminoácidos (aa) foram observadas nos ectodomínios I e III da proteína E e foram associadas à separação das linhagens. Uma substituição em E297 diferenciou a linhagem 1a das linhagens 1b e 2. As alterações observadas em E338, E394 (ectodomínio III), E428 e E430 (região “stem”) diferenciaram as linhagens...

This study presents the phylogeny and molecular characterization based onenvelope gene (E) analysis of DENV-1 (n=48) isolated during epidemics occurred since this serotype introduction in 1986 to 2011. From those strains, six were fullysequenced (coding region) and possible genomic recombination events wereanalyzed. The results of the phylogenetic analysis based on the E gene showed that DENV-1 isolates from the states of Rio de Janeiro, Espírito Santo, Minas Gerais, Mato Grosso do Sul, Alagoas, Ceará, Piauí and Rio Grande do Norte belong to genotype V (America/Africa), but grouping in distinct clades. Three groups wereidentified, one dating from 1986 to 2002 (lineage 1a), a second group isolated between 2009 and 2011 and a representative strain isolated in 2002 (lineage 2) and a group of strains isolated in 2010 and 2011 (lineage 1b). Lineage 2 has a high bootstrap support ensuring their separation from the other lineages. The lineages 1aand 1b, despite in distinct branches, were characterized in a same group supported by a bootstrap of 75 percent. Furthermore, the lineages 1a and 1b were more closely related to the American strains, while lineage 2 to the Asian strains. Amino acids (aa)substitutions were observed in the ectodomains I and III of the E protein and were associated to the lineages separation. A substitution on E297 differentiated the lineage...

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 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.