Characterization of Dengue Virus Type 2: New Insights on the 2010 Brazilian Epidemic

Dengue viruses (DENV) serotypes 1, 2, and 3 have been causing yearly outbreaks in Brazil. In this study, we report the re-introduction of DENV2 in the coast of São Paulo State. Partial envelope viral genes were sequenced from eighteen patients with dengue fever during the 2010 epidemic. Phylogenetic analysis showed this strain belongs to the American/Asian genotype and was closely related to the virus that circulated in Rio de Janeiro in 2007 and 2008. The phylogeny also showed no clustering by clinical presentation, suggesting that the disease severity could not be explained by distinct variants or genotypes. The time of the most recent common ancestor of American/Asian genotype and the São Paulo and Rio de Janeiro (SP/RJ) monophyletic cluster was estimated to be around 40 and 10 years, respectively. Since this virus was first identified in Brazil in 2007, we suggest that it was already circulating in the country before causing the first documented outbreak. This is the first description of the 2010 outbreak in the State of São Paulo, Brazil, and should contribute to efforts to control and monitor the spread of DENVs in endemic areas

Phenotypic profile of CD8+ T cells in acute dengue infection

A dengue é uma doença infecciosa aguda causada pelo vírus DEN do gênero Flavivirus e é transmitida pela picada de um mosquito vetor, principalmente o Aedes aegypti. Existem quatro sorotipos do vírus da dengue (DEN-1, DEN-2, DEN-3 e DEN-4) e sua incidência tem aumentado dramaticamente nos últimos 50 anos, inclusive no Brasil. O objetivo deste trabalho é caracterizar subpopulações de linfócitos T, principalmente linfócitos T CD8+, provenientes de pacientes infectados quanto a sua capacidade proliferativa, seu estado de ativação e memória celular. Os pacientes foram recrutados no Hospital Ana Costa de Santos, SP, no ano de 2010, após assinarem o Termo de Consentimento Livre e Esclarecido. O diagnóstico de dengue foi realizado utilizando o teste rápido Dengue Duo e os parâmetros imunológicos foram analisados no citômetro de fluxo. Foi coletado sangue periférico para criopreservação de células mononucleares e separação de soro para detecção da carga viral. Pacientes com dengue apresentaram maior proliferação de linfócitos T CD8+ quando comparados com indivíduos saudáveis. Foi ainda observado que tal proliferação celular foi evidente nos dias cinco e seis de sintomas. Quando marcadores de ativação celular foram analisados por citometria de fluxo, observou-se um aumento de linfócitos T CD8+ expressando CD38 e HLA-DR nos pacientes, quando comparados com indivíduos saudáveis. Da mesma forma, a ativação celular também aumentou com o passar dos dias de sintomas com destaque para o quinto e sexto dia. Este aumento na ativação das células juntamente com os dias de sintomas, foi igualmente observado em várias subpopulações de células T de memória Além disso, foi observada uma correlação negativa entre o número absoluto de linfócitos T CD8+ e a carga viral. Juntos, os resultados desse estudo sugerem que a infecção por DEN leva a um aumento da ativação de células T CD8+Dengue is an acute disease caused by DEN, a Flavivirus transmitted by a mosquito vector, primarily Aedes aegypti. There are four serotypes of dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) and their incidences have increased dramatically over the past 50 years, including in Brazil. The goal of this study is to characterize subpopulations of T lymphocytes, mainly CD8+ T cells, from infected patients. Status of cell activation, and memory cell profiles were assessed. Patients were recruited at Hospital Ana Costa, Santos-SP, Brazil, in 2010, after having signed an informed consent form. The serologic diagnosis of dengue was carryied out using a rapid test and immunological parameters were analyzed in flow cytometer. Peripheral blood was collected for cryopreservation of mononuclear cells and separation of serum for viral load testing. Dengue patients showed higher proliferation of CD8+ T cells compared to healthy subjects. Cell proliferation was more evident in the fifth and sixth days of symptoms. We observed increased frequency of CD8+ T cells expressing activation markers CD38 and HLA-DR in patients, when compared to healthy subjects. Similarly, T cell activation also increased along with the passing days of symptoms hitting on the fifth and the sixth days. Such augment in cellular activation along with the days of symptoms was equally observed in the several memory T cell compartments. Furthermore, we observed a negative correlation between the absolute number of CD8+ T lymphocytes and viral load. Together, the results of this study suggest that dengue virus infection leads to an increased activation of CD8+ T cell

CD8+ T Lymphocyte Expansion, Proliferation and Activation in Dengue Fever

<div><p>Dengue fever induces a robust immune response, including massive T cell activation. The level of T cell activation may, however, be associated with more severe disease. In this study, we explored the level of CD8+ T lymphocyte activation in the first six days after onset of symptoms during a DENV2 outbreak in early 2010 on the coast of São Paulo State, Brazil. Using flow cytometry we detected a progressive increase in the percentage of CD8+ T cells in 74 dengue fever cases. Peripheral blood mononuclear cells from 30 cases were thawed and evaluated using expanded phenotyping. The expansion of the CD8+ T cells was coupled with increased Ki67 expression. Cell activation was observed later in the course of disease, as determined by the expression of the activation markers CD38 and HLA-DR. This increased CD8+ T lymphocyte activation was observed in all memory subsets, but was more pronounced in the effector memory subset, as defined by higher CD38 expression. Our results show that most CD8+ T cell subsets are expanded during DENV2 infection and that the effector memory subset is the predominantly affected sub population.</p></div

Activation of CD8+ T cells in dengue fever.

<p>(A) Percentage and (B) absolute numbers of three subpopulations (HLADR+, CD38+HLADR+, and CD38+) within CD8+ T cells in the peripheral blood from healthy controls (n = 17) and dengue fever patients (n = 74) at different days of symptoms: HLADR+, CD38+HLADR+, and CD38+. Only the dual positive subpopulation significantly increased during the course of dengue fever, either in their percentage or absolute numbers. *p< 0.05, ***p< 0.0001.</p

CD8+ T lymphocytes in the course of dengue fever.

<p>(A) Percentage within T cells and (B) absolute numbers of CD8+ T lymphocytes in the peripheral blood from healthy controls (n = 17) and dengue fever patients at different days of symptoms (n = 74). A significant change in the percentage of CD8+ T lymphocytes was observed, between days 5 and 6 after the onset of symptoms when compared to healthy controls and days 3 and 4 of symptoms, as shown in A. However, decreased absolute CD8+ T lymphocyte numbers were documented in the first four days of symptoms when compared to healthy controls, returning to higher levels later in the disease’s course, as shown in B. *p< 0.05, ** p<0.01.</p

Activation of CD8+ T cells subpopulations in dengue fever.

<p>Percentage and absolute numbers of naïve (A and B), central memory (C and D), effector memory (E and F), and terminal effector memory (G and H) T CD8+ cells are shown. Increased percentages of activated cells were observed mostly in dual positive CD38+HLA-DR+ central memory (TCM) (p<0.001) effector memory (TEM) (p<0.0001) and terminal effector memory (TEMRA) (p<0.01) T cells, although the percentage of CD38+HLA-DR- effector memory (p<0.01) and terminal effector (p<0.01) T cells were also augmented in dengue fever.</p

Correlation of CD8+ T lymphocyte numbers and DENV viral load.

<p>In A, viral load was determined on different days of symptoms and a significant decrease was observed in days 5 and 6 when compared to days 1 and 2 after the onset of symptoms (p value = 0.0136). In B, a negative correlation was observed between 1 and 2 days of symptoms (r = -0.5900, p = 0.0019). There is no correlation between 3 and 4 days of symptoms (C) and viral loads; In D, a negative correlation was observed between viral loads and 5 and 6 days of symptoms (r = - 0.4705, p = 0.0488). Dashed lines are arbitrarily set at 450 CD8+ T lymphocytes/μL and 1050 copies of RNA/ml.</p

Demographic and hematologic characteristics of study participants.

<p>* DENV: dengue virus</p><p>Demographic and hematologic characteristics of study participants.</p