Comparative assessment of the replication efficiency of dengue, yellow fever, and chikungunya arboviruses in some insect and mammalian cell lines

Abstract INTRODUCTION: Insect cell cultures play an essential role in understanding arboviral replication. However, the replicative efficiency of some of these viruses such as dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) in a new cellular substrate (Lulo) and in the other two recognized cell lines has not been comparatively assessed. METHODS: Vero, C6/36, and Lulo cell lines were infected with DENV, YFV, and CHIKV. The viral progeny was quantified through plaque assays and quantitative reverse transcription-polymerase chain reaction, while for DENV2, the findings were confirmed by immunofluorescence antibody assay. RESULTS: The higher DENV2 titer (from multiplicity of infection 0.001) was obtained on day four post-infection in C6/36 and on day six in Vero cells, while the Lulo cell line was almost impossible to infect under the same conditions. However, C6/36 showed the highest values of viral RNA production compared to Vero cells, while the quantification of the viral RNA in Lulo cells showed high levels of viral genomes, which had no correlation to the infectious viral particles. CONCLUSIONS: C6/36 was the most efficient cell line in the alpha and flavivirus production, followed by Vero cells. Thus, Lulo cells may be a useful substrate to study the mechanisms by which cells evade viral replication.

Nuclei ultrastructural changes of C6/36 cells infected with virus dengue type 2 / Cambios ultraestructurales en núcleos de células C6/36 infectadas con virus dengue de tipo 2

ABSTRACT Introduction: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. Objective: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. Materials and methods: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. Results: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. Conclusions: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism.

RESUMEN Introducción. La replicación del virus del dengue se ha considerado principalmente citoplásmica; sin embargo, en diversos estudios se ha informado que algunos flavivirus pueden utilizar factores intranucleares como parte de la maquinaria que utilizan para aumentar la capacidad de infección en la célula huésped. En este trabajo se describen las alteraciones a nivel nuclear en células infectadas con dengue, probablemente involucradas en procesos de replicación viral. Objetivo. Presentar las observaciones ultraestructurales de células C6/36 de Aedes albopictus infectadas con el virus del dengue de tipo 2. Materiales y métodos. Se infectaron células C6/36 con suero de un paciente con diagnóstico de dengue 2; posteriormente, se mantuvieron en medio de cultivo durante 10 días y se evaluó el efecto citopático. Las células se procesaron para los ensayos de inmunofluorescencia y microscopía electrónica de transmisión, con el fin de hacer el estudio ultraestructural. Resultados. Los ensayos de inmunofluorescencia confirmaron la presencia de la proteína E viral asociada con sincitios celulares en el cultivo. En el estudio ultraestructural, las células infectadas tenían estructuras vesiculares y tubulares, y cisternas dilatadas del retículo endoplásmico en el citoplasma. Las partículas virales se encontraron exclusivamente en vacuolas localizadas en el citoplasma. Los núcleos de los sincitios celulares contenían estructuras de membrana dispuestas en forma circular y, en algunos casos, dichos sincitios presentaban lisis. En ningún caso se observaron partículas virales en el núcleo. Conclusiones. No se habían reportado alteraciones ultraestructurales en los núcleos de células infectadas con el virus del dengue detectadas mediante técnicas de microscopia electrónica. Es probable que tales modificaciones estén asociadas con procesos intranucleares de replicación como un mecanismo alternativo.

Differences in the replicative capacities of clinical isolates of dengue virus in C6/36 cells and in urban populations of Aedes aegypti from Colombia, South America

ABSTRACT Dengue, the most prevalent arboviral disease worldwide, is caused by any of the four dengue virus (DENV) serotypes that co-circulate constantly in hyperendemic areas such as Medellin (Colombia), and these serotypes are transmitted by mosquitoes of the genus Aedes. In this study, we evaluated the replicative capacity of strains isolated in Medellin between 2003 and 2007 in C6/36 cells and in colonies of Aedes aegypti collected during 2010-2011 from high or low-incidence areas within the same city. The phylogenetic analysis grouped isolates according to the predominant genotypes found in the Americas, and the in vitro characterization showed differences in the morphological changes induced by the isolates of each of the isolated serotypes compared to the reference serotypes. In vitro replicative capacity studies demonstrated that genomic copy number increased at four days post-infection and that cell viability decreased significantly compared to the control for all serotypes. The largest number of genomic copies in C6/36 was produced by DENV-2, followed by DENV-1 and DENV-4; DENV-3 produced the smallest number of genomic copies and had the smallest negative effect on cell viability. Finally, differences in the in vivo replication of intercolonial serotypes between the Rockefeller colony and the field colonies and among the intracolonial serotypes were found. The replication of DENV-2 at 7 and 14 days in both high- and low-incidence colonies was higher than that of the other serotypes, and replication of DENV-3 in the mosquito colonies was the most stable on the days evaluated. Our results support the notion that replication and, possibly, DENV transmission and severity depend on many factors, including serotype and vector characteristics.

Dengue virus infection induces chromatin remodeling at locus AAEL006536 in the midgut of Aedes aegypti / La infección por virus dengue induce remodelación de la cromatina en el locus AAEL006536 en el intestino medio de Aedes aegypti

Abstract: Objective: To identify and characterize Aedes aegypti's AAEL006536 gene proximal upstream cis-regulatory sequences activated by dengue virus infection. Materials and methods: A. aegypti Rockefeller strain mosquitoes were blood fed or infected with dengue virus 2. Open chromatin profiling was then carried out in pools of midguts from each group of mosquitoes. Results: The proximal upstream region does not contain open chromatin sites in the midguts of blood-fed mosquitoes as detected by FAIRE-qPCR. In contrast, two cis-regulatory sites were identified in the same upstream region of dengue virus-infected mosquito midguts. The distal sequence contains STAT-, REL- and C/EBP-type transcription factor binding sites. Conclusion: The activation of two proximal cis-regulatory sequences, induced by dengue virus infection, is mediated by chromatin remodeling mechanisms. Binding sites suggest a dengue virus infection-induced participation of immunity transcription factors in the up-regulation of this gene. This suggests the participation of the AAEL006536 gene in the mosquito's antiviral innate immune response.

Resumen: Objetivo: Identificar y caracterizar las secuencias reguladoras activadas por la infección por virus dengue en la región proximal del gen AAEL006536 de Aedes aegypti. Material y métodos: Mosquitos de la cepa Rockefeller de A. aegypti se infectaron con virus dengue o se alimentaron con sangre. Se obtuvieron los perfiles de cromatina abierta del locus en los intestinos de cada uno de los grupos. Resultados: Se identificaron dos sitios reguladores solo en los intestinos de mosquitos infectados por virus dengue. El sitio distal contiene sitios de unión a factores de transcripción tipo REL, STAT y C/EBP. Conclusiones: La activación de dos sitios reguladores proximales está mediada por la remodelación de la cromatina. Los sitios de unión a factores de transcripción en el sitio regulador distal sugieren la participación de las vías de inmunidad en la regulación del gen. Esto sugiere la participación de este gen en la respuesta inmune del mosquito frente a la infección viral.

Pathogen-insect interaction candidate molecules for transmission-blocking control strategies of vector borne diseases / Moléculas candidatas para el control de enfermedades transmitidas por vector mediante el bloqueo de interacciones patógeno-insecto

Abstract: Objective: To analyze the current knowledge of pathogen-insect interactions amenable for the design of molecular-based control strategies of vector-borne diseases. Materials and methods: We examined malaria, dengue, and Chagas disease pathogens and insect molecules that participate in interactions during their vectors infection. Results: Pathogen molecules that participate in the insect intestine invasion and induced vector immune molecules are presented, and their inclusion in transmission blocking vaccines (TBV) and in genetically modify insect (GMI) vectors or symbiotic bacteria are discussed. Conclusion: Disruption of processes by blocking vector-pathogen interactions provides several candidates for molecular control strategies, but TBV and GMI efficacies are still limited and other secondary effects of GMI (improving transmission of other pathogens, affectation of other organisms) should be discarded.

Resumen: Objetivo: Analizar el conocimiento actual de las interacciones patógeno-insecto susceptibles a incluirse en el diseño de estrategias moleculares para el control de enfermedades transmitidas por vectores. Material y métodos: Se examinaron los agentes causales de la malaria, el dengue y la enfermedad de Chagas, y las moléculas de insectos que participan en interacciones durante la infección de sus vectores. Resultados: Se presentan moléculas de patógenos que participan en la invasión del intestino del insecto y moléculas inmunes inducidas en los vectores. Se discute su inclusión en vacunas bloqueadoras de transmisión (VBT) y en la modificación genética de vectores (MGI) o de sus bacterias simbióticas. Conclusión: La interrupción de procesos mediante el bloqueo de las interacciones patógeno-vector proporciona varios candidatos para las estrategias de control molecular, pero la eficacia de VBT y MGI es aún limitada y los efectos secundarios de MGI (aumento de la transmisión de otros patógenos y afectación de otros organismos) deben descartase.

Overexpression of miR-484 and miR-744 in Vero cells alters Dengue virus replication

BACKGROUND Dengue is considered one of the world’s most important mosquito-borne diseases. MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that play an important role in the regulation of gene expression in eukaryotes. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in Dengue virus (DENV) replication remains poorly understood. OBJECTIVE To determine the role of miR-484 and miR-744 in DENV infection and to examine whether DENV infection alters the expression of both miRNAs. METHODS We used bioinformatics tools to explore the relationship between DENV and cellular miRNAs. We then overexpressed miR-484 or miR-744 in Vero cells to examine their role in DENV replication using flow cytometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and western blotting. FINDINGS We found several cellular miRNAs that target a conserved region within the 3′ untranslated region (3′ UTR) of the genome of the four DENV serotypes and found that overexpression of miR-484 or miR-744 inhibits infection by DENV-1 to DENV-4. Furthermore, we observed that DENV RNA might be involved in the downregulation of endogenous miR-484 and miR-744. CONCLUSION Our study identifies miR-484 and miR-744 as two possible restriction host factors against DENV infection. However, further studies are needed to directly verify whether miR-484 and miR-744 both have an anti-DENV effect in vivo.

Mosquito-transmitted viruses - the great Brazilian challenge

ABSTRACT Arboviruses pose a serious threat to public health worldwide, overloading the healthcare system and causing economic losses. These viruses form a very diverse group, and in Brazil, arboviruses belonging to the families Flaviviridae and Togaviridae are predominant. Unfortunately, the number of arboviruses increases in proportion with factors such as deforestation, poor sanitation, climate changes, and introduction of new viruses like Chikungunya virus and Zika virus. In Brazil, dengue is endemic, along with the presence of other arboviruses. The situation is complicated by the scarcity of diagnostic infrastructure and the absence of approved vaccines for these diseases. Disease control, thus, relies solely on vector control. Therefore, enhanced clinical knowledge and improved general awareness about these arboviruses are indispensable to tackle diagnostic inadequacies.

Camundongos inoculados com DENV2 por via intracerebralhistopatologia, detecção viral e avaliação de proteção mediada por uma vacina de DNA / Mice intracerebrally inoculated with DENV2: histopathology, virus detection and evaluation of protection mediated by a DNA vaccine

A dengue constitui um sério problema de saúde pública, principalmente em regiões tropicais e subtropicais do mundo. [...] Apesar disso, um dos modelos mais utilizados para testes de vacinas contra a dengue se baseia na inoculação em camundongos por via intracerebral (i.c.) de vírus neuroadaptado. No entanto, poucos estudos avaliaram o efeito da infecção i.c. e/ou proteção gerada por protótipos vacinais em diferentes órgãos, tais como fígado, um dosórgãos comprometidos pela dengue em humanos. O nosso grupo construiu a vacina de DNA, pcTPANS1, que induziu altos níveis de sobrevivência em camundongos desafiados por via i.c. com vírus da dengue 2 (DENV2). Diante disso, o presente trabalho se propõe avaliar aspectos da patogênese no cérebro, cerebelo, fígado e pulmão, no modelo de camundongos BALB/c inoculados pela via i.c. com uma dose letal de DENV2, em diferentes dias após infecção (d.p.i.). Adicionalmente, tais análises foram estendidas para animais imunizados com a vacina pcTPANS1 e desafiados com DENV2. Detectamos alterações histopatológicas no cérebro/cerebelo (edema,hemorragia, gliose reacional, microglia hiperplásica e hipertrofiada e infiltrado mononuclear na pia-máter, no neurópilo e perivasculares), no fígado (edema,hemorragia, balonização hepatocitária, infiltrado mononuclear, hiperplasia e hipertrofiade células de Kupffer) e no pulmão (edema, hemorragia, infiltrados mononuclear esperibronquiolares, aumento do número de macrófagos alveolares e espessamento de septo interalveolar). Alguns destes danos foram quantificados, utilizando uma escala subjetiva com atribuição de diferentes graus, revelando diferenças significativas...

The dengue is a serious public health problem, mainly in tropical and subtropicalregions of the world. [...] Nevertheless, oneof the most widely used model for vaccine tests against dengue is based on theinoculation of mice by the intracerebral route (i.c.) with neuroadapted virus. However,few studies have evaluated the effects of i.c. infection and/or protection generated byvaccine prototypes in different organs, such as the liver, one of the compromised organin dengue in humans. Our group have constructed a DNA vaccine, pcTPANS1, whichinduced high survival rates in mice challenged by the i.c. inoculation with dengue virus2 (DENV2). Therefore, the present work aim to evaluate aspects of the pathogenesis inthe brain, cerebellum, liver and lung in the BALB/c mouse model intracerebrallyinoculated with a lethal dose of DENV2, at different days post infection (d.p.i.). Inaddition, these analyzes were extended to pcTPANS1-immunized animals challengedwith DENV2. We detect histopathological changes in the brain/cerebellum(hemorrhage, edema, reactive gliosis, hyperplasic and hypertrophied microglia andmononuclear infiltrate in the pia-mater, neuropile and perivascular), the liver(hemorrhage, edema, hepatocyte ballooning, mononuclear infiltrate, hyperplasia andhypertrophy of Kupffer cells) and the lung (hemorrhage, edema, peribronchialmononuclear infiltrates, increased number of alveolar macrophages and thickening ofinteralveolar septa). Some of these damages were quantified using a subjective scalewith different degrees and revealing significant differences...

Dengue e imunologia inata: estudo dos fatores antivirais e citotóxicos em células dendríticas plasmacitoides e células NK durante a infecção pelo vírus Dengue / Dengue and innate immunology: study of antiviral and cytotoxic fators in plasmacytoid dendritic cells and nk cells during dengue virus infection

O vírus dengue é um flavivírus causador de síndrome febril hemorrágica, sendoalguns casos graves evidenciados pelo acúmulo de plasma nas cavidades. Sugereseque a intensa replicação viral na fase aguda resultaria na intensa produção demediadores inflamatórios, levando ao extravasamento plasmático. A imunidade inataé uma importante barreira na limitação da dispersão viral. As células dendríticasplasmacitoides (PDCs) e as células NK são fundamentais durante a fase inicial dasinfecções por suas características antivirais e citotóxicas contra células infectadas. Ofenótipo Killer das PDCs (IKPDCs) resultaria da ativação viral e é caracterizadopela expressão membranar do ligante indutor de apoptose relacionado ao fator denecrose tumoral (TRAIL) e pela produção de interferons do tipo I. As células NKpodem ser diretamente ativadas reconhecendo as células alvo ou, indiretamenteativadas pelas citocinas (como os interferons) tornando-se citotóxica, expressandoTRAIL membranar. Apesar do papel antiviral, pouco se sabe sobre os mecanismosde ação dessas populações celulares durante a febre do dengue (FD). Nossoobjetivo foi estudar o envolvimento das PDCs e células NK ativadas naimunopatologia da FD. Analisando as células de pacientes ex vivo, observamosmaior frequência de IKPDCs nos casos brandos de FD, assim como de células NKTRAIL+. Outros marcadores de ativação como o marcador de degranulação CD107ae o receptor de reconhecimento padrão TLR3 foram expressos em maiores níveisnos pacientes comparando aos controles saudáveis...

Dengue virus is a flavivirus that can cause a hemorrhagic febrile syndrome, in whichsome severe cases are characterized by plasma accumulation in cavities. It issuggested that a high viral burden in the acute phase would lead to an enhancedproduction of inflammatory mediators, leading to plasma leakage. Innate immunity isan important barrier limiting viral spread. Plasmacytoid dendritic cells (PDCs) and NKcells are main actors in the beginning of infection because of their antiviral andcytotoxic features against infected cells. PDC killer phenotype (IKPDCs) is inducedby viral activation and main profile is membrane expression of TNF-relatedapoptosis-inducer ligand (TRAIL) and type I interferon production. NK cells can beactivated directly, by target recognition, or indirectly, by cytokines (like interferons)and become cytotoxic, expressing membrane TRAIL. Despite its antiviral role, thefunction of those cell populations during dengue fever (DF) is not largely known. Ouraim was to study activated PDCs and NK cells involvement in DF immunopathology.DF patients’ cells analysis ex vivo presented higher frequency of IKPDCs and alsohigher frequency of TRAIL+NK cells in mild DF cases. NK activation markers such asCD107a degranulation marker and pattern recognition receptor TLR3 wereupregulated in patients’ cells compared to healthy donors...

Estudo dos mediadores inflamatórios e de coagulação na epidemia 2013 pelo DENV-4 / Evaluation of inflammatory and coagulation mediators in 2013 dengue virus type 4 outbreak

A dengue se tornou a arbovirose mais difundida no mundo, atingindo mais de 100 países. O sorotipo DENV-4 foi reintroduzido no Brasil em 2010, se espalhou para as diferentes regiões brasileiras sendo responsável por uma das maiores epidemias de dengue relatadas no país em 2013. O objetivo deste estudo foi avaliar o perfilclínico-laboratorial, citocinas/quimiocinas inflamatórias e mediadores da coagulação em pacientes infectados pelo DENV durante esta epidemia. Para isto, foram avaliados 265 casos confirmados de dengue atendidos nos centros de saúde dos estados do RJ e MS no ano de 2013. Os pacientes foram classificados de acordo com a nova classificação da OMS, 2009 nos grupos: 158 (70,2 por cento) dengue sem sinais de alarme (DSSA), 65 (28,9 por cento) dengue com sinais de alarme (DCSA) e 2 (0,9 por cento)dengue grave. Os sinais de alarme mais frequentes foram dor abdominal persistente(61,5 por cento) e sangramento de mucosas (32,3 por cento). O DENV-4 foi o sorotipo de maior incidência e 85,2 por cento dos pacientes apresentaram infecção secundária. Pacientes DCSA/DG apresentaram baixa contagem de leucócitos totais, de plaquetas e altos níveis séricos de AST/TGO e ALT/TGP quando comparados aos pacientes DSSA e aqueles ODF (outras doenças febris). A quantificação dos mediadores inflamatórios por ensaios imunoenzimáticos indicou: (i) níveis aumentados de citocinas (TNF-alfa, IL-6 e IL-10) e quimiocinas (IL-8/CXCL8, IP-10/CXCL10 e MCP-1/CCL2) nos pacientes DSSA e DCSA/DG quando comparados com o grupo controle; (ii) níveis aumentados de IL-10 e IL-6 nos pacientes DCSA/DG enquanto que IL-8/CXCL8 foimaior nos pacientes DSSA...

Dengue has become the most widespread arbovirus in the world, reaching more than100 countries. The DENV-4 serotype was reintroduced in Brazil in 2010, and spreadto the different Brazilian regions being responsible for one of the largest epidemics ofdengue reported in the country in 2013. The objective of this study was to evaluatethe clinical and laboratory profile, and inflammatory cytokines/chemokines andcoagulation mediators in patients infected with DENV during this epidemic. For this,were evaluated 265 confirmed cases of dengue attended at health centers in thestates of RJ and MS in the year 2013. Patients were classified according to the newWHO classification, 2009 in groups: 158 (70.2 percent) dengue without warning signs(DwoWS), 65 (28.9 percent) dengue with warning signs (DwWS) and 2 (0.9 percent) severedengue (SD). The most frequent warning signals were persistent abdominal pain(61.5 percent) and mucosal bleeding (32.3 percent). The DENV-4 was the most prevalentserotype and 85,2 percent of dengue patients showed secondary infection. DwWS/SDpatients showed lower white blood cell and platelet counts, and higher AST and ALTserum levels compared to DwoWS and other febrile illnesses patients. Quantificationof inflammatory mediators by immunoassays indicated: (i) increased levels ofcytokines (TNF-alpha, IL-6 and IL-10) and chemokines (IL-8/CXCL8, IP-10/CXCL10 andMCP-1/CCL2) in DwoWS and DwWS patients compared with healthy individuals; (ii)increased levels of IL-10 and IL-6 in DwWS/SD patients while IL-8/CXCL8 wasincreased in DSSA patients; In respect to coagulation parameters: (iii) DwoWS andDwWS/SD patients showed lower levels of sTF and higher of TFPI compared withthe control group; (iv) Lower levels of fibrinogen were found in DwWS/SD patientsand higher levels of thrombomodulin in patients between 4-7 days of illness...

Vector biology prospects in dengue research

We argue that using more natural blood feeding methods to study mosquito vector competence for dengue viruses and exploring the effect of viral infection on other mosquito life-history traits that influence vectorial capacity will significantly advance our understanding of dengue epidemiology.

Propiedades biológicas de cepas de dengue virus-3 aisladas durante la epidemia ocurrida en La Habana, 2001-2002 / Biological properties of virus dengue-3 strains isolated during the epidemic ocurred in Havana, 2001-2002

Introducción: durante epidemias de dengue ocurridas en Cuba se ha observado de forma reiterada, un incremento de severidad clínica con la progresión de las epidemias en el tiempo, particularmente en infecciones secundarias. Se considera que este incremento pudiera estar relacionado con cambios genéticos del virus circulante. Objetivo: estudiar algunas propiedades biológicas de cepas aisladas en diferentes momentos de la epidemia de La Habana, 2001-2002. Métodos: se estudiaron 9 cepas de dengue virus-3, se evaluó el efecto citopatogénico y el crecimiento viral en las líneas celulares C6/36 HT y Vero, tamaño de las placas virales, sensibilidad a la temperatura, neurovirulencia en ratones lactantes y la influencia del pH en la unión del virus a la célula, así como en el medio de multiplicación de este. Resultados: las cepas de dengue virus-3 resultaron más citopatogénicas en células Vero, sin embargo, en C6/36 HT se obtuvieron títulos superiores. El conjunto de cepas mostró reducción del título viral y del tamaño de placa al aumentar la temperatura y fueron poco neurovirulentas. En cuanto a la unión del virus a la célula, a pH básico se observaron los mejores títulos, mientras que a pH ácido solo se observó el crecimiento de algunas cepas aisladas al final de la epidemia. El medio de multiplicación del virus a pH 6,5-7 favoreció el crecimiento de las cepas del inicio de la epidemia, mientras que las cepas del final tuvieron títulos superiores a pH 7-8. Conclusiones: se pudo comprobar la existencia de cambios fenotípicos entre cepas de diferentes momentos de la epidemia, que pudieran estar asociados con diferencias en cuanto a su adecuación viral o en su potencial virulento. No obstante, algunas de las propiedades biológicas estudiadas sugieren que las cepas de dengue virus-3 son menos virulentas que las cepas cubanas de dengue virus-2 de 1997.

Introduction: during dengue epidemics in Cuba, an increase in clinical severity with the epidemics progression in time, particularly in secondary infections, have been frequently observed. It is considered that this increase could be related with genetic changes in the circulating virus. Objective: to study some biological attributes related to strains isolated at different points of time during the dengue epidemic occurred in Havana city, 2001-2002. Methods: nine DENV-3 strains were studied. Cytopathogenic effect, viral growth in C6/36 HT and Vero cell lines, viral plaque sizes, temperature sensitivity, neurovirulence in newborn mice and pH influence in the binding of the virus and the cell as well as in the multiplication medium were evaluated. Results: DENV-3 strains were more cytopathogenic in Vero Cells. However, higher titres were obtained in C6/36 HT cells. All the strains showed reduction of viral titres and plaque size with temperature increasing and low neurovirulence. Basic pH favoured virus-cell binding whereas acid pH was only permissive for some strains isolated at the end of the epidemic. On the other hand, at pH 6.5-7, the viral multiplication medium favoured the growth of strains isolated at the beginning of the epidemic whereas the growth of those isolated at the endof the epidemic was noticeable at pH 7-8. Conclusions: this study proved the phenotypical changes among strains isolated at different points of time in the epidemic. They might be related to differences in viral fitness or in virulent potential. Nevertheless, some of the studied biological properties suggest that dengue virus-3 strains are less virulent than the Cuban dengue virus 2 strains isolated in 1997.

Epidemiología molecular de los virus Dengue / Molecular epidemiology of Dengue virus

El dengue es la enfermedad viral más importante transmitida por mosquitos a humanos por su alta morbimortalidad y el potencial de diseminación de su vector Aedes aegypti. Además, la falta de una vacuna y medicamentos antivirales específicos, así como el incremento progresivo de las infecciones secundarias y la hiperendemicidad en diferentes países, hacen de esta enfermedad un problema de salud pública. Existen cuatro serotipos del virus del dengue (DENV), dentro de cada serotipo se han descrito varios genotipos, constituidos a su vez por diferentes linajes o clados. La epidemiología molecular combina los análisis filogenéticos de los DENV detectados en un área geográfica, en un tiempo definido, con la información clínica y epidemiológica disponible. El objetivo de estos estudios es tratar de establecer asociaciones entre genotipos o linajes virales con el origen (ancestros), procedencia geográfica, ruta de transmisión viral, severidad de la enfermedad, grupos poblacionales afectados, y la intensidad y extensión de los brotes epidémicos. La epidemiología molecular ha generado información relevante como la etiología del DENV genotipo Asiático en los casos graves de dengue de la epidemia ocurrida en Venezuela en 1989, y la identificación de cambios nucleotídicos puntuales en el genoma viral asociados a propiedades biológicas fundamentales. En la actualidad se hace necesario realizar análisis exhaustivos del genoma viral completo, conjuntamente con el análisis bioinformático, biológico, clínico y epidemiológico de los cuatro serotipos circulantes en los países endémicos, así como instaurar en los laboratorios adscritos a los sistemas de vigilancia epidemiológica del dengue, la vigilancia molecular para la identificación de genotipos (o linajes) circulantes, lo que contribuiría entre otros aspectos al control efectivo de la enfermedad por DENV.

Dengue is the most important viral disease transmitted by mosquitoes to humans in tropical and subtropical regions of the world. This is the result of its high morbidity and mortality, the spread potential of the vector Aedes aegypti, the lack of effective vaccines and specific antiviral drugs, the gradual increase in secondary infections and hyperendemicity differences in distinct countries. There are four serotypes of dengue virus which are phylogenetically grouped in genotypes and subdivided in lineages or clades. Molecular epidemiology combines phylogenetic analysis of DENV detected in particular geographic areas within a defined time with the available clinical and epidemiologic information. The objective of these studies is to look for relationships between genotypes or lineages, viral origin, geographical spreading and routes of viral transmission, disease severity, population groups affected, and the intensity, speed and extent of outbreaks. Also, molecular epidemiology has generated relevant information such as the Asian genotype DENV etiology in cases of the severe dengue epidemic in Venezuela in 1989, and the identification of specific nucleotide changes in the viral genome associated with its fundamental biological properties. However, analysis of the complete viral genome, together with bioinformatic, biological, clinical and epidemiological analysis corresponding to the four serotypes circulating in endemic countries should be performed. Molecular surveillance for the identification of genotypes (or strains) circulating should be implemented in the laboratories responsible for the epidemiological surveillance of dengue, which would improve the effective control of DENV.

Potential impact of a presumed increase in the biting activity of dengue-virus-infected Aedes aegypti (Diptera: Culicidae) females on virus transmission dynamics

Recently, we showed that infection with dengue virus increases the locomotor activity of Aedes aegypti females. We speculate that the observed increased locomotor activity could potentially increase the chances of finding a suitable host and, as a consequence, the relative biting rate of infected mosquitoes. We used a mathematical model to investigate the impact of the increased locomotor activity by assuming that this activity translated into an increased biting rate for infected mosquitoes. The results show that the increased biting rate resulted in dengue outbreaks with greater numbers of primary and secondary infections and with more severe biennial epidemics.

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

Assessing key safety concerns of a Wolbachia-based strategy to control dengue transmission by Aedes mosquitoes

Mosquito-borne diseases such as dengue fever, chikungunya or malaria affect millions of people each year and control solutions are urgently needed. An international research program is currently being developed that relies on the introduction of the bacterial endosymbiont Wolbachia pipientis into Aedes aegypti to control dengue transmission. In order to prepare for open-field testing releases of Wolbachia-infected mosquitoes, an intensive social research and community engagement program was undertaken in Cairns, Northern Australia. The most common concern expressed by the diverse range of community members and stakeholders surveyed was the necessity of assuring the safety of the proposed approach for humans, animals and the environment. To address these concerns a series of safety experiments were undertaken. We report in this paper on the experimental data obtained, discuss the limitations of experimental risk assessment and focus on the necessity of including community concerns in scientific research.

Incidencia de infecciones sintomáticas y asintomáticas por virus dengue en Maracay, Venezuela: 2006-2007 / Incidence of symptomatic and asymptomatic dengue virus infections in Maracay, Venezuela: 2006-2007

Con el objetivo de estimar las tasas de incidencia de infecciones sintomáticas y asintomáticas por virus dengue (DENV) durante un año (octubre 2006-septiembre 2007) en cuatro barrios de Maracay (Venezuela) se realizó un estudio prospectivo consistente de visitas domiciliarias, tres veces por semana, para detectar casos de dengue y de encuestas serológicas semestrales para determinar infecciones asintomáticas probables por DENV. Los sujetos de estudio pertenecían a una cohorte de 2663 personas ≥5 años de edad. El diagnóstico confirmatorio de DENV se realizó mediante la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR). Las pruebas serológicas se realizaron mediante el ensayo inmunoenzimático de captura de IgM anti-dengue (MAC-ELISA). Los resultados determinaron tasas de incidencia de 5,7 y 18,6 por 100 000 personas/día (p/d) para las infecciones sintomáticas y asintomáticas, respectivamente. La tasa de incidencia de las infecciones sintomáticas observada en las personas <15 años de edad fue significativamente mayor que la encontrada en los sujetos ≥15 años (15,8 versus 2,9 por 100 000 p/d). Por otro lado, las tasas de incidencia de las infecciones asintomáticas en ambos grupos de edad fueron similares (17,3 y 18,9 por 100 000 p/d, respectivamente). Se detectaron los cuatro serotipos del DENV en tres de los barrios estudiados. Se observó que la edad y la hiperendemicidad fueron probablemente los factores que más contribuyeron a la incidencia del dengue en los cuatro barrios investigados. Seguramente, las infecciones asintomáticas contribuyeron a incrementar la transmisión viral en el área de estudio.

The incidence rates of symptomatic and asymptomatic dengue virus (DENV) infections in four "barrios" of Maracay, Venezuela, during one-year (October 2006-September 2007) were estimated. A prospective study consisting of house visits three-times a week to detect dengue cases, and semiannual serological surveys to determine probable asymptomatic dengue virus (DENV) infections was conducted. The study subjects belonged to a cohort of 2,663 people ≥5 year-old. Confirmatory diagnosis of DENV infections was carried out by reverse-transcriptase polymerase chain reaction (RT-PCR). Serological surveys were performed by anti-dengue IgM-capture immunoassay (MAC-ELISA). The results showed that the incidence rates for symptomatic and asymptomatic infections were determined to be 5.7 and 18.6 per 100,000 persons/day (p/d), respectively. The incidence rate of symptomatic infections was significantly higher in persons <15 year-old than that found in subjects ≥15 years (15.8 versus 2.9 per 100,000 p/d). On the other hand, the incidence rates of asymptomatic infections in both age groups were similar (17.3 and 18.9 per 100,000 p/d, respectively). All four DENV serotypes were detected in three of the four "barrios" studied. Finally, age and hyperendemicity were probably the contributing factors to the incidence of dengue in the four "barrios" investigated. Surely, the asymptomatic infections contributed to increase the viral transmission in the study area.

Dengue-2 infection and the induction of apoptosis in human primary monocytes

Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-á and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

Dengue virus-induced regulation of the host cell translational machinery

Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5 percent of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score ≥ ±2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.