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Sample records for dengue virus denv

  1. Dengue Virus (DENV) Neutralizing Antibody Kinetics in Children After Symptomatic Primary and Postprimary DENV Infection.

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    Clapham, Hannah E; Rodriguez-Barraquer, Isabel; Azman, Andrew S; Althouse, Benjamin M; Salje, Henrik; Gibbons, Robert V; Rothman, Alan L; Jarman, Richard G; Nisalak, Ananda; Thaisomboonsuk, Butsaya; Kalayanarooj, Siripen; Nimmannitya, Suchitra; Vaughn, David W; Green, Sharone; Yoon, In-Kyu; Cummings, Derek A T

    2016-05-01

    The immune response to dengue virus (DENV) infection is complex and not fully understood. Using longitudinal data from 181 children with dengue in Thailand who were followed for up to 3 years, we describe neutralizing antibody kinetics following symptomatic DENV infection. We observed that antibody titers varied by serotype, homotypic vs heterotypic responses, and primary versus postprimary infections. The rates of change in antibody titers over time varied between primary and postprimary responses. For primary infections, titers increased from convalescence to 6 months. By comparing homotypic and heterotypic antibody titers, we saw an increase in type specificity from convalescence to 6 months for primary DENV3 infections but not primary DENV1 infections. In postprimary cases, there was a decrease in titers from convalescence up until 6 months after infection. Beginning 1 year after both primary and postprimary infections, there was evidence of increasing antibody titers, with greater increases in children with lower titers, suggesting that antibody titers were boosted due to infection and that higher levels of neutralizing antibody may be more likely to confer a sterilizing immune response. These findings may help to model virus transmission dynamics and provide baseline data to support the development of vaccines and therapeutics.

  2. Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1

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    Messer, William B.; Yount, Boyd L.; Royal, Scott R.; de Alwis, Ruklanthi; Widman, Douglas G.; Smith, Scott A.; Crowe, James E.; Pfaff, Jennifer M.; Kahle, Kristen M.; Doranz, Benjamin J.; Ibarra, Kristie D.; Harris, Eva

    2016-01-01

    ABSTRACT The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. IMPORTANCE Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they

  3. Circulation of Dengue virus-1 (DENV-1 serotype in Delhi, during 2010–11 after Dengue virus-3 (DENV-3 predominance: A single centre hospital-based study

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    Ekta Gupta , Sweta Mohan , Meenu Bajpai , Aashish Choudhary & Gaurav Singh

    2012-06-01

    Full Text Available Background: Delhi, a city in north India, has so far witnessed several reported outbreaks of dengue. Dengue inDelhi from being epidemic is slowly changing towards being endemic and hyper-endemic. Circulating type ofthe virus is also changing over the years. In the absence of an effective vaccine, dengue prevention to a majorextent relies on virological surveillance, and development of effective, locally adapted control programmes. Inthe present study, we tried to identify the between-year non-epidemic serotype of dengue virus circulating inDelhi, during 2010–11.Methods: Acute-phase samples were collected from the patients attending the Institute of Liver & Biliary Sciences,New Delhi, India. Dengue diagnosis was done using WHO case definitions. All the samples were subjected toDengue NS1 Ag ELISA and modified nested RT-PCR.Results: A total of 75 acute-phase samples were received, of which 19 (25.3% were positive for dengue NS1antigen. Dengue RT-PCR was positive in 14.6% (11/75 samples. All the RT-PCR isolates were of DENV-1serotype. No case of concomitant infection with more than one serotype was observed. Median age of involvementwas 23 yr (range10–86. Maximum number of cases were seen in the age group of 21–30 yr. Male to female ratiowas 1.2 : 1. Maximum number of suspected dengue cases (n=79 was seen during September and October.Conclusions: DENV-1 was circulating in Delhi in the year 2010–11 in non-epidemic period following reportedpredominance of DENV-3 and co-circulation of all dengue serotypes in the epidemic years 2003, 2006 and 2007.

  4. Construction of self-replicating subgenomic dengue virus 4 (DENV4) replicon.

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    Alcaraz-Estrada, Sofia L; Del Angel, Rosa; Padmanabhan, Radhakrishnan

    2014-01-01

    Dengue virus serotypes 1-4 are members of mosquito-borne flavivirus genus of Flaviviridae family that encode one long open reading frame (ORF) that is translated to a polyprotein. Both host and virally encoded proteases function in the processing of the polyprotein by co-translational and posttranslational mechanisms to yield 10 mature proteins prior to viral RNA replication. To study cis- and trans-acting factors involved in viral RNA replication, many groups [1-8] have constructed cDNAs encoding West Nile virus (WNV), DENV, or yellow fever virus reporter replicon RNAs. The replicon plasmids constructed in our laboratory for WNV [9] and the DENV4 replicon described here are arranged in the order of 5'-untranslated region (UTR), the N-terminal coding sequence of capsid (C), Renilla luciferase (Rluc) reporter gene with a translation termination codon, and an internal ribosome entry site (IRES) element from encephalomyocarditis virus (EMCV) for cap-independent translation of the downstream ORF that codes for a polyprotein precursor, CterE-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, followed by the 3'-UTR. In the second DENV4 replicon, the Rluc gene is fused sequentially downstream to the 20 amino acid (aa) FMDV 2A protease coding sequence, neomycin resistance gene (Neo(r)), a termination codon, and the EMCV leader followed by the same polyprotein coding sequence and 3'-UTR as in the first replicon. The first replicon is useful to study by transient transfection experiments the cis-acting elements and trans-acting factors involved in viral RNA replication. The second DENV4 replicon is used to establish a stable monkey kidney (Vero) cell line by transfection of replicon RNA and selection in the presence of the G418, an analog of neomycin. This replicon is useful for screening and identifying antiviral compounds that are potential inhibitors of viral replication.

  5. Virus del dengue de serotipo 1 (DENV-1 de Colombia: su contribución a la presentación del dengue en el departamento de Santander

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    Raquel E. Ocazionez-Jiménez

    2013-08-01

    Full Text Available Introducción. Los cuatro serotipos del virus del dengue circularon en el departamento de Santander entre 1998 y 2008. No existe información sobre el papel del serotipo 1 (DENV-1 en la epidemiología de la enfermedad. Objetivo. Analizar la relación entre el cambio de predominancia del (DENV-1 con su diversificación genética, predominancia de los otros serotipos y presentación del dengue grave. Materiales y métodos. La diversificación genética se estudió por análisis filogenético usando la secuencia del gen E de 12 cepas del virus. Para el análisis se utilizaron datos sobre predominancia delos serotipos obtenidos en estudios previos y datos oficiales de incidencia del dengue. Resultados. Los virus seleccionados se agruparon en el genotipo V junto a (DENV-1 de países de Latinoamérica y se evidenció segregación en cuatro linajes. Los cambios en la predominancia del virus coincidieron con el reemplazo de linaje y esto, a su vez, con incremento en la prevalencia de DENV-2y DENV-3, e incremento del dengue grave. Conclusión. La diversificación genética podría contribuir a cambios de predominancia de (DENV-1, y la relación del virus con el DENV-2 y DENV-3 en situaciones que favorecen la presentación de casos graves. Se necesitan más estudios para precisar el papel de los serotipos en la epidemiología del dengue. doi: http://dx.doi.org/10.7705/biomedica.v33i0.717

  6. Identification of continuous human B-cell epitopes in the envelope glycoprotein of dengue virus type 3 (DENV-3.

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    Andréa N M Rangel da Silva

    Full Text Available BACKGROUND: Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. METHODOLOGY/PRINCIPAL FINDINGS: Synthetic peptides were used to identify B-cell epitopes in the envelope (E glycoprotein of dengue virus type 3 (DENV-3. Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa positions: 51-65, 71-90, 131-170, 196-210 and 246-260 were identified by employing an enzyme- linked immunosorbent assay (ELISA, using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51-65, 27 and 28 (aa131-150 also reacted with dengue 1 (DENV-1 and dengue 2 (DENV-2 patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51-65, 131-170, 196-210 and 246-260 elicited the highest antibody response and epitopes131-170, 196-210 and 246-260, elicited IFN-gamma production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. CONCLUSIONS/SIGNIFICANCE: Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine.

  7. Dengue virus surveillance: Detection of DENV-4 in the city of São José do Rio Preto, SP, Brazil.

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    Colombo, Tatiana Elias; Vedovello, Danila; Pacca-Mazaro, Carolina Colombelli; Mondini, Adriano; Araújo, João Pessoa; Cabrera, Eliana; Lopes, José Carlos; Penha Dos Santos, Izalco Nuremberg; Negri Reis, Andréia Francesli; Costa, Fabiana Rodrigues; Antônio Cruz, Lilian Elisa Arão; Ferreira, Jurandir; de Oliveira Rocha, Eliseu Soares; Kroon, Erna Geessien; de Morais Bronzoni, Roberta Vieira; Vasilakis, Nikos; Nogueira, Maurício Lacerda

    2016-12-01

    Dengue viruses are the most common arbovirus infection worldwide and are caused by four distinct serotypes of the dengue virus (DENV). In the present study, we assessed DENV transmission in São José do Rio Preto (SJRP) from 2010 to 2014. We analyzed blood samples from febrile patients who were attended at health care centers in SJRP. DENV detection was performed using multiplex RT-PCR, using flavivirus generic primers, based on the genes of the non-structural protein (NS5), followed by nested-PCR assay with species-specific primers. We analyzed 1549 samples, of which 1389 were positive for NS1 by rapid test. One thousand and eight-seven samples (78%) were confirmed as positive by multiplex RT-PCR: DENV-4, 48.5% (528/1087); DENV-1, 41.5% (449/1087); DENV-2, 9.5% (104/1087); and co-infection (5 DENV-1/DENV-4, 1 DENV-1/DENV-2), 0.5% (6/1087). Phylogenetic analysis of the DENV-4 grouped the isolates identified in this study with the American genotype and the showed a relationship between isolates from SJRP and isolates from the northern region of South America. Taken together, our data shows the detection and emergence of new dengue genotype in a new region and reiterate the importance of surveillance programs to detect and trace the evolution of DENV. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. [Co-infection by Chikungunya virus (CHIK-V) and dengue virus (DEN-V) during a recent outbreak in Cali, Colombia: Report of a fatal case].

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    Rosso, Fernando; Pacheco, Robinson; Rodríguez, Sarita; Bautista, Diego

    2016-08-01

    The recent outbreaks of Chikungunya (CHIK-V) virus in endemic areas of dengue (DEN-V) could increase the risk of co-infection. CHIK infection has been considered not severe and with very unusual mortality, however DEN is associated with severe manifestations and increased mortality. Little is known about coinfection. It is possible that co-infection could generate severe cases. We present a case report of co-infection DEN-V -3 and CHIK-V in an elderly patient who developed acute renal failure, dengue shock syndrome (DSS), progresses to multiple organ failure and died. With the recent emergence of CHIK-V in Colombia, the possibility of co-infection with DEN-V should be suspected, especially in severe cases.

  9. Electrical detection of dengue virus (DENV) DNA oligomer using silicon nanowire biosensor with novel molecular gate control.

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    Nuzaihan M N, M; Hashim, U; Md Arshad, M K; Kasjoo, S R; Rahman, S F A; Ruslinda, A R; Fathil, M F M; Adzhri, R; Shahimin, M M

    2016-09-15

    In this paper, a silicon nanowire biosensor with novel molecular gate control has been demonstrated for Deoxyribonucleic acid (DNA) detection related to dengue virus (DENV). The silicon nanowire was fabricated using the top-down nanolithography approach, through nanostructuring of silicon-on-insulator (SOI) layers achieved by combination of the electron-beam lithography (EBL), plasma dry etching and size reduction processes. The surface of the fabricated silicon nanowire was functionalized by means of a three-step procedure involving surface modification, DNA immobilization and hybridization. This procedure acts as a molecular gate control to establish the electrical detection for 27-mers base targets DENV DNA oligomer. The electrical detection is based on the changes in current, resistance and conductance of the sensor due to accumulation of negative charges added by the immobilized probe DNA and hybridized target DNA. The sensitivity of the silicon nanowire biosensors attained was 45.0µAM(-1), which shows a wide-range detection capability of the sensor with respect to DNA. The limit of detection (LOD) achieved was approximately 2.0fM. The demonstrated results show that the silicon nanowire has excellent properties for detection of DENV with outstanding repeatability and reproducibility performances.

  10. Computational prediction and analysis of envelop glycoprotein epitopes of DENV-2 and DENV-3 Pakistani isolates: a first step towards Dengue vaccine development.

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    Hafsa Amat-ur-Rasool

    Full Text Available Dengue fever of tropics is a mosquito transmitted devastating disease caused by dengue virus (DENV. There is no effective vaccine available, so far, against any of its four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4. There is a need for the development of preventive and therapeutic vaccines against DENV to decrease the prevalence of dengue fever, especially in Pakistan. In this research, linear and conformational B-cell epitopes of envelope glycoprotein of DENV-2 and DENV-3 (the most prevalent serotypes in Pakistan were predicted. We used Kolaskar and Tongaonkar method for linear epitope prediction, Emini's method for surface accessibility prediction and Karplus and Schulz's algorithm for flexibility determination. To propose three dimensional epitopes, the E proteins for both serotypes were homology modeled by using Phyre2 V 2.0 server, and ElliPro was used for the prediction of surface epitopes on their globular structure. Total 21 and 19 linear epitopes were predicted for DENV-2 and DENV-3 Pakistani isolates respectively. Whereas, 5 and 4 discontinuous epitopes were proposed for DENV-2 and DENV-3 Pakistani isolates respectively. Moreover, the values of surface accessibility, flexibility and solvent-accessibility can be helpful in analyzing vaccines against DENV-2 and DENV-3. In conclusion, the proposed continuous and discontinuous antigenic peptides can be valuable candidates for diagnostic and therapeutics of DENV.

  11. Quasispecies of dengue virus.

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    Kurosu, Takeshi

    2011-12-01

    Pathogenic viruses have RNA genomes that cause acute and chronic infections. These viruses replicate with high mutation rates and exhibit significant genetic diversity, so-called viral quasispecies. Viral quasispecies play an important role in chronic infectious diseases, but little is known about their involvement in acute infectious diseases such as dengue virus (DENV) infection. DENV, the most important human arbovirus, is a causative agent of dengue fever (DF) and dengue hemorrhagic fever (DHF). Accumulating observations suggest that DENV exists as an extremely diverse virus population, but its biological significance is unclear. In other virus diseases, quasispecies affect the therapeutic strategies using drugs and vaccines. Here, I describe the quasispecies of DENV and discuss the possible role of quasispecies in the pathogenesis of and therapeutic strategy against DENV infection in comparison with other viruses such as Hepatitis C virus, human immunodeficiency virus type 1, and poliovirus.

  12. Dengue virus vaccine development.

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    Yauch, Lauren E; Shresta, Sujan

    2014-01-01

    Dengue virus (DENV) is a significant cause of morbidity and mortality in tropical and subtropical regions, causing hundreds of millions of infections each year. Infections range from asymptomatic to a self-limited febrile illness, dengue fever (DF), to the life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The expanding of the habitat of DENV-transmitting mosquitoes has resulted in dramatic increases in the number of cases over the past 50 years, and recent outbreaks have occurred in the United States. Developing a dengue vaccine is a global health priority. DENV vaccine development is challenging due to the existence of four serotypes of the virus (DENV1-4), which a vaccine must protect against. Additionally, the adaptive immune response to DENV may be both protective and pathogenic upon subsequent infection, and the precise features of protective versus pathogenic immune responses to DENV are unknown, complicating vaccine development. Numerous vaccine candidates, including live attenuated, inactivated, recombinant subunit, DNA, and viral vectored vaccines, are in various stages of clinical development, from preclinical to phase 3. This review will discuss the adaptive immune response to DENV, dengue vaccine challenges, animal models used to test dengue vaccine candidates, and historical and current dengue vaccine approaches.

  13. First report of sylvatic DENV-2-associated dengue hemorrhagic fever in West Africa.

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    Leticia Franco

    2011-08-01

    Full Text Available Dengue virus (DENV circulates in human and sylvatic cycles. Sylvatic strains are both ecologically and evolutionarily distinct from endemic viruses. Although sylvatic dengue cycles occur in West African countries and Malaysia, only a few cases of mild human disease caused by sylvatic strains and one single case of dengue hemorrhagic fever in Malaysia have been reported. Here we report a case of dengue hemorrhagic fever (DHF with thrombocytopenia (13000/µl, a raised hematocrit (32% above baseline and mucosal bleeding in a 27-year-old male returning to Spain in November 2009 after visiting his home country Guinea Bissau. Sylvatic DENV-2 West African lineage was isolated from blood and sera. This is the first case of DHF associated with sylvatic DENV-2 in Africa and the second case worldwide of DHF caused by a sylvatic strain.

  14. First report of sylvatic DENV-2-associated dengue hemorrhagic fever in West Africa.

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    Franco, Leticia; Palacios, Gustavo; Martinez, José Antonio; Vázquez, Ana; Savji, Nazir; De Ory, Fernando; Sanchez-Seco, María Paz; Martín, Dolores; Lipkin, W Ian; Tenorio, Antonio

    2011-08-01

    Dengue virus (DENV) circulates in human and sylvatic cycles. Sylvatic strains are both ecologically and evolutionarily distinct from endemic viruses. Although sylvatic dengue cycles occur in West African countries and Malaysia, only a few cases of mild human disease caused by sylvatic strains and one single case of dengue hemorrhagic fever in Malaysia have been reported. Here we report a case of dengue hemorrhagic fever (DHF) with thrombocytopenia (13000/µl), a raised hematocrit (32% above baseline) and mucosal bleeding in a 27-year-old male returning to Spain in November 2009 after visiting his home country Guinea Bissau. Sylvatic DENV-2 West African lineage was isolated from blood and sera. This is the first case of DHF associated with sylvatic DENV-2 in Africa and the second case worldwide of DHF caused by a sylvatic strain.

  15. DENV-2NGC株诱导HUVEC凋亡及与Fas/FasL表达的关系%Apoptosis and expression of Fas/FasL on HUVEC induced by Dengue virus type 2 NGC strain

    Institute of Scientific and Technical Information of China (English)

    王念雪; 左丽

    2013-01-01

    目的:观察登革2型病毒NGC株(Dengue virus-2 NGC strain,DENV-2 NGC)诱导的HUVEC凋亡及其Fas/FasL表达的变化.方法:利用BALB/c乳鼠脑内接种以及C6/36细胞增殖病毒,RT-PCR鉴定病毒,细胞半数感染量(TCID50)测定法确定病毒滴度.直接免疫荧光法观察DENV-2吸附HUVEC;AO/EB染色法检测DENV-2感染后HUVEC细胞凋亡形态;流式细胞术动态测定不同滴度DENV-2诱导HUVEC凋亡率的变化,检测不同剂量病毒感染48小时的细胞感染情况及HUVEC膜表面Fas/FasL的表达水平.组间比较采用独立样本t检验.结果:DENV-2能够吸附于HUVEC并诱导HUVEC凋亡,凋亡率达(18.44±1.29)%,与未接种病毒组比较(5.78±1.43)%,差异有显著性(P<0.05),但凋亡率并未随着时间延长和病毒剂量增加,呈现出明显的上升或下降的趋势.DENV-2对HUVEC的感染率并未与细胞凋亡率呈现明显的相关性.与对照组相比,接种病毒组的HUVEC膜表面Fas的表达水平均有明显的增加,但FasL表达水平仅在接种病毒的1 000TCID50组显著增加.结论:DENV-2 NGC能够感染HUVECs并诱导其凋亡,可能是DHF/DSS发病机制中血管内皮屏障损伤导致血浆外渗的主要原因之一,Fas/FasL激活的凋亡死亡受体途径可能在DENV-2 NGC诱导的HUVECs凋亡中发挥了作用.

  16. Investigating the efficacy of monovalent and tetravalent dengue vaccine formulations against DENV-4 challenge in AG129 mice.

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    Fuchs, Jeremy; Chu, Haiyan; O'Day, Peter; Pyles, Richard; Bourne, Nigel; Das, Subash C; Milligan, Gregg N; Barrett, Alan D T; Partidos, Charalambos D; Osorio, Jorge E

    2014-11-12

    Dengue (DEN) is the most important mosquito-borne viral disease, with a major impact on global health and economics, caused by four serologically and distinct viruses termed DENV-1 to DENV-4. Currently, there is no licensed vaccine to prevent DEN. We have developed a live attenuated tetravalent DENV vaccine candidate (TDV) (formally known as DENVax) that has shown promise in preclinical and clinical studies and elicits neutralizing antibody responses to all four DENVs. As these responses are lowest to DENV-4 we have used the AG129 mouse model to investigate the immunogenicity of monovalent TDV-4 or tetravalent TDV vaccines, and their efficacy against lethal DENV-4 challenge. Since the common backbone of TDV is based on an attenuated DENV-2 strain (TDV-2) we also tested the efficacy of TDV-2 against DENV-4 challenge. Single doses of the tetravalent or monovalent vaccines elicited neutralizing antibodies, anti-NS1 antibodies, and cellular responses to both envelope and nonstructural proteins. All vaccinated animals were protected against challenge at 60 days post-immunization, whereas all control animals died. Investigation of DENV-4 viremias post-challenge showed that only the control animals had high viremias on day 3 post-challenge, whereas vaccinated mice had no detectable viremia. Overall, these data highlight the excellent immunogenicity and efficacy profile of our candidate dengue vaccine in AG129 mice.

  17. Identifizierung neuer Dengue Virus Typ-2 Proteaseinhibitoren

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    Snitko, Mariya

    2015-01-01

    Weltweit leben ca. 2,5 Mrd. Menschen im Dengue Virus Verbreitungsgebiet. Dengue Virus Infektionen führen zum Dengue Fieber und können bei Re-Infektionen mit anderen Serotypen das sog. Dengue Schocksyndrom mit einer Letalität von 10% verursachen. Momentan stehen jedoch weder Impfstoffe noch antivirale Substanzen zur Verfügung. In der vorliegenden Arbeit sollten DENV2-Proteaseinhibitoren entwickelt werden. Dazu wurde ein in vitro DENV Proteasetest etabliert, für den die DENV Protease i...

  18. A recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype 2 backbone.

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    Osorio, Jorge E; Wallace, Derek; Stinchcomb, Dan T

    2016-01-01

    Dengue fever is caused by infection with one of four dengue virus (DENV) serotypes (DENV-1-4), necessitating tetravalent dengue vaccines that can induce protection against all four DENV. Takeda's live attenuated tetravalent dengue vaccine candidate (TDV) comprises an attenuated DENV-2 strain plus chimeric viruses containing the prM and E genes of DENV-1, -3 and -4 cloned into the attenuated DENV-2 'backbone'. In Phase 1 and 2 studies, TDV was well tolerated by children and adults aged 1.5-45 years, irrespective of prior dengue exposure; mild injection-site symptoms were the most common adverse events. TDV induced neutralizing antibody responses and seroconversion to all four DENV as well as cross-reactive T cell-mediated responses that may be necessary for broad protection against dengue fever.

  19. Dengue viruses cluster antigenically but not as discrete serotypes

    NARCIS (Netherlands)

    L. Katzelnick (Leah); J.M. Fonville (Judith); G.D. Gromowski (Gregory D.); J.B. Arriaga (Jose Bustos); A. Green (Angela); S.L. James (Sarah ); L. Lau (Louis); M. Montoya (Magelda); C. Wang (Chunling); L.A. Van Blargan (Laura A.); C.A. Russell (Colin); H.M. Thu (Hlaing Myat); T.C. Pierson (Theodore C.); P. Buchy (Philippe); J.G. Aaskov (John G.); J.L. Muñoz-Jordán (Jorge L.); N. Vasilakis (Nikos); R.V. Gibbons (Robert V.); R.B. Tesh (Robert B.); A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); A. Durbin (Anna); C.P. Simmons (Cameron P.); E.C. Holmes (Edward C.); E. Harris (Eva); S.S. Whitehead (Stephen S.); D.J. Smith (Derek James)

    2015-01-01

    textabstractThe four genetically divergent dengue virus (DENV) types are traditionally classified as serotypes. Antigenic and genetic differences among the DENV types influence disease outcome, vaccine-induced protection, epidemic magnitude, and viral evolution.We scharacterized antigenic diversity

  20. Dengue virus-like particles: construction and application.

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    Shang, Weilong; Liu, Jie; Yang, Jie; Hu, Zhen; Rao, Xiancai

    2012-04-01

    Virus-like particles (VLPs) are shell-like viruses that lack virus-specific genetic materials. Many viral-structured proteins can assemble into VLPs, which mimic the overall structure of virus particles and can elicit strong immune responses in a host. Dengue viruses (DENVs), from the genus Flavivirus, are transmitted to humans through the bites of an infected Aedes mosquito. DENVs cause several diseases that prevailed mainly in tropical and subtropical areas. However, effective treatment measures and preventive strategies for dengue diseases are still lacking. The present minireview summarized the assembly and maturation of DENVs, the strategies and effective factors for dengue VLP construction, and the application of DENV VLPs.

  1. Altered immune response of immature dendritic cells upon dengue virus infection in the presence of specific antibodies

    NARCIS (Netherlands)

    Torres, Silvia; Flipse, Jacky; Upasani, Vinit C; van der Ende-Metselaar, Heidi; Urcuqui-Inchima, Silvio; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A

    2016-01-01

    Dengue virus (DENV) replication is known to prevent maturation of infected DCs thereby impeding the development of adequate immunity. During secondary DENV infection, dengue-specific antibodies can suppress DENV replication in immature DCs (immDCs), however how dengue-antibody complexes (DENV-IC) in

  2. Human genetic determinants of dengue virus susceptibility.

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    Coffey, Lark L; Mertens, Eva; Brehin, Anne-Claire; Fernandez-Garcia, Maria Dolores; Amara, Ali; Després, Philippe; Sakuntabhai, Anavaj

    2009-02-01

    Dengue virus (DENV) is an emerging mosquito-borne pathogen that produces significant morbidity worldwide resulting in an estimated 50-100 million infections annually. DENV causes a spectrum of illness ranging from inapparent infection to life-threatening hemorrhagic fever and shock. The varied DENV disease outcome is determined by complex interactions between immunopathologic, viral, and human genetic factors. This review summarizes these interactions with a focus on human genetic determinants of DENV susceptibility, including human leukocyte antigens, blood type, and single nucleotide polymorphisms in immune response genes that have been associated with DENV disease. We also discuss other factors related to DENV outcome including viral genetic determinants, age, ethnicity, and nutritional status as they relate to DENV susceptibility. We emphasize the need for functional genetics studies to complement association-based data and we call for controlled study designs and standard clinical DENV disease definitions that will strengthen conclusions based on human genetic DENV studies.

  3. Dengue Virus Seroconversion in Travelers to Dengue-Endemic Areas.

    Science.gov (United States)

    Olivero, Rosemary M; Hamer, Davidson H; MacLeod, William B; Benoit, Christine M; Sanchez-Vegas, Carolina; Jentes, Emily S; Chen, Lin H; Wilson, Mary E; Marano, Nina; Yanni, Emad A; Ooi, Winnie W; Karchmer, Adolf W; Kogelman, Laura; Barnett, Elizabeth D

    2016-11-02

    We conducted a prospective study to measure dengue virus (DENV) antibody seroconversion in travelers to dengue-endemic areas. Travelers seen in the Boston Area Travel Medicine Network planning to visit dengue-endemic countries for ≥ 2 weeks were enrolled from 2009 to 2010. Pre- and post-travel blood samples and questionnaires were collected. Post-travel sera were tested for anti-DENV IgG by indirect IgG enzyme-linked immunosorbent assay (ELISA) and anti-DENV IgM by capture IgM ELISA. Participants with positive post-travel anti-DENV IgG or IgM were tested for pre-travel anti-DENV IgG and IgM; they were excluded from the seroconversion calculation if either pre-travel anti-DENV IgG or IgM were positive. Paired sera and questionnaires were collected for 62% (589/955) of enrolled travelers. Most participants were 19-64 years of age, female, and white. The most common purposes of travel were tourism and visiting friends and relatives; most trips were to Asia or Africa. Median length of travel was 21 days. DENV antibody seroconversion by either anti-DENV IgM or IgG ELISA was 2.9-6.8%; lower range percent excluded potential false-positive anti-DENV IgG due to receipt of yellow fever or Japanese encephalitis vaccines at enrollment; upper range percent excluded proven false-positive anti-DENV IgM. Eighteen percent of those with seroconversion reported dengue-like symptoms. Seroconversion was documented for travel to Africa as well as countries and regions known to be highly dengue endemic (India, Brazil, southeast Asia). Given widespread risk of dengue, travel medicine counseling should include information on risk of dengue in endemic areas and advice on preventing insect bites and seeking prompt medical attention for febrile illness.

  4. Superinfection interference between dengue-2 and dengue-4 viruses in Aedes aegypti mosquitoes.

    Science.gov (United States)

    Muturi, Ephantus J; Buckner, Eva; Bara, Jeffrey

    2017-04-01

    Dengue virus consists of four antigenically distinct serotypes (DENV 1-4) that are transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes. In many dengue-endemic regions, co-circulation of two or more DENV serotypes is fairly common increasing the likelihood for exposure of the two vectors to multiple serotypes. We used a model system of DENV-2 and DENV-4 to investigate how prior exposure of Aedes aegypti to one DENV serotype affects its susceptibility to another serotype. Aedes aegypti mosquitoes were sequentially infected with DENV-2 and DENV-4 and the infection and dissemination rates for each virus determined. We found that prior infection of Ae. aegypti mosquitoes with DENV-4 rendered them significantly less susceptible to secondary infection with DENV-2. Although the results were not statistically significant, mosquitoes infected with DENV-2 were also less susceptible to secondary infection with DENV-4. The midgut dissemination and population dissemination rates for DENV-2 were significantly higher than those of DENV-4 when either virus was administered 7 days after administration of either a non-infectious blood meal or a blood meal containing a heterologous dengue serotype. These results demonstrate that superinfection interference between DENV serotypes is possible within Ae. aegypti mosquitoes, but its effect on DENV epidemiology may be dependent on the fitness of interacting serotypes. © 2017 John Wiley & Sons Ltd.

  5. Imunocompetent Mice Model for Dengue Virus Infection

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    Denise Gonçalves

    2012-01-01

    Full Text Available Dengue fever is a noncontagious infectious disease caused by dengue virus (DENV. DENV belongs to the family Flaviviridae, genus Flavivirus, and is classified into four antigenically distinct serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The number of nations and people affected has increased steadily and today is considered the most widely spread arbovirus (arthropod-borne viral disease in the world. The absence of an appropriate animal model for studying the disease has hindered the understanding of dengue pathogenesis. In our study, we have found that immunocompetent C57BL/6 mice infected intraperitoneally with DENV-1 presented some signs of dengue disease such as thrombocytopenia, spleen hemorrhage, liver damage, and increase in production of IFNγ and TNFα cytokines. Moreover, the animals became viremic and the virus was detected in several organs by real-time RT-PCR. Thus, this animal model could be used to study mechanism of dengue virus infection, to test antiviral drugs, as well as to evaluate candidate vaccines.

  6. Recombinant Production of the Amino Terminal Cytoplasmic Region of Dengue Virus Non-Structural Protein 4A for Structural Studies

    OpenAIRE

    Yu-Fu Hung; Olga Valdau; Sven Schünke; Omer Stern; Koenig, Bernd W.; Dieter Willbold; Silke Hoffmann

    2014-01-01

    BACKGROUND: Dengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. There is no specific treatment and no anti-DENV vaccines. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A)...

  7. Dengue virus life cycle : viral and host factors modulating infectivity

    NARCIS (Netherlands)

    Rodenhuis-Zybert, Izabela A.; Wilschut, Jan; Smit, Jolanda M.

    2010-01-01

    Dengue virus (DENV 1-4) represents a major emerging arthropod-borne pathogen. All four DENV serotypes are prevalent in the (sub) tropical regions of the world and infect 50-100 million individuals annually. Whereas the majority of DENV infections proceed asymptomatically or result in self-limited de

  8. Dengue virus life cycle : viral and host factors modulating infectivity

    NARCIS (Netherlands)

    Rodenhuis-Zybert, Izabela A.; Wilschut, Jan; Smit, Jolanda M.

    Dengue virus (DENV 1-4) represents a major emerging arthropod-borne pathogen. All four DENV serotypes are prevalent in the (sub) tropical regions of the world and infect 50-100 million individuals annually. Whereas the majority of DENV infections proceed asymptomatically or result in self-limited

  9. Dengue virus antibodies enhance Zika virus infection

    Science.gov (United States)

    Paul, Lauren M; Carlin, Eric R; Jenkins, Meagan M; Tan, Amanda L; Barcellona, Carolyn M; Nicholson, Cindo O; Michael, Scott F; Isern, Sharon

    2016-01-01

    For decades, human infections with Zika virus (ZIKV), a mosquito-transmitted flavivirus, were sporadic, associated with mild disease, and went underreported since symptoms were similar to other acute febrile diseases. Recent reports of severe disease associated with ZIKV have greatly heightened awareness. It is anticipated that ZIKV will continue to spread in the Americas and globally where competent Aedes mosquito vectors are found. Dengue virus (DENV), the most common mosquito-transmitted human flavivirus, is both well-established and the source of outbreaks in areas of recent ZIKV introduction. DENV and ZIKV are closely related, resulting in substantial antigenic overlap. Through antibody-dependent enhancement (ADE), anti-DENV antibodies can enhance the infectivity of DENV for certain classes of immune cells, causing increased viral production that correlates with severe disease outcomes. Similarly, ZIKV has been shown to undergo ADE in response to antibodies generated by other flaviviruses. We tested the neutralizing and enhancing potential of well-characterized broadly neutralizing human anti-DENV monoclonal antibodies (HMAbs) and human DENV immune sera against ZIKV using neutralization and ADE assays. We show that anti-DENV HMAbs, cross-react, do not neutralize, and greatly enhance ZIKV infection in vitro. DENV immune sera had varying degrees of neutralization against ZIKV and similarly enhanced ZIKV infection. Our results suggest that pre-existing DENV immunity may enhance ZIKV infection in vivo and may lead to increased disease severity. Understanding the interplay between ZIKV and DENV will be critical in informing public health responses and will be particularly valuable for ZIKV and DENV vaccine design and implementation strategies. PMID:28090318

  10. Characterization of the 2013 dengue epidemic in Myanmar with dengue virus 1 as the dominant serotype.

    Science.gov (United States)

    Ngwe Tun, Mya Myat; Kyaw, Aung Kyaw; Makki, Nader; Muthugala, Rohitha; Nabeshima, Takeshi; Inoue, Shingo; Hayasaka, Daisuke; Moi, Meng Ling; Buerano, Corazon C; Thwe, Saw Myat; Thant, Kyaw Zin; Morita, Kouichi

    2016-09-01

    In 2013 in Myanmar, dengue epidemic occurred with 20,255 cases including 84 deaths. This study aimed to determine the serological and molecular characteristics of dengue virus (DENV) infection among children with clinical diagnosis of dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) during this period. Single acute serum samples were collected from 300 children in Mandalay Children Hospital, Mandalay, Myanmar. Out of the 300 children, 175 (58.3%) and 183 (61%) were positive for anti-dengue IgM and anti-dengue IgG, respectively. Among the IgM positives, 41 (23.4%) had primary DENV infection. Thirty-nine DENV strains (23 DENV-1, 10 DENV-2 and 6 DENV-4) were successfully isolated after inoculation of the patient serum samples onto C6/36 cells. DENV 1 was the dominant serotype in the 2013 epidemic. There was no correlation between the infecting serotypes and clinical severities. The DENV-1 strains belonged to three lineages of the genotype 1; the DENV-2 strains were of the Asian I genotype and were separated into two lineages; and DENV-4 strains belonged to the same lineage of genotype I. It is of interest to note the diversity of DENV-1 and -2 circulating in the same location during June-August 2013. These DENV isolates were genetically close (98%-100%) to the other previously reported isolates from Myanmar and its neighboring countries, namely China, Thailand, Sri Lanka, Cambodia and Vietnam. Primary DENV infection was still high among the severe dengue cases. Different serotypes of DENV were co-circulating in 2013, however, genotype shift was not observed. Additionally, amino acid mutations were detected in the study strains not seen in the previously reported strains from other countries and Myanmar. This paper provided information on the circulating serotypes for the last 15years and the recent dengue situation in Mandalay, Myanmar after 2006. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Characterization of Dengue Virus Infections Among Febrile Children Clinically Diagnosed With a Non-Dengue Illness, Managua, Nicaragua.

    Science.gov (United States)

    Waggoner, Jesse J; Gresh, Lionel; Mohamed-Hadley, Alisha; Balmaseda, Angel; Soda, K James; Abeynayake, Janaki; Sahoo, Malaya K; Liu, Yuanyuan; Kuan, Guillermina; Harris, Eva; Pinsky, Benjamin A

    2017-06-15

    We sought to characterize dengue virus (DENV) infections among febrile children enrolled in a pediatric cohort study who were clinically diagnosed with a non-dengue illness ("C cases"). DENV infections were detected and viral load quantitated by real-time reverse transcription-polymerase chain reaction in C cases presenting between January 2007 and January 2013. One hundred forty-one of 2892 C cases (4.88%) tested positive for DENV. Of all febrile cases in the study, DENV-positive C cases accounted for an estimated 52.0% of patients with DENV viremia at presentation. Compared with previously detected, symptomatic dengue cases, DENV-positive C cases were significantly less likely to develop long-lasting humoral immune responses to DENV, as measured in healthy annual serum samples (79.7% vs 47.8%; P dengue. These findings have important implications for DENV transmission modeling, immunology, and epidemiologic surveillance.

  12. How antibodies alter the cell entry pathway of dengue virus particles in macrophages

    NARCIS (Netherlands)

    Ayala-Nunez, Nilda V.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Sjollema, Klaas A.; Flipse, Jacky; van der Schaar, Hilde M.; Smit, Jolanda M.

    2016-01-01

    Antibody-dependent enhancement of dengue virus (DENV) infection plays an important role in the exacerbation of DENV-induced disease. To understand how antibodies influence the fate of DENV particles, we explored the cell entry pathway of DENV in the absence and presence of antibodies in macrophage-l

  13. Dengue virus serotype in Aceh Province

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    Paisal

    2015-06-01

    Full Text Available WHO estimated 50 million dengue infections happen every year in the world. In Indonesia, there were 90,245 DHF cases on 2012 with 816 deaths. In the Province of Aceh, 2,269 cases happened in the same year. This study aimed to identify dengue virus serotype in Aceh. Sampling was done in Kota Banda Aceh Hospital, Kota Lhokseumawe Hospital, Kabupaten Aceh Tamiang Hospital, Kabupaten Aceh Barat Hospital, and Kabupaten Simeulue Hospital between May to December 2012. This was a clinical laboratory research with observation design using cross sectional approach. Research’s population was sample from patients with dengue clinical symptom. Using purposive sampling technique, we have collected 100 samples from the five hospitals (20 samples from each hospital. From RT-PCR, we found 16 positive samples (9 samples were DENV-4, 3 samples were DENV-1, 2 samples were DENV-2, and 2 samples were DENV-3.

  14. Dengue virus serotype 2 from a sylvatic lineage isolated from a patient with dengue hemorrhagic fever.

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    Jane Cardosa

    Full Text Available Dengue viruses circulate in both human and sylvatic cycles. Although dengue viruses (DENV infecting humans can cause major epidemics and severe disease, relatively little is known about the epidemiology and etiology of sylvatic dengue viruses. A 20-year-old male developed dengue hemorrhagic fever (DHF with thrombocytopenia (12,000/ul and a raised hematocrit (29.5% above baseline in January 2008 in Malaysia. Dengue virus serotype 2 was isolated from his blood on day 4 of fever. A phylogenetic analysis of the complete genome sequence revealed that this virus was a member of a sylvatic lineage of DENV-2 and most closely related to a virus isolated from a sentinel monkey in Malaysia in 1970. This is the first identification of a sylvatic DENV circulating in Asia since 1975.

  15. Immune Activation in the Pathogenesis of Dengue Virus Infection

    NARCIS (Netherlands)

    C.A.M. van de Weg (Cornelia A.M.)

    2014-01-01

    markdownabstract__Abstract__ Dengue virus (DENV) is a positive-stranded RNA virus and belongs to the Flaviviridae family. The virus is transmitted by the bite of an infected Aedes-mosquito and circulates in tropical and subtropical areas around the world. The incidence of dengue has risen dramatica

  16. Dengue viruses – an overview

    Directory of Open Access Journals (Sweden)

    Anne Tuiskunen Bäck

    2013-08-01

    Full Text Available Dengue viruses (DENVs cause the most common arthropod-borne viral disease in man with 50–100 million infections per year. Because of the lack of a vaccine and antiviral drugs, the sole measure of control is limiting the Aedes mosquito vectors. DENV infection can be asymptomatic or a self-limited, acute febrile disease ranging in severity. The classical form of dengue fever (DF is characterized by high fever, headache, stomach ache, rash, myalgia, and arthralgia. Severe dengue, dengue hemorrhagic fever (DHF, and dengue shock syndrome (DSS are accompanied by thrombocytopenia, vascular leakage, and hypotension. DSS, which can be fatal, is characterized by systemic shock. Despite intensive research, the underlying mechanisms causing severe dengue is still not well understood partly due to the lack of appropriate animal models of infection and disease. However, even though it is clear that both viral and host factors play important roles in the course of infection, a fundamental knowledge gap still remains to be filled regarding host cell tropism, crucial host immune response mechanisms, and viral markers for virulence.

  17. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

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    Milly M Choy

    2015-11-01

    Full Text Available The mosquito-borne dengue virus (DENV is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.

  18. Molecular characterization of dengue and chikungunya virus strains circulating in New Delhi, India.

    Science.gov (United States)

    Afreen, Nazia; Deeba, Farah; Khan, Wajihul H; Haider, Shakir H; Kazim, Syed Naqui; Ishrat, Romana; Naqvi, Irshad Hussain; Shareef, Mohammad Y; Broor, Shobha; Ahmed, Anwar; Parveen, Shama

    2014-12-01

    Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. Both diseases are transmitted by common mosquito vectors resulting in their co-circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were performed on 87 acute phase blood samples collected from patients with suspected dengue/chikungunya infections in Delhi from September to December, 2011. RT-PCR and IgM ELISA were performed to detect dengue virus (DENV) and chikungunya virus (CHIKV). NS1 and IgG ELISA were also performed to detect DENV specific antigen and secondary DENV infection. DENV infection was detected in 49%, CHIKV infection in 29% and co-infection with DENV and CHIKV in 10% of the samples by RT-PCR. DENV serotypes 1, 2 and 3 were detected in this study. Nine DENV-1 strains, six DENV-2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of their respective envelope protein genes. DENV-1 strains grouped in the American African genotype, DENV-2 strains in the Cosmopolitan genotype and CHIKV strains in the East Central South African genotype by phylogenetic analysis. This is one of the few studies reporting the phylogeny of two dengue virus serotypes (DENV-1 and DENV-2) and CHIKV. Surveillance and monitoring of DENV and CHIKV strains are important for design of strategies to control impending epidemics.

  19. The Interplay of Dengue Virus Morphological Diversity and Human Antibodies.

    Science.gov (United States)

    Lok, Shee-Mei

    2016-04-01

    Dengue virus (DENV) infects ∼400 million people annually, and there is no available vaccine or therapeutics. It is not clear why candidate vaccines provide only modest protection. In addition to the presence of four different dengue serotypes, there is also structural heterogeneity in DENV infectious particles, even within a strain. This severely complicates the development of vaccines and therapeutics. The currently known different morphologies of DENV are: immature, partially mature, compact mature, and expanded mature forms of the virus. In this review I describe these forms of the virus, their infectivity, and how antibodies could recognize these morphologies. I also discuss possible vaccine and antibody therapeutic formulations to protect against all morphologies.

  20. Evaluation of dengue virus strains for human challenge studies.

    Science.gov (United States)

    Mammen, M P; Lyons, A; Innis, B L; Sun, W; McKinney, D; Chung, R C Y; Eckels, K H; Putnak, R; Kanesa-thasan, N; Scherer, J M; Statler, J; Asher, L V; Thomas, S J; Vaughn, D W

    2014-03-14

    Discordance between the measured levels of dengue virus neutralizing antibody and clinical outcomes in the first-ever efficacy study of a dengue tetravalent vaccine (Lancet, Nov 2012) suggests a need to re-evaluate the process of pre-screening dengue vaccine candidates to better predict clinical benefit prior to large-scale vaccine trials. In the absence of a reliable animal model and established correlates of protection for dengue, a human dengue virus challenge model may provide an approach to down-select vaccine candidates based on their ability to reduce risk of illness following dengue virus challenge. We report here the challenge of flavivirus-naïve adults with cell culture-passaged dengue viruses (DENV) in a controlled setting that resulted in uncomplicated dengue fever (DF). This sets the stage for proof-of-concept efficacy studies that allow the evaluation of dengue vaccine candidates in healthy adult volunteers using qualified DENV challenge strains well before they reach field efficacy trials involving children. Fifteen flavivirus-naïve adult volunteers received 1 of 7 DENV challenge strains (n=12) or placebo (n=3). Of the twelve volunteers who received challenge strains, five (two DENV-1 45AZ5 and three DENV-3 CH53489 cl24/28 recipients) developed DF, prospectively defined as ≥2 typical symptoms, ≥48h of sustained fever (>100.4°F) and concurrent viremia. Based on our study and historical data, we conclude that the DENV-1 and DENV-3 strains can be advanced as human challenge strains. Both of the DENV-2 strains and one DENV-4 strain failed to meet the protocol case definition of DF. The other two DENV-4 strains require additional testing as the illness approximated but did not satisfy the case definition of DF. Three volunteers exhibited effusions (1 pleural/ascites, 2 pericardial) and 1 volunteer exhibited features of dengue (rash, lymphadenopathy, neutropenia and thrombocytopenia), though in the absence of fever and symptoms. The occurrence of

  1. Factors contributing to the disturbance of coagulation and fibrinolysis in dengue virus infection

    Directory of Open Access Journals (Sweden)

    Yung-Chun Chuang

    2013-01-01

    Full Text Available Hemorrhage is one of the hallmarks of dengue hemorrhagic fever. However, the mechanisms that cause hemorrhage are unclear. In this review we focus on the possible factors that may be involved in the disturbance of coagulation and fibrinolysis during dengue virus (DENV infection. Factors such as autoantibodies and cytokines induced by DENV infection as well as hemostatic molecules expressed on DENV-infected cells, and DENV viral proteins may all contribute to the defect of hemostasis during DENV infection. It is the combination of these viral and host factors that may tilt the balance of coagulation and fibrinolysis toward bleeding in dengue patients.

  2. Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.

    Science.gov (United States)

    Mattia, Kimberly; Puffer, Bridget A; Williams, Katherine L; Gonzalez, Ritela; Murray, Meredith; Sluzas, Emily; Pagano, Dan; Ajith, Sandya; Bower, Megan; Berdougo, Eli; Harris, Eva; Doranz, Benjamin J

    2011-01-01

    The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.

  3. Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.

    Directory of Open Access Journals (Sweden)

    Kimberly Mattia

    Full Text Available The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.

  4. Mouse models of dengue virus infection for vaccine testing.

    Science.gov (United States)

    Sarathy, Vanessa V; Milligan, Gregg N; Bourne, Nigel; Barrett, Alan D T

    2015-12-10

    Dengue is a mosquito-borne disease caused by four serologically and genetically related viruses termed DENV-1 to DENV-4. With an annual global burden of approximately 390 million infections occurring in the tropics and subtropics worldwide, an effective vaccine to combat dengue is urgently needed. Historically, a major impediment to dengue research has been development of a suitable small animal infection model that mimics the features of human illness in the absence of neurologic disease that was the hallmark of earlier mouse models. Recent advances in immunocompromised murine infection models have resulted in development of lethal DENV-2, DENV-3 and DENV-4 models in AG129 mice that are deficient in both the interferon-α/β receptor (IFN-α/β R) and the interferon-γ receptor (IFN-γR). These models mimic many hallmark features of dengue disease in humans, such as viremia, thrombocytopenia, vascular leakage, and cytokine storm. Importantly AG129 mice develop lethal, acute, disseminated infection with systemic viral loads, which is characteristic of typical dengue illness. Infected AG129 mice generate an antibody response to DENV, and antibody-dependent enhancement (ADE) models have been established by both passive and maternal transfer of DENV-immune sera. Several steps have been taken to refine DENV mouse models. Viruses generated by peripheral in vivo passages incur substitutions that provide a virulent phenotype using smaller inocula. Because IFN signaling has a major role in immunity to DENV, mice that generate a cellular immune response are desired, but striking the balance between susceptibility to DENV and intact immunity is complicated. Great strides have been made using single-deficient IFN-α/βR mice for DENV-2 infection, and conditional knockdowns may offer additional approaches to provide a panoramic view that includes viral virulence and host immunity. Ultimately, the DENV AG129 mouse models result in reproducible lethality and offer multiple

  5. A Protective Role for Dengue Virus-Specific CD8+ T Cells

    OpenAIRE

    Yauch, Lauren E.; Zellweger, Raphaël M.; Kotturi, Maya F.; Qutubuddin, Afrina; Sidney, John; Peters, Bjoern; Prestwood, Tyler R.; Sette, Alessandro; Shresta, Sujan

    2009-01-01

    Infection with one of the four serotypes of dengue virus (DENV1-4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8+ T cells in ...

  6. n Silico Analysis of Envelope Dengue Virus-2 and Envelope Dengue Virus-3 Protein as the Backbone of Dengue Virus Tetravalent Vaccine by Using Homology Modeling Method

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    Rizky I. Taufik

    2009-01-01

    Full Text Available Problem statement: Dengue fever, which was caused by Dengue virus infection, had became a major public health problem in the tropic and subtropical countries. Dengue virus (DENV had four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4, based on their immunogenic in the human body. Preventive measure will be necessary to decrease the prevalence of dengue fever, by developing modern vaccine. Approach: This research was focused on in silico study of dengue virus vaccines, by using envelope (E protein of DENV-2 and DENV-3 as their backbones. T cell epitope prediction was determined by using MULTIPRED server and B cell epitope prediction was determined by using Conformational Epitope Prediction server (CEP. Homology modeling study of E DENV-3 protein as the vaccine backbone had produced six dengue vaccine peptides (HMM Vaccine 1-6. Moreover, homology modeling study of E DENV-2 protein as vaccine backbone had produced six dengue vaccine peptides (ANN vaccine 1-6. Results: The BLAST analysis of HMM and ANN vaccines had produced 93% and 91% identity, respectively. The Ramachandran Plot of both vaccines had shown less than 15% non glycine residue in the disallowed region, therefore it showed the solid stability of the proteins. The VAST analysis of E DENV-3 backbone vaccines had determined, that HMM4 and HMM6 had the highest structure similarity with native E DENV-3. HMM4 and HMM6 had the highest VAST score of 64.5. Moreover, the VAST analysis of E DENV-2 backbone vaccines had determined, that ANN1, ANN3, ANN4, ANN5 and ANN6 had the highest structure similarity with native E DENV-2. ANN1, ANN3, ANN4, ANN5 and ANN6 have the highest VAST score of 64.7. Conclusion/Recommendation: It could be inferred from this research that HMM4; HMM6; ANN1; ANN3; ANN4; ANN5; and ANN6 were the best in silico vaccine design, based on their similarity with native E DENV Proteins. This research could be applied for the wet

  7. Aedes aegypti D7 Saliva Protein Inhibits Dengue Virus Infection

    Science.gov (United States)

    Conway, Michael J.; Londono-Renteria, Berlin; Troupin, Andrea; Watson, Alan M.; Klimstra, William B.; Fikrig, Erol; Colpitts, Tonya M.

    2016-01-01

    Aedes aegypti is the primary vector of several medically relevant arboviruses including dengue virus (DENV) types 1–4. Ae. aegypti transmits DENV by inoculating virus-infected saliva into host skin during probing and feeding. Ae. aegypti saliva contains over one hundred unique proteins and these proteins have diverse functions, including facilitating blood feeding. Previously, we showed that Ae. aegypti salivary gland extracts (SGEs) enhanced dissemination of DENV to draining lymph nodes. In contrast, HPLC-fractionation revealed that some SGE components inhibited infection. Here, we show that D7 proteins are enriched in HPLC fractions that are inhibitory to DENV infection, and that recombinant D7 protein can inhibit DENV infection in vitro and in vivo. Further, binding assays indicate that D7 protein can directly interact with DENV virions and recombinant DENV envelope protein. These data reveal a novel role for D7 proteins, which inhibits arbovirus transmission to vertebrates through a direct interaction with virions. PMID:27632170

  8. Aedes aegypti D7 Saliva Protein Inhibits Dengue Virus Infection.

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    Michael J Conway

    2016-09-01

    Full Text Available Aedes aegypti is the primary vector of several medically relevant arboviruses including dengue virus (DENV types 1-4. Ae. aegypti transmits DENV by inoculating virus-infected saliva into host skin during probing and feeding. Ae. aegypti saliva contains over one hundred unique proteins and these proteins have diverse functions, including facilitating blood feeding. Previously, we showed that Ae. aegypti salivary gland extracts (SGEs enhanced dissemination of DENV to draining lymph nodes. In contrast, HPLC-fractionation revealed that some SGE components inhibited infection. Here, we show that D7 proteins are enriched in HPLC fractions that are inhibitory to DENV infection, and that recombinant D7 protein can inhibit DENV infection in vitro and in vivo. Further, binding assays indicate that D7 protein can directly interact with DENV virions and recombinant DENV envelope protein. These data reveal a novel role for D7 proteins, which inhibits arbovirus transmission to vertebrates through a direct interaction with virions.

  9. Characterization of an efficient dengue virus replicon for development of assays of discovery of small molecules against dengue virus.

    Science.gov (United States)

    Yang, Chi-Chen; Tsai, Ming-Han; Hu, Han-Shu; Pu, Szu-Yuan; Wu, Ren-Huang; Wu, Szu-Huei; Lin, Hui-Mei; Song, Jen-Shin; Chao, Yu-Sheng; Yueh, Andrew

    2013-05-01

    Dengue virus (DENV) is a public health threat to approximately 40% of the global population. At present, neither licensed vaccines nor effective therapies exist, and the mechanism of viral RNA replication is not well understood. Here, we report the development of efficient Renilla luciferase reporter-based DENV replicons that contain the full-length capsid sequence for transient and stable DENV RNA replication. A comparison of the transient and stable expression of this RNA-launched replicon to replicons containing various deletions revealed dengue replicon containing entire mature capsid RNA element has higher replicon activity. An efficient DNA-launched DENV replicon, pCMV-DV2Rep, containing a full-length capsid sequence, was created and successfully applied to evaluate the potency of known DENV inhibitors. Stable cell lines harboring the DENV replicon were easily established by transfecting pCMV-DV2Rep into BHK21 cells. Steady and high replicon reporter signals were observed in the stable DENV replicon cells, even after 30 passages. The stable DENV replicon cells were successfully used to determine the potency of known DENV inhibitors. A high-throughput screening assay based on stable DENV replicon cells was evaluated and shown to have an excellent Z' factor of 0.74. Altogether, the development of our efficient DENV replicon system will facilitate the study of virus replication and the discovery of antiviral compounds.

  10. Lack of Durable Cross-Neutralizing Antibodies Against Zika Virus from Dengue Virus Infection.

    Science.gov (United States)

    Collins, Matthew H; McGowan, Eileen; Jadi, Ramesh; Young, Ellen; Lopez, Cesar A; Baric, Ralph S; Lazear, Helen M; de Silva, Aravinda M

    2017-05-01

    Cross-reactive antibodies elicited by dengue virus (DENV) infection might affect Zika virus infection and confound serologic tests. Recent data demonstrate neutralization of Zika virus by monoclonal antibodies or human serum collected early after DENV infection. Whether this finding is true in late DENV convalescence (>6 months after infection) is unknown. We studied late convalescent serum samples from persons with prior DENV or Zika virus exposure. Despite extensive cross-reactivity in IgG binding, Zika virus neutralization was not observed among primary DENV infections. We observed low-frequency (23%) Zika virus cross-neutralization in repeat DENV infections. DENV-immune persons who had Zika virus as a secondary infection had distinct populations of antibodies that neutralized DENVs and Zika virus, as shown by DENV-reactive antibody depletion experiments. These data suggest that most DENV infections do not induce durable, high-level Zika virus cross-neutralizing antibodies. Zika virus-specific antibody populations develop after Zika virus infection irrespective of prior DENV immunity.

  11. Genetic relatedness of dengue viruses in Key West, Florida, USA, 2009-2010.

    Science.gov (United States)

    Muñoz-Jordán, Jorge L; Santiago, Gilberto A; Margolis, Harold; Stark, Lillian

    2013-04-01

    Sequencing of dengue virus type 1 (DENV-1) strains isolated in Key West/Monroe County, Florida, indicate endemic transmission for >2 years of a distinct and predominant sublineage of the American-African genotype. DENV-1 strains isolated elsewhere in Florida grouped within a separate Central American lineage. Findings indicate endemic transmission of DENV into the continental United States.

  12. The immunogenicity of tetravalent dengue DNA vaccine in mice pre-exposed to Japanese encephalitis or Dengue virus antigens.

    Science.gov (United States)

    Prompetchara, Eakachai; Ketloy, Chutitorn; Keelapang, Poonsook; Sittisombut, Nopporn; Ruxrungtham, Kiat

    2015-09-01

    Asian countries are an endemic area for both dengue (DENV) and Japanese encephalitis viruses (JEV). While JEV vaccines have been used extensively in this region, DENV vaccines remains under development. Whether preexisting naturally acquired or vaccination-induced immunity against JEV may affect the immune response to dengue vaccine candidate is unclear. In this study we used mice previously immunized with JEV vaccines to evaluate the impact on dengue-specific neutralizing antibody responses to a tetravalent dengue DNA vaccine candidate (TDNA). A tetravalent cocktail of plasmids encoding pre-membrane and envelope proteins from each dengue serotype was administered into mice which had been previously primed with inactivated or live-attenuated JEV vaccines, or dengue serotype2 virus (DENV-2). Neutralizing antibody response was measured employing a plaque reduction neutralization test at two weeks after the priming and at four weeks after the second dose of the dengue tetravalent plasmids. Inactivated or live-attenuated JEV vaccines, or DENV-2 induced low levels of neutralizing antibodies against the homologous viruses (JE and dengue virus, respectively). DENV-2 injection induced also low levels of cross-reactive antibodies against DENV-1, -3 and -4. JEV vaccines have no effect on the dengue-specific neutralizing antibody responses to the subsequent TDNA immunization. Pre-exposure to DENV-2 infection increased DENV-2 specific response neutralizing antibody to two doses of TDNA plasmids by six folds, but did not affect antibody response to other serotypes. Priming with JEV vaccines did not impact on dengue virus-specific neutralizing antibody response to a dengue TDNA vaccine candidate in mice.

  13. Meningitis Associated with Simultaneous Infection by Multiple Dengue Virus Serotypes in Children, Brazil

    Science.gov (United States)

    Marinho, Paula Eillanny Silva; Bretas de Oliveira, Danilo; Candiani, Talitah Michel Sanchez; Crispim, Ana Paula Correia; Alvarenga, Pedro Paulo Martins; Castro, Fabrizia Cristina dos Santos; Abrahão, Jonatas Santos; Rios, Maria; Coimbra, Roney Santos

    2017-01-01

    To determine the causes of viral meningitis, we analyzed 22 cerebrospinal fluid samples collected during the 2014–2015 dengue epidemics in Brazil. We identified 3 serotypes of dengue virus (DENV-1, -2, and -3), as well as co-infection with 2 or 3 serotypes. We also detected the Asian II genotype of DENV-2. PMID:27983492

  14. Characterization of dengue virus 2 growth in megakaryocyte–erythrocyte progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Clark, Kristina B. [Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA (United States); Hsiao, Hui-Mien; Bassit, Leda [Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, GA (United States); Crowe, James E. [Departments of Pediatrics, Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN (United States); Schinazi, Raymond F. [Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, GA (United States); Perng, Guey Chuen [Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Villinger, Francois [Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA (United States); New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA (United States)

    2016-06-15

    Megakaryocyte–erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis. - Highlights: • DenV replicates efficiently in undifferentiated megakaryocyte–erythrocyte progenitors. • MEP produced DenV differs in protein content from Vero produced DenV. • MEP produced DenV may be more difficult to neutralize relative to Vero DenV.

  15. Genome sequence analysis of dengue virus 1 isolated in Key West, Florida.

    Science.gov (United States)

    Shin, Dongyoung; Richards, Stephanie L; Alto, Barry W; Bettinardi, David J; Smartt, Chelsea T

    2013-01-01

    Dengue virus (DENV) is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL), including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1) and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR) to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW), FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI) and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs.

  16. Genome sequence analysis of dengue virus 1 isolated in Key West, Florida.

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    Dongyoung Shin

    Full Text Available Dengue virus (DENV is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL, including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1 and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW, FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs.

  17. New Inhibitors of the DENV-NS5 RdRp from Carpolepis laurifolia as Potential Antiviral Drugs for Dengue Treatment

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    Paul Coulerie

    2014-05-01

    Full Text Available Since a few decades the dengue virus became a major public health concern and no treatment is available yet. In order to propose potential antidengue compounds for chemotherapy we focused on DENV RNA polymerase (DENV-NS5 RdRp which is specific and essential for the virus replication. Carpolepis laurifolia belongs to the Myrtaceae and is used as febrifuge in traditional kanak medicine. Leaf extract of this plant has been identified as a hit against the DENV-NS5 RdRp. Here we present a bioguided fractionation of the leaf extract of C. laurifolia which is also the first phytochemical evaluation of this plant. Five flavonoids, namely quercetin (1, 6-methyl-7-methoxyapigenin (2, avicularin (3, quercitrin (4 and hyperoside (5, together with betulinic acid (6, were isolated from the leaf extract of C. laurifolia. All isolated compounds were tested individually against the DENV-NS5 RdRp and compared with four other commercial flavonoids: isoquercitrin (7, spiraeoside (8, quercetin-3,4’-di-O-glucoside (9 and rutine (10. Compounds 3, 4, 6, 8 and 10 displayed IC 50 ranging from 1.7 to 2.1 µM, and were the most active against the DENV-NS5 RdRp.

  18. Novel dengue virus type 1 from travelers to Yap State, Micronesia.

    Science.gov (United States)

    Nukui, Yoko; Tajima, Shigeru; Kotaki, Akira; Ito, Mikako; Takasaki, Tomohiko; Koike, Kazuhiko; Kurane, Ichiro

    2006-02-01

    Dengue virus type 1 (DENV-1), which was responsible for the dengue fever outbreak in Yap State, Micronesia, in 2004, was isolated from serum samples of 4 dengue patients in Japan. Genome sequencing demonstrated that this virus belonged to genotype IV and had a 29-nucleotide deletion in the 3 noncoding region.

  19. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

    NARCIS (Netherlands)

    van der Schaar, Hilde M.; Rust, Michael J.; Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, an

  20. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

    NARCIS (Netherlands)

    van der Schaar, Hilde M.; Rust, Michael J.; Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking,

  1. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

    NARCIS (Netherlands)

    van der Schaar, Hilde M.; Rust, Michael J.; Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, an

  2. Dengue virus binding and replication by platelets.

    Science.gov (United States)

    Simon, Ayo Y; Sutherland, Michael R; Pryzdial, Edward L G

    2015-07-16

    Dengue virus (DENV) infection causes ∼200 million cases of severe flulike illness annually, escalating to life-threatening hemorrhagic fever or shock syndrome in ∼500,000. Although thrombocytopenia is typical of both mild and severe diseases, the mechanism triggering platelet reduction is incompletely understood. As a probable initiating event, direct purified DENV-platelet binding was followed in the current study by quantitative reverse transcription-polymerase chain reaction and confirmed antigenically. Approximately 800 viruses specifically bound per platelet at 37°C. Fewer sites were observed at 25°C, the blood bank storage temperature (∼350 sites), or 4°C, known to attenuate virus cell entry (∼200 sites). Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and heparan sulfate proteoglycan were implicated as coreceptors because only the combination of anti-DC-SIGN and low-molecular-weight heparin prevented binding. Interestingly, at 37°C and 25°C, platelets replicated the positive sense single-stranded RNA genome of DENV by up to ∼4-fold over 7 days. Further time course experiments demonstrated production of viral NS1 protein, which is known to be highly antigenic in patient serum. The infectivity of DENV intrinsically decayed in vitro, which was moderated by platelet-mediated generation of viable progeny. This was shown using a transcription inhibitor and confirmed by freeze-denatured platelets being incapable of replicating the DENV genome. For the first time, these data demonstrate that platelets directly bind DENV saturably and produce infectious virus. Thus, expression of antigen encoded by DENV is a novel consideration in the pathogen-induced thrombocytopenia mechanism. These results furthermore draw attention to the possibility that platelets may produce permissive RNA viruses in addition to DENV.

  3. Dengue virus inhibits immune responses in Aedes aegypti cells.

    Directory of Open Access Journals (Sweden)

    Shuzhen Sim

    Full Text Available The ability of many viruses to manipulate the host antiviral immune response often results in complex host-pathogen interactions. In order to study the interaction of dengue virus (DENV with the Aedes aegypti immune response, we have characterized the DENV infection-responsive transcriptome of the immune-competent A. aegypti cell line Aag2. As in mosquitoes, DENV infection transcriptionally activated the cell line Toll pathway and a variety of cellular physiological systems. Most notably, however, DENV infection down-regulated the expression levels of numerous immune signaling molecules and antimicrobial peptides (AMPs. Functional assays showed that transcriptional induction of AMPs from the Toll and IMD pathways in response to bacterial challenge is impaired in DENV-infected cells. In addition, Escherichia coli, a gram-negative bacteria species, grew better when co-cultured with DENV-infected cells than with uninfected cells, suggesting a decreased production of AMPs from the IMD pathway in virus-infected cells. Pre-stimulation of the cell line with gram-positive bacteria prior to DENV infection had no effect on DENV titers, while pre-stimulation with gram-negative bacteria resulted in an increase in DENV titers. These results indicate that DENV is capable of actively suppressing immune responses in the cells it infects, a phenomenon that may have important consequences for virus transmission and insect physiology.

  4. Dengue Virus Glycosylation: What Do We Know?

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    Sally S. L. Yap

    2017-07-01

    Full Text Available In many infectious diseases caused by either viruses or bacteria, pathogen glycoproteins play important roles during the infection cycle, ranging from entry to successful intracellular replication and host immune evasion. Dengue is no exception. Dengue virus glycoproteins, envelope protein (E and non-structural protein 1 (NS1 are two popular sub-unit vaccine candidates. E protein on the virion surface is the major target of neutralizing antibodies. NS1 which is secreted during DENV infection has been shown to induce a variety of host responses through its binding to several host factors. However, despite their critical role in disease and protection, the glycosylated variants of these two proteins and their biological importance have remained understudied. In this review, we seek to provide a comprehensive summary of the current knowledge on protein glycosylation in DENV, and its role in virus biogenesis, host cell receptor interaction and disease pathogenesis.

  5. Dengue Virus in Bats from Southeastern Mexico

    Science.gov (United States)

    Sotomayor-Bonilla, Jesús; Chaves, Andrea; Rico-Chávez, Oscar; Rostal, Melinda K.; Ojeda-Flores, Rafael; Salas-Rojas, Mónica; Aguilar-Setien, Álvaro; Ibáñez-Bernal, Sergio; Barbachano-Guerrero, Arturo; Gutiérrez-Espeleta, Gustavo; Aguilar-Faisal, J. Leopoldo; Aguirre, A. Alonso; Daszak, Peter; Suzán, Gerardo

    2014-01-01

    To identify the relationship between landscape use and dengue virus (DENV) occurrence in bats, we investigated the presence of DENV from anthropogenically changed and unaltered landscapes in two Biosphere Reserves: Calakmul (Campeche) and Montes Azules (Chiapas) in southern Mexico. Spleen samples of 146 bats, belonging to 16 species, were tested for four DENV serotypes with standard reverse transcriptase polymerase chain reaction (RT-PCR) protocols. Six bats (4.1%) tested positive for DENV-2: four bats in Calakmul (two Glossophaga soricina, one Artibeus jamaicensis, and one A. lituratus) and two bats in Montes Azules (both A. lituratus). No effect of anthropogenic disturbance on the occurrence of DENV was detected; however, all three RT-PCR–positive bat species are considered abundant species in the Neotropics and well-adapted to disturbed habitats. To our knowledge, this study is the first study conducted in southeastern Mexico to identify DENV-2 in bats by a widely accepted RT-PCR protocol. The role that bats play on DENV's ecology remains undetermined. PMID:24752688

  6. Role of CD137 signaling in dengue virus-mediated apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Nagila, Amar [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Netsawang, Janjuree [Faculty of Medical Technology, Rangsit University, Bangkok (Thailand); Srisawat, Chatchawan [Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Noisakran, Sansanee [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Morchang, Atthapan; Yasamut, Umpa [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Puttikhunt, Chunya [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Kasinrerk, Watchara [Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai (Thailand); Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at Chiang Mai University, Chiang Mai (Thailand); and others

    2011-07-08

    Highlights: {yields} For the first time the role of CD137 in dengue virus (DENV) infection. {yields} Induction of DENV-mediated apoptosis by CD137 signaling. {yields} Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). {yields} Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

  7. Simultaneous circulation of genotypes I and III of dengue virus 3 in Colombia

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    Domingo Cristina

    2008-09-01

    Full Text Available Abstract Background Dengue is a major health problem in tropical and subtropical regions. In Colombia, dengue viruses (DENV cause about 50,000 cases annually, 10% of which involve Dengue Haemorrhagic Fever/Dengue Shock Syndrome. The picture is similar in other surrounding countries in the Americas, with recent outbreaks of severe disease, mostly associated with DENV serotype 3, strains of the Indian genotype, introduced into the Americas in 1994. Results The analysis of the 3'end (224 bp of the envelope gene from 32 DENV-3 strains recently recovered in Colombia confirms the circulation of the Indian genotype, and surprisingly the co-circulation of an Asian-Pacific genotype only recently described in the Americas. Conclusion These results have important implications for epidemiology and surveillance of DENV infection in Central and South America. Molecular surveillance of the DENV genotypes infecting humans could be a very valuable tool for controlling/mitigating the impact of the DENV infection.

  8. HLA-B*44 Is Associated with Dengue Severity Caused by DENV-3 in a Brazilian Population

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    Liciana Xavier Eurico de Alencar

    2013-01-01

    Full Text Available Human leukocyte antigen (HLA alleles have been correlated with susceptibility or resistance to severe dengue; however, few immunogenetic studies have been performed in Latin American (LA populations. We have conducted immunogenetic studies of HLA class I and II alleles in a cohort of 187 patients with DENV-3 infection and confirmed clinical diagnosis of either severe dengue, known as dengue hemorrhagic fever (DHF, or the less severe form, dengue fever (DF, in Recife, Pernambuco, Brazil. An association analysis was performed using Fisher’s association test, with odds ratios (ORs calculated using conditional maximum likelihood estimates. HLA-B*44 (P=0.047, OR = 2.025, 95% CI = 0.97–4.24 was found to be associated with increased susceptibility to DHF in response to DENV-3 infection. In addition, HLA-B*07 (P=0.048, OR = 0.501, one-sided 95% CI = 0–0.99 and HLA-DR*13 (P=0.028, OR = 0.511, one-sided 95% CI = 0–0.91 were found to be associated with resistance to secondary dengue infection by DENV-3. These results suggest that HLA-B*44 supertype alleles and their respective T-cell responses might be involved in susceptibility to severe dengue infections, whereas the HLA-B*07 supertype alleles and DR*13 might be involved in cross-dengue serotype immunity.

  9. Evidence for the Inhibition of Dengue Virus Binding in the Presence of Silver Nanoparticles

    Science.gov (United States)

    2015-03-26

    pretreatment with AgNP (6-10nm, 25µg/mL). These results suggest that similarly to other viruses , DENV2 can be inhibited at the first stage of the virus ...closely related viruses known as dengue virus -1 through -4 (DENV1-DENV4). A fifth serotype (DENV5) was discovered from a serological sample from 2007...various viruses at the binding and entry phase of viral replication. However, this is the first time a Group IV virus (ss(-)RNA) has been inhibited by

  10. Meta-Analysis of Dengue Severity during Infection by Different Dengue Virus Serotypes in Primary and Secondary Infections.

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    Kuan-Meng Soo

    Full Text Available Dengue virus (DENV infection is currently a major cause of morbidity and mortality in the world; it has become more common and virulent over the past half-century and has gained much attention. Thus, this review compared the percentage of severe cases of both primary and secondary infections with different serotypes of dengue virus.Data related to the number of cases involving dengue fever (DF, dengue hemorrhagic fever (DHF, dengue shock syndrome (DSS or severe dengue infections caused by different serotypes of dengue virus were obtained by using the SCOPUS, the PUBMED and the OVID search engines with the keywords "(dengue* OR dengue virus* AND (severe dengue* OR severity of illness index* OR severity* OR DF* OR DHF* OR DSS* AND (serotypes* OR serogroup*", according to the MESH terms suggested by PUBMED and OVID.Approximately 31 studies encompassing 15,741 cases reporting on the dengue serotypes together with their severity were obtained, and meta-analysis was carried out to analyze the data. This study found that DENV-3 from the Southeast Asia (SEA region displayed the greatest percentage of severe cases in primary infection (95% confidence interval (CI, 31.22-53.67, 9 studies, n = 598, I2 = 71.53%, whereas DENV-2, DENV-3, and DENV-4 from the SEA region, as well as DENV-2 and DENV-3 from non-SEA regions, exhibited the greatest percentage of severe cases in secondary infection (95% CI, 11.64-80.89, 4-14 studies, n = 668-3,149, I2 = 14.77-96.20%. Moreover, DENV-2 and DENV-4 from the SEA region had been found to be more highly associated with dengue shock syndrome (DSS (95% CI, 10.47-40.24, 5-8 studies, n = 642-2,530, I2 = 76.93-97.70%, while DENV-3 and DENV-4 from the SEA region were found to be more highly associated with dengue hemorrhagic fever (DHF (95% CI, 31.86-54.58, 9 studies, n = 674-2,278, I2 = 55.74-88.47%, according to the 1997 WHO dengue classification. Finally, DENV-2 and DENV-4 from the SEA region were discovered to be more highly

  11. Molecular surveillance of dengue in Semarang, Indonesia revealed the circulation of an old genotype of dengue virus serotype-1.

    Directory of Open Access Journals (Sweden)

    Sukmal Fahri

    Full Text Available Dengue disease is currently a major health problem in Indonesia and affects all provinces in the country, including Semarang Municipality, Central Java province. While dengue is endemic in this region, only limited data on the disease epidemiology is available. To understand the dynamics of dengue in Semarang, we conducted clinical, virological, and demographical surveillance of dengue in Semarang and its surrounding regions in 2012. Dengue cases were detected in both urban and rural areas located in various geographical features, including the coastal and highland areas. During an eight months' study, a total of 120 febrile patients were recruited, of which 66 were serologically confirmed for dengue infection using IgG/IgM ELISA and/or NS1 tests. The cases occurred both in dry and wet seasons. Majority of patients were under 10 years old. Most patients were diagnosed as dengue hemorrhagic fever, followed by dengue shock syndrome and dengue fever. Serotyping was performed in 31 patients, and we observed the co-circulation of all four dengue virus (DENV serotypes. When the serotypes were correlated with the severity of the disease, no direct correlation was observed. Phylogenetic analysis of DENV based on Envelope gene sequence revealed the circulation of DENV-2 Cosmopolitan genotype and DENV-3 Genotype I. A striking finding was observed for DENV-1, in which we found the co-circulation of Genotype I with an old Genotype II. The Genotype II was represented by a virus strain that has a very slow mutation rate and is very closely related to the DENV strain from Thailand, isolated in 1964 and never reported in other countries in the last three decades. Moreover, this virus was discovered in a cool highland area with an elevation of 1,001 meters above the sea level. The discovery of this old DENV strain may suggest the silent circulation of old virus strains in Indonesia.

  12. [Dengue virus infection in neotropical forest mammals: incidental hosts or potential reservoirs?].

    Science.gov (United States)

    Lavergne, A; Lacoste, V; Germain, A; Matheus, S; Dussart, P; Deparis, X; de Thoisy, B

    2009-08-01

    The arboviral disease with the highest human incidence in South America is dengue fever. In French Guiana, where all four dengue serotypes, i.e., DENV-1, DENV-2, DENV-3 and DENV-4, are present, the disease is endemic with epidemic outbreaks. Though previous serological studies have suggested a sylvatic cycle, involvement of wild mammals in the dengue cycle in the neotropics has never been confirmed. The purpose of this study was to search for the presence of DENV in wild animals captured at two different sites between 2001 and 2007. About 10,000 trap/nights were performed leading to the capture of 464 non-flying mammals (rodents and marsupials). In addition, mistnests placed in the same zone yielded 152 bats. Reverse transcription-polymerase chain reaction amplification to detect infection by any of the four dengue serotypes demonstrated viral RNA in the livers and/or sera of 92 captured animals. Sequence analysis of amplification products revealed that the DENV-1, DENV-3 and DENV-4 serotypes were distinct from those circulating in humans at the same periods. Analysis for DENV-2 showed that some strains were divergent from concurrent human strains but that others were identical. The latter finding suggests that wild neotropical mammals living in periurban area can be infected by dengue virus strains circulating in humans. However, further investigation will be needed to determine if neotropical mammals are incidental hosts or potential reservoirs of dengue virus.

  13. Innate immunity evasion by Dengue virus.

    Science.gov (United States)

    Morrison, Juliet; Aguirre, Sebastian; Fernandez-Sesma, Ana

    2012-03-01

    For viruses to productively infect their hosts, they must evade or inhibit important elements of the innate immune system, namely the type I interferon (IFN) response, which negatively influences the subsequent development of antigen-specific adaptive immunity against those viruses. Dengue virus (DENV) can inhibit both type I IFN production and signaling in susceptible human cells, including dendritic cells (DCs). The NS2B3 protease complex of DENV functions as an antagonist of type I IFN production, and its proteolytic activity is necessary for this function. DENV also encodes proteins that antagonize type I IFN signaling, including NS2A, NS4A, NS4B and NS5 by targeting different components of this signaling pathway, such as STATs. Importantly, the ability of the NS5 protein to bind and degrade STAT2 contributes to the limited host tropism of DENV to humans and non-human primates. In this review, we will evaluate the contribution of innate immunity evasion by DENV to the pathogenesis and host tropism of this virus.

  14. Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus

    Directory of Open Access Journals (Sweden)

    J. A. Swanstrom

    2016-07-01

    Full Text Available Zika virus (ZIKV is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73% failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], 1:100 serum dilution; 9% levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV.

  15. Detection of Saint Louis encephalitis virus in dengue-suspected cases during a dengue 3 outbreak.

    Science.gov (United States)

    Terzian, Ana Carolina Bernardes; Mondini, Adriano; Bronzoni, Roberta Vieira de Moraes; Drumond, Betânia Paiva; Ferro, Bianca Piovezan; Cabrera, Eliana Márcia Sotello; Figueiredo, Luis Tadeu Moraes; Chiaravalloti-Neto, Francisco; Nogueira, Maurício Lacerda

    2011-03-01

    Arboviruses are frequently associated with outbreaks in humans and represent a serious public health problem. Among the Brazilian arboviruses, Mayaro virus, Dengue virus (DENV), Yellow Fever virus, Rocio virus, Saint Louis Encephalitis virus (SLEV), and Oropouche virus are responsible for most of human cases. All these arboviruses usually produce undistinguishable acute febrile illness, especially in the acute phase of infection. In this study we investigated the presence of arboviruses in sera of 519 patients presenting acute febrile illness, during a dengue outbreak in São José do Rio Preto City (São Paulo, Brazil). A multiplex-nested RT-polymerase chain reaction assay was applied to detect and identify the main Brazilian arboviruses (Flavivirus, Alphavirus, and Orthobunyavirus genera). The molecular analysis showed that 365 samples were positive to DENV-3, 5 to DENV-2, and 8 to SLEV. Among the positive samples, one coinfection was detected between DENV-2 and DENV-3. The phylogenetic analysis of the SLEV envelope gene indicated that the virus circulating in city is related to lineage V strains. These results indicated that during that large DENV-3 outbreak in 2006, different arboviruses cocirculated causing human disease. Thus, it is necessary to have an efficient surveillance system to control the dissemination of these arboviruses in the population.

  16. Immature Dengue Virus Is Infectious in Human Immature Dendritic Cells via Interaction with the Receptor Molecule DC-SIGN

    NARCIS (Netherlands)

    Richter, Mareike K. S.; Da Silva-Voorham, Júlia M.; Torres Pedraza, Silvia; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Rodenhuis-Zybert, Izabela A.; Wilschut, Jan; Smit, Jolanda M.

    2014-01-01

    Background: Dengue Virus (DENV) is the most common mosquito-borne viral infection worldwide. Important target cells during DENV infection are macrophages, monocytes, and immature dendritic cells (imDCs). DENV-infected cells are known to secrete a large number of partially immature and fully immature

  17. Asian genotypes of dengue virus 4 in Brazil.

    Science.gov (United States)

    Pinho, A C O; Sardi, S I; Paula, F L; Peixoto, I B; Brandão, C J; Fernandez, F M C; Campos, G S

    2015-10-01

    Dengue virus, commonly transmitted by mosquitoes, causes a human disease of significant social impact and presents a serious public health problem in Brazil. This report describes the unusual emergence of DENV-4 in northern Brazil after a nearly 30-year-long absence. DENV-4 genotype I is of Asian origin and was identified in the serum of patients receiving treatment at a hospital serving the Salvador area (Brazilian state of Bahia). The identification of dengue virus serotypes through molecular and phylogenetic analysis is essential for predicting disease severity or fatal illness, principally in endemic countries such as Brazil.

  18. Cellufine sulfate column chromatography as a simple, rapid, and effective method to purify dengue virus.

    Science.gov (United States)

    Kanlaya, Rattiyaporn; Thongboonkerd, Visith

    2016-08-01

    Conventional method to purify/concentrate dengue virus (DENV) is time-consuming with low virus recovery yield. Herein, we applied cellufine sulfate column chromatography to purify/concentrate DENV based on the mimicry between heparan sulfate and DENV envelope protein. Comparative analysis demonstrated that this new method offered higher purity (as determined by less contamination of bovine serum albumin) and recovery yield (as determined by greater infectivity). Moreover, overall duration used for cellufine sulfate column chromatography to purify/concentrate DENV was approximately 1/20 of that of conventional method. Therefore, cellufine sulfate column chromatography serves as a simple, rapid, and effective alternative method for DENV purification/concentration.

  19. Single-virus tracking approach to reveal the interaction of Dengue virus with autophagy during the early stage of infection

    Science.gov (United States)

    Chu, Li-Wei; Huang, Yi-Lung; Lee, Jin-Hui; Huang, Long-Ying; Chen, Wei-Jun; Lin, Ya-Hsuan; Chen, Jyun-Yu; Xiang, Rui; Lee, Chau-Hwang; Ping, Yueh-Hsin

    2014-01-01

    Dengue virus (DENV) is one of the major infectious pathogens worldwide. DENV infection is a highly dynamic process. Currently, no antiviral drug is available for treating DENV-induced diseases since little is known regarding how the virus interacts with host cells during infection. Advanced molecular imaging technologies are powerful tools to understand the dynamics of intracellular interactions and molecular trafficking. This study exploited a single-virus particle tracking technology to address whether DENV interacts with autophagy machinery during the early stage of infection. Using confocal microscopy and three-dimensional image analysis, we showed that DENV triggered the formation of green fluorescence protein-fused microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta, and DENV-induced autophagosomes engulfed DENV particles within 15-min postinfection. Moreover, single-virus particle tracking revealed that both DENV particles and autophagosomes traveled together during the viral infection. Finally, in the presence of autophagy suppressor 3-methyladenine, the replication of DENV was inhibited and the location of DENV particles spread in cytoplasma. In contrast, the numbers of newly synthesized DENV were elevated and the co-localization of DENV particles and autophagosomes was detected while the cells were treated with autophagy inducer rapamycin. Taken together, we propose that DENV particles interact with autophagosomes at the early stage of viral infection, which promotes the replication of DENV.

  20. Dengue virus compartmentalization during antibody-enhanced infection

    Science.gov (United States)

    Ong, Eugenia Z.; Zhang, Summer L.; Tan, Hwee Cheng; Gan, Esther S.; Chan, Kuan Rong; Ooi, Eng Eong

    2017-01-01

    Secondary infection with a heterologous dengue virus (DENV) serotype increases the risk of severe dengue, through a process termed antibody-dependent enhancement (ADE). During ADE, DENV is opsonized with non- or sub-neutralizing antibody levels that augment entry into monocytes and dendritic cells through Fc-gamma receptors (FcγRs). We previously reported that co-ligation of leukocyte immunoglobulin-like receptor-B1 (LILRB1) by antibody-opsonized DENV led to recruitment of SH2 domain-containing phosphatase-1 (SHP-1) to dephosphorylate spleen tyrosine kinase (Syk) and reduce interferon stimulated gene induction. Here, we show that LILRB1 also signals through SHP-1 to attenuate the otherwise rapid acidification for lysosomal enzyme activation following FcγR-mediated uptake of DENV. Reduced or slower trafficking of antibody-opsonized DENV to lytic phagolysosomal compartments, demonstrates how co-ligation of LILRB1 also permits DENV to overcome a cell-autonomous immune response, enhancing intracellular survival of DENV. Our findings provide insights on how antiviral drugs that modify phagosome acidification should be used for viruses such as DENV. PMID:28084461

  1. Detection of dengue virus type 4 in Easter Island, Chile.

    Science.gov (United States)

    Fernández, J; Vera, L; Tognarelli, J; Fasce, R; Araya, P; Villagra, E; Roos, O; Mora, J

    2011-10-01

    We report the detection of dengue virus type 4 (DENV-4) for the first time in Easter Island, Chile. The virus was detected in serum samples of two patients treated at the Hospital in Easter Island. The two samples were IgM positive, and the infection was confirmed by RT-PCR and genetic sequencing; viral isolation was possible with one of them. The Easter Island isolates were most closely related to genotype II of dengue type 4.

  2. Incomplete Protection against Dengue Virus Type 2 Re-infection in Peru.

    Directory of Open Access Journals (Sweden)

    Brett M Forshey

    2016-02-01

    Full Text Available Nearly half of the world's population is at risk for dengue, yet no licensed vaccine or anti-viral drug is currently available. Dengue is caused by any of four dengue virus serotypes (DENV-1 through DENV-4, and infection by a DENV serotype is assumed to provide life-long protection against re-infection by that serotype. We investigated the validity of this fundamental assumption during a large dengue epidemic caused by DENV-2 in Iquitos, Peru, in 2010-2011, 15 years after the first outbreak of DENV-2 in the region.We estimated the age-dependent prevalence of serotype-specific DENV antibodies from longitudinal cohort studies conducted between 1993 and 2010. During the 2010-2011 epidemic, active dengue cases were identified through active community- and clinic-based febrile surveillance studies, and acute inapparent DENV infections were identified through contact tracing studies. Based on the age-specific prevalence of DENV-2 neutralizing antibodies, the age distribution of DENV-2 cases was markedly older than expected. Homologous protection was estimated at 35.1% (95% confidence interval: 0%-65.2%. At the individual level, pre-existing DENV-2 antibodies were associated with an incomplete reduction in the frequency of symptoms. Among dengue cases, 43% (26/66 exhibited elevated DENV-2 neutralizing antibody titers for years prior to infection, compared with 76% (13/17 of inapparent infections (age-adjusted odds ratio: 4.2; 95% confidence interval: 1.1-17.7.Our data indicate that protection from homologous DENV re-infection may be incomplete in some circumstances, which provides context for the limited vaccine efficacy against DENV-2 in recent trials. Further studies are warranted to confirm this phenomenon and to evaluate the potential role of incomplete homologous protection in DENV transmission dynamics.

  3. Correlation of Serotype-Specific Dengue Virus Infection with Clinical Manifestations

    Science.gov (United States)

    Halsey, Eric S.; Marks, Morgan A.; Gotuzzo, Eduardo; Fiestas, Victor; Suarez, Luis; Vargas, Jorge; Aguayo, Nicolas; Madrid, Cesar; Vimos, Carlos; Kochel, Tadeusz J.; Laguna-Torres, V. Alberto

    2012-01-01

    Background Disease caused by the dengue virus (DENV) is a significant cause of morbidity throughout the world. Although prior research has focused on the association of specific DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) with the development of severe outcomes such as dengue hemorrhagic fever and dengue shock syndrome, relatively little work has correlated other clinical manifestations with a particular DENV serotype. The goal of this study was to estimate and compare the prevalence of non-hemorrhagic clinical manifestations of DENV infection by serotype. Methodology and Principal Findings Between the years 2005–2010, individuals with febrile disease from Peru, Bolivia, Ecuador, and Paraguay were enrolled in an outpatient passive surveillance study. Detailed information regarding clinical signs and symptoms, as well as demographic information, was collected. DENV infection was confirmed in patient sera with polyclonal antibodies in a culture-based immunofluorescence assay, and the infecting serotype was determined by serotype-specific monoclonal antibodies. Differences in the prevalence of individual and organ-system manifestations were compared across DENV serotypes. One thousand seven hundred and sixteen individuals were identified as being infected with DENV-1 (39.8%), DENV-2 (4.3%), DENV-3 (41.5%), or DENV-4 (14.4%). When all four DENV serotypes were compared with each other, individuals infected with DENV-3 had a higher prevalence of musculoskeletal and gastrointestinal manifestations, and individuals infected with DENV-4 had a higher prevalence of respiratory and cutaneous manifestations. Conclusions/Significance Specific clinical manifestations, as well as groups of clinical manifestations, are often overrepresented by an individual DENV serotype. PMID:22563516

  4. Complement-mediated neutralization of dengue virus requires mannose-binding lectin

    DEFF Research Database (Denmark)

    Avirutnan, Panisadee; Hauhart, Richard E; Marovich, Mary A

    2011-01-01

    -dependent activation of the complement cascade neutralized insect cell-derived West Nile virus (WNV) in cell culture and restricted pathogenesis in mice. Here, we investigated the antiviral activity of MBL in infection by dengue virus (DENV), a related flavivirus. Using a panel of naïve sera from mouse strains...... with lower levels. Our studies suggest that allelic variation of MBL in humans may impact complement-dependent control of DENV pathogenesis. IMPORTANCE Dengue virus (DENV) is a mosquito-transmitted virus that causes a spectrum of clinical disease in humans ranging from subclinical infection to dengue...... hemorrhagic fever and dengue shock syndrome. Four serotypes of DENV exist, and severe illness is usually associated with secondary infection by a different serotype. Here, we show that mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation...

  5. Vector dynamics and transmission of dengue virus: implications for dengue surveillance and prevention strategies: vector dynamics and dengue prevention.

    Science.gov (United States)

    Scott, Thomas W; Morrison, Amy C

    2010-01-01

    Accounting for variation in mosquito vector populations will improve dengue surveillance and prevention. Because Aedes aegypti, the principle dengue virus (DENV) vector, transmit the virus with remarkable efficiency, entomological thresholds are especially low. Assessing risk of human infection based on immature mosquito indices has proven difficult. Greater emphasis should be placed on relative abundance of adult vectors in relation to human serotype-specific herd immunity, introduction of unique viruses, mosquito-human contact and weather. The most appropriate spatial scale for assessing entomological risk is the individual household. The scale for measuring DENV transmission risk has yet to be determined but is clearly larger than the household and likely to exceed several city blocks. Because households are expected to be a primary site for human DENV infection, intradomicile vector control strategies should be a priority, especially when the force of transmission is high. The most effective intervention strategy will combine vector control with vaccine delivery for rapid and sustained disease prevention.

  6. Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus.

    Science.gov (United States)

    Moi, Meng Ling; Ami, Yasushi; Shirai, Kenji; Lim, Chang-Kweng; Suzaki, Yuriko; Saito, Yuka; Kitaura, Kazutaka; Saijo, Masayuki; Suzuki, Ryuji; Kurane, Ichiro; Takasaki, Tomohiko

    2015-02-01

    Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells.

  7. Inhibition of dengue virus production and cytokine/chemokine expression by ribavirin and compound A.

    Science.gov (United States)

    Rattanaburee, Thidarath; Junking, Mutita; Panya, Aussara; Sawasdee, Nunghathai; Songprakhon, Pucharee; Suttitheptumrong, Aroonroong; Limjindaporn, Thawornchai; Haegeman, Guy; Yenchitsomanus, Pa-thai

    2015-12-01

    Dengue virus (DENV) infection is a worldwide public health problem with an increasing magnitude. The severity of disease in the patients with DENV infection correlates with high viral load and massive cytokine production - the condition referred to as "cytokine storm". Thus, concurrent inhibition of DENV and cytokine production should be more effective for treatment of DENV infection. In this study, we investigated the effects of the antiviral agent - ribavirin (RV), and the anti-inflammatory compound - compound A (CpdA), individually or in combination, on DENV production and cytokine/chemokine transcription in human lung epithelial carcinoma (A549) cells infected with DENV. Initially, the cells infected with DENV serotype 2 (DENV2) was studied. The results showed that treatment of DENV-infected cells with RV could significantly reduce both DENV production and cytokine (IL-6 and TNF-α) and chemokine (IP-10 and RANTES) transcription while treatment of DENV-infected cells with CpdA could significantly reduce cytokine (IL-6 and TNF-α) and chemokine (RANTES) transcription. Combined RV and CpdA treatment of the infected cells showed greater reduction of DENV production and cytokine/chemokine transcription. Similar results of this combined treatment were observed for infection with any one of the four DENV (DENV1, 2, 3, and 4) serotypes. These results indicate that combination of the antiviral agent and the anti-inflammatory compound offers a greater efficiency in reduction of DENV and cytokine/chemokine production, providing a new therapeutic approach for DENV infection.

  8. Dengue virus infection-enhancing antibody activities against Indonesian strains in inhabitants of central Thailand.

    Science.gov (United States)

    Yamanaka, Atsushi; Oddgun, Duangjai; Chantawat, Nantarat; Okabayashi, Tamaki; Ramasoota, Pongrama; Churrotin, Siti; Kotaki, Tomohiro; Kameoka, Masanori; Soegijanto, Soegeng; Konishi, Eiji

    2016-04-01

    Dengue virus (DENV) infection-enhancing antibodies are a hypothetic factor to increase the dengue disease severity. In this study, we investigated the enhancing antibodies against Indonesian strains of DENV-1-4 in 50 healthy inhabitants of central Thailand (Bangkok and Uthai Thani). Indonesia and Thailand have seen the highest dengue incidence in Southeast Asia. The infection history of each subject was estimated by comparing his/her neutralizing antibody titers against prototype DENV-1-4 strains. To resolve the difficulty in obtaining foreign live viruses for use as assay antigens, we used a recombinant system to prepare single-round infectious dengue viral particles based on viral sequence information. Irrespective of the previously infecting serotype(s), most serum samples showed significantly higher enhancement titers against Indonesian DENV-2 strains than against Thai DENV-2 strains, whereas the opposite effect was observed for the DENV-3 strains. Equivalent enhancing activities were observed against both DENV-1 and DENV-4. These results suggest that the genotype has an impact on enhancing antibody activities against DENV-2 and DENV-3, because the predominant circulating genotypes of each serotype differ between Indonesia and Thailand.

  9. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

    Directory of Open Access Journals (Sweden)

    Noda M

    2012-11-01

    Full Text Available Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4 are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the

  10. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    OpenAIRE

    Alvaro Díaz-Badillo; María de Lourdes Muñoz; Gerardo Perez-Ramirez; Victor Altuzar; Juan Burgueño; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Alejandro Cisneros; Joel Navarrete-Espinosa; Feliciano Sanchez-Sinencio

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybrid...

  11. Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014.

    Science.gov (United States)

    Zangmo, Sangay; Klungthong, Chonticha; Chinnawirotpisan, Piyawan; Tantimavanich, Srisurang; Kosoltanapiwat, Nathamon; Thaisomboonsuk, Butsaya; Phuntsho, Kelzang; Wangchuk, Sonam; Yoon, In-Kyu; Fernandez, Stefan

    2015-01-01

    Dengue is one of the most significant public health problems in tropical and subtropical countries, and is increasingly being detected in traditionally non-endemic areas. In Bhutan, dengue virus (DENV) has only recently been detected and limited information is available. In this study, we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013-2014. During this period, 379 patients were clinically diagnosed with suspected dengue, of whom 119 (31.4%) were positive for DENV infection by NS1 ELISA and/or nested RT-PCR. DENV serotypes 1, 2 and 3 were detected with DENV-1 being predominant. Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V, closely related to strains from northern India.

  12. Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014.

    Directory of Open Access Journals (Sweden)

    Sangay Zangmo

    Full Text Available Dengue is one of the most significant public health problems in tropical and subtropical countries, and is increasingly being detected in traditionally non-endemic areas. In Bhutan, dengue virus (DENV has only recently been detected and limited information is available. In this study, we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013-2014. During this period, 379 patients were clinically diagnosed with suspected dengue, of whom 119 (31.4% were positive for DENV infection by NS1 ELISA and/or nested RT-PCR. DENV serotypes 1, 2 and 3 were detected with DENV-1 being predominant. Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V, closely related to strains from northern India.

  13. Dengue virus neutralizing antibody levels associated with protection from infection in thai cluster studies.

    Directory of Open Access Journals (Sweden)

    Darunee Buddhari

    2014-10-01

    Full Text Available Long-term homologous and temporary heterologous protection from dengue virus (DENV infection may be mediated by neutralizing antibodies. However, neutralizing antibody titers (NTs have not been clearly associated with protection from infection.Data from two geographic cluster studies conducted in Kamphaeng Phet, Thailand were used for this analysis. In the first study (2004-2007, cluster investigations of 100-meter radius were triggered by DENV-infected index cases from a concurrent prospective cohort. Subjects between 6 months and 15 years old were evaluated for DENV infection at days 0 and 15 by DENV PCR and IgM ELISA. In the second study (2009-2012, clusters of 200-meter radius were triggered by DENV-infected index cases admitted to the provincial hospital. Subjects of any age ≥6 months were evaluated for DENV infection at days 0 and 14. In both studies, subjects who were DENV PCR positive at day 14/15 were considered to have been "susceptible" on day 0. Comparison subjects from houses in which someone had documented DENV infection, but the subject remained DENV negative at days 0 and 14/15, were considered "non-susceptible." Day 0 samples were presumed to be from just before virus exposure, and underwent plaque reduction neutralization testing (PRNT. Seventeen "susceptible" (six DENV-1, five DENV-2, and six DENV-4, and 32 "non-susceptible" (13 exposed to DENV-1, 10 DENV-2, and 9 DENV-4 subjects were evaluated. Comparing subjects exposed to the same serotype, receiver operating characteristic (ROC curves identified homotypic PRNT titers of 11, 323 and 16 for DENV-1, -2 and -4, respectively, to differentiate "susceptible" from "non-susceptible" subjects.PRNT titers were associated with protection from infection by DENV-1, -2 and -4. Protective NTs appeared to be serotype-dependent and may be higher for DENV-2 than other serotypes. These findings are relevant for both dengue epidemiology studies and vaccine development efforts.

  14. Establishment and preliminary application of Dengue virus envelope domain Ⅲ IgG antibody capture enzyme-linked immuno-absorbent assay

    Institute of Scientific and Technical Information of China (English)

    胡冬梅

    2013-01-01

    Objective To establish a highly sensitive and specific assay to detect Dengue virus(DENV) envelope protein domainⅢ(EDⅢ) IgG antibody,and to explore its value in the diagnosis and seroepidemiological survey of dengue

  15. The cellular bases of antibody responses during dengue virus infection

    Directory of Open Access Journals (Sweden)

    Juan Carlos Yam-Puc

    2016-06-01

    Full Text Available Dengue virus (DENV is one of the most significant human viral pathogens transmitted by mosquitoes and can cause from an asymptomatic disease to mild undifferentiated fever, classical dengue, and severe dengue. Neutralizing memory antibody (Ab responses are one of the most important mechanisms that counteract reinfections and are therefore the main aim of vaccination. However, it has also been proposed that in dengue, some of these class-switched (IgG memory Abs might worsen the disease. Although these memory Abs derive from B cells by T-cell dependent processes, we know rather little about the (acute, chronic or memory B cell responses and the complex cellular mechanisms generating these Abs during DENV infections.This review aims to provide an updated and comprehensive perspective of the B cell responses during DENV infection, starting since the very early events like the cutaneous DENV entrance and the arrival into draining lymph nodes, to the putative B cell activation, proliferation and germinal centers (GCs formation (the source of affinity-matured class-switched memory Abs, till the outcome of GC reactions such as the generation of plasmablasts, Ab-secreting plasma cells and memory B cells. We discuss topics very poorly explored such as the possibility of B cell infection by DENV or even activation-induced B cell death. The current information about the nature of the Ab responses to DENV is also illustrated.

  16. T-cell responses to dengue virus in humans.

    Science.gov (United States)

    Kurane, Ichiro; Matsutani, Takaji; Suzuki, Ryuji; Takasaki, Tomohiko; Kalayanarooj, Siripen; Green, Sharone; Rothman, Alan L; Ennis, Francis A

    2011-12-01

    Dengue virus (DENV) is a leading cause of morbidity and mortality in most tropical and subtropical areas of the world. Dengue virus infection induces specific CD4+CD8- and CD8+CD4- T cells in humans. In primary infection, T-cell responses to DENV are serotype cross-reactive, but the highest response is to the serotype that caused the infection. The epitopes recognized by DENV-specific T cells are located in most of the structural and non-structural proteins, but NS3 is the protein that is most dominantly recognized. In patients with dengue hemorrhagic fever (DHF) caused by secondary DENV infection, T cells are highly activated in vivo. These highly activated T cells are DENV-specific and oligoclonal. Multiple kinds of lymphokines are produced by the activated T cells, and it has been hypothesized that these lymphokines are responsible for induction of plasma leakage, one of the most characteristic features of DHF. Thus, T-cells play important roles in the pathogenesis of DHF and in the recovery from DENV infection.

  17. Molecular characterization of dengue viruses circulating during 2009-2012 in Uttar Pradesh, India.

    Science.gov (United States)

    Mishra, Gitika; Jain, Amita; Prakash, Om; Prakash, Shantanu; Kumar, Rashmi; Garg, Ravindra K; Pandey, Nidhi; Singh, Mastan

    2015-01-01

    Dengue is the most rapidly spreading mosquito-borne viral disease in the world; in India it has taken endemic proportion implicating all the four known dengue virus serotypes. Dengue infection is caused by a small, single stranded RNA virus comprising of four antigenically distinct virus serotypes designated as dengue virus type 1-4 (DENV-1-4). On the basis of genomic variations, each serotype is classified further into its genotypes. Epidemiological studies have shown that the emergence of a newer dengue serotype/genotype after an interval always leads to a major outbreak; therefore a continuous epidemiological surveillance is needed to monitor the epidemiology of dengue viruses. The present study was planned to identify the serotype/genotype of dengue viruses circulating in Uttar Pradesh, India. Of 433 dengue suspected patients, tested by reverse transcriptase PCR (RT-PCR), 136 were positive for dengue virus RNA. Of these, DENV-1, 2, and 3 were detected in 26 (19.1%), 77 (56.6%), and 33 (24.3%) patients, respectively. Of 136 RT-PCR positive samples, 24 samples were sequenced to identify their genotypes. For sequencing C-prM gene junction of dengue virus genome was chosen. Phylogenetic analysis of sequenced dengue strains revealed that all the 12 DENV-1 strains were genotype III, all the eight DENV-2 strains were genotype IV (Cosmopolitan genotype) and among four DENV-3 strains, three were genotype III and one was genotype I. In conclusion, the co-circulation of multiple dengue virus serotypes and genotypes is alarming in U.P., India.

  18. Serological characterization of dengue virus infections observed among dengue hemorrhagic fever/dengue shock syndrome cases in upper Myanmar.

    Science.gov (United States)

    Ngwe Tun, Mya Myat; Thant, Kyaw Zin; Inoue, Shingo; Kurosawa, Yae; Lwin, Yee Yee; Lin, Sanda; Aye, Kay Thi; Thet Khin, Pe; Myint, Tin; Htwe, Khin; Mapua, Cynthia A; Natividad, Filipinas F; Hirayama, Kenji; Morita, Kouichi

    2013-07-01

    In Myanmar, dengue fever (DF)/dengue hemorrhagic fever (DHF) is one of the leading causes of morbidity and mortality among children. From Pyinmana Hospital in 2004 and Mandalay Children Hospital in 2006, 160 patients diagnosed clinically to have DHF/dengue shock syndrome (DSS) were examined for immunoglobulin M (IgM) and IgG levels. A focus reduction neutralization test was also used to determine primary or secondary dengue virus (DENV) infection. By using IgM-capture ELISA, 139 cases were confirmed as DENV infections. Of these IgM-positives, 94 samples were collected 7-24 days from the onset of illness, to which 13 (14%) and 81 (86%) were determined to be primary and secondary DENV infections, respectively. The 13 primary DENV infection cases were spread among the various severity groups (DHF grade I-IV and DSS) and represented age groups ranging from <1 year of age to 9 years of age. The patients in these primary infection cases showed a remarkably high IgM with a low IgG titer response compared with the secondary infection cases. No significant differences were observed in IgG titers with clinical severity. The data obtained in this study suggest that primary DENV infection cases exist certainly among DHF/DSS cases in Myanmar, and that additional mechanism(s) aside from the antibody-dependent enhancement mechanism could have influenced the clinical severity in DHF/DSS cases. Copyright © 2013 Wiley Periodicals, Inc.

  19. High Anti-Dengue Virus Activity of the OAS Gene Family Is Associated With Increased Severity of Dengue.

    Science.gov (United States)

    Simon-Loriere, Etienne; Lin, Ren-Jye; Kalayanarooj, Sita Mint; Chuansumrit, Ampaiwan; Casademont, Isabelle; Lin, Shyr-Yi; Yu, Han-Pang; Lert-Itthiporn, Worachart; Chaiyaratana, Wathanee; Tangthawornchaikul, Nattaya; Tangnararatchakit, Kanchana; Vasanawathana, Sirijitt; Chang, Bi-Lan; Suriyaphol, Prapat; Yoksan, Sutee; Malasit, Prida; Despres, Philipe; Paul, Richard; Lin, Yi-Ling; Sakuntabhai, Anavaj

    2015-12-15

    Dengue is a mosquito-borne viral disease that afflicts millions of individuals worldwide every year. Infection by any of the 4 dengue virus (DENV) serotypes can result in a spectrum of disease severity. We investigated the impact of variants of interferon-regulated innate immunity genes with a potent antiviral effect on the outcome of DENV infection. We compared the effect of OAS gene family variants on 2 DENV serotypes in cell culture. While both OAS1-p42 and p46 showed antiviral activity against DENV-2, only OAS1-p42 presented anti-DENV-1 activity. Conversely, whereas both OAS3_S381 and R381 variants were able to block DENV-1 infection, the anti-DENV-2 activity observed for OAS3_S381 was largely lost for the R381 variant. By means of an allelic association study of a cohort of 740 patients with dengue, we found a protective effect of OAS3_R381 against shock (odds ratio [OR], 0.37; P dengue has long been associated with a cytokine storm of unclear origin. This work identifies an early innate immunity process that could lead to the immune overreaction observed in severe dengue and could be triggered by a specific host genotype-pathogen genotype interaction.

  20. Emergence of New Lineage of Dengue virus 3 (genotype III in Lucknow, India

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    Sanjeev Tripathi

    2013-03-01

    Full Text Available Dengue has re-emerged as an important arboviral disease causing significant morbidity. It has become hyperendemic in the Indian subcontinent with all the four known dengue serotypes circulating. In the present study we have characterized dengue virus type 3 (DENV-3 circulating in Lucknow, Uttar Pradesh, India. Multiple sequence alignment and phylogenetic tree were constructed to determine the extent of genetic heterogeneity and phylogeny of the dengue virus isolated. Sequencing and phylogenetic analysis of the C-prM gene junction revealed the active circulation and persistence of a new lineage of DENV-3 (genotype III circulating in this region of India.

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

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    Marize Pereira Miagostovich

    2006-05-01

    Full Text Available 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.

  2. Development, characterization and application of monoclonal antibodies against Brazilian Dengue virus isolates.

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    Camila Zanluca

    Full Text Available Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV detection through the production and characterization of 22 monoclonal antibodies (mAbs against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3 and dengue serotype-specific (DENV-2 or -3. Additionally, some mAbs cross-reacted with yellow fever virus (YFV, West Nile virus (WNV and Saint Louis encephalitis virus (SLEV. None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV. Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.

  3. Dengue virus cell entry : Unraveling the role of antibodies, maturation status, and antiviral drugs

    NARCIS (Netherlands)

    Ayala Nunez, Vanesa

    2014-01-01

    Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus-induced disease during a heterologous re-infection. Pre-existing cross-reactive anti-dengue antibodies are generally believed to bind to the newly infecting DENV and target the antibody-virus

  4. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

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    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  5. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Science.gov (United States)

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  6. Dengue Virus Specific Immune Response: Implications for laboratory diagnosis and vaccine development

    NARCIS (Netherlands)

    P. Koraka (Penelope)

    2007-01-01

    textabstractDengue viruses (DENV 1-4) belong to the family Flaviviridae, genus Flavivirus. They are transmitted to humans through the bite of infected mosquitoes of the Aedes species. An estimated 100 million people are annually infected with DENV and over two billion people are at risk in acquiring

  7. Dengue Virus Specific Immune Response: Implications for laboratory diagnosis and vaccine development

    NARCIS (Netherlands)

    P. Koraka (Penelope)

    2007-01-01

    textabstractDengue viruses (DENV 1-4) belong to the family Flaviviridae, genus Flavivirus. They are transmitted to humans through the bite of infected mosquitoes of the Aedes species. An estimated 100 million people are annually infected with DENV and over two billion people are at risk in

  8. Deeper understanding about the genetic structure of dengue virus using SVM

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    Choi Subin

    2016-01-01

    Full Text Available Dengue fever, mainly found in the tropical and subtropical regions, is carried by mosquitoes. With the help of greenhouse effect, places considered to be a Dengue safe-zone are becoming more and more dangerous. Dengue fever shows similar aspects to MERS, which caused heavy casualties in South Korea; Dengue virus does not have clear treatments nor vaccines like MERS. Development of Dengue vaccine is actively investigated lately. However, it is not easy to succeed; the fact that Dengue’s 4 serotypes have different properties and that repeated infections worsen the symptoms. This research aims to analyze the 4 serotypes (DENV1, DENV2, DENV3, DENV4 using SVM and ANN algorithms to investigate the constraints in the development of Dengue’s vaccines and treatments.

  9. The first case of laboratory-confirmed dengue virus infection in Mimika, Papua province, Indonesia

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    Agustiningsih Agustiningsih

    2016-07-01

    Full Text Available Abstrak Latar belakang: Dengue merupakan penyakit bersumber vektor yang berkontribusi cukup besar dalam menyebabkan masalah kesehatan baik di negara tropis maupun subtropis. Hingga saat ini virus dengue telah menyebar ke seluruh provinsi di Indonesia sejak pertama kali ditemukan di Surabaya pada tahun 1968. Kabupaten Mimika di propinsi Papua, Indonesia, merupakan daerah non-endemis dengue dan tidak pernah melaporkan munculnya kasus dengue. Walau begitu, pada tahun 2012 ditemukan 13 kasus tersangka dengue yang dirawat di Rumah Sakit Umum di Mimika. Studi ini bertujuan memberi gambaran karakteristik genetik virus dengue dari kasus terkonfirmasi (laboratory-confirmed pertama di kabupaten Mimika, propinsi Papua, Indonesia. Metode: Isolasi virus pada sel nyamuk C6/36, RT-PCR dan penentuan serotipe dilakukan untuk mengkonfirmasi adanya virus dengue (DENV di dalam serum pasien tersangka dengue dari kabupaten Mimika, propinsi Papua, Indonesia. Sekuensing dan analisis pohon filogenetik terhadap complete-coding sequence (CDS gen E dilakukan terhadap sampel yang telah positif DENV untuk penentuan genotipe virus. Hasil: Sebanyak 4 kasus tersangka dengue terkonfirmasi positif DENV berdasarkan pemeriksaan RT-PCR, sedangkan 2 sampel berhasil dilakukan kultur pada sel C6/36. Hasil penentuan serotipe menunjukkan bahwa virus DENV dari kabupaten Mimika, propinsi Papua, Indonesia, termasuk ke dalam serotipe DENV 3.  Analisis CDS gen E menunjukkan DENV 3 termasuk ke dalam genotipe I. Kesimpulan: Studi ini melaporkan kasus pertama dengue yang terkonfirmasi secara laboratorium dari kabupaten Mimika, propinsi Papua, Indonesia, yang merupakan daerah non-endemis dengue. Kata Kunci: dengue, penentuan serotipe, penentuan genotipe, kabupaten Mimika Abstract Background: Dengue is the most important vector-borne disease that poses serious health problem both in tropical and subtropical countries. Since the first outbreak in Surabaya in 1968, dengue infection has spread in

  10. Targeting of highly conserved Dengue virus sequences with anti-Dengue virus trans-splicing group I introns

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    Fraser Tresa S

    2010-11-01

    Full Text Available Abstract Background Dengue viruses (DENV are one of the most important viral diseases in the world with approximately 100 million infections and 200,000 deaths each year. The current lack of an approved tetravalent vaccine and ineffective insecticide control measures warrant a search for alternatives to effectively combat DENV. The trans-splicing variant of the Tetrahymena thermophila group I intron catalytic RNA, or ribozyme, is a powerful tool for post-transcriptional RNA modification. The nature of the ribozyme and the predictability with which it can be directed makes it a powerful tool for modifying RNA in nearly any cell type without the need for genome-altering gene therapy techniques or dependence on native cofactors. Results Several anti-DENV Group I trans-splicing introns (αDENV-GrpIs were designed and tested for their ability to target DENV-2 NGC genomes in situ. We have successfully targeted two different uracil bases on the positive sense genomic strand within the highly conserved 5'-3' cyclization sequence (CS region common to all serotypes of DENV with our αDENV-GrpIs. Our ribozymes have demonstrated ability to specifically trans-splice a new RNA sequence downstream of the targeted site in vitro and in transfected insect cells as analyzed by firefly luciferase and RT-PCR assays. The effectiveness of these αDENV-GrpIs to target infecting DENV genomes is also validated in transfected or transformed Aedes mosquito cell lines upon infection with unattenuated DENV-2 NGC. Conclusions Analysis shows that our αDENV-GrpIs have the ability to effectively trans-splice the DENV genome in situ. Notably, these results show that the αDENV-GrpI 9v1, designed to be active against all forms of Dengue virus, effectively targeted the DENV-2 NGC genome in a sequence specific manner. These novel αDENV-GrpI introns provide a striking alternative to other RNA based approaches for the transgenic suppression of DENV in transformed mosquito cells and

  11. NK cells during dengue disease and their recognition of dengue virus-infected cells

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    Davis Alexander Beltrán

    2014-05-01

    Full Text Available The innate immune response, in addition to the B and T cell response, plays a role in protection against dengue virus (DENV infection and the degree of disease severity. Early activation of NK cells and type-I interferon-dependent immunity may be important in limiting viral replication during the early stages of DENV infection and thus reducing subsequent pathogenesis. NK cells may also produce cytokines that reduce inflammation and tissue injury. On the other hand, NK cells are also capable of inducing liver injury at early-time points of DENV infection. In vitro, NK cells can kill antibody-coated DENV-infected cells through antibody-dependent cell-mediated cytotoxicity (ADCC. In additional, NK cells may directly recognize DENV-infected cells through their activating receptors, although the increase in HLA class I expression may allow infected cells to escape the NK response. Recently, genome-wide association studies (GWAS have shown an association between MICB and MICA, which encode ligands of the activating NK receptor NKG2D, and dengue disease outcome. This review focuses on recognition of DENV-infected cells by NK cells and on the regulation of expression of NK cell ligands by DENV.

  12. Vector competence of Malaysian Aedes albopictus with and without Wolbachia to four dengue virus serotypes.

    Science.gov (United States)

    Joanne, Sylvia; Vythilingam, Indra; Teoh, Boon-Teong; Leong, Cherng-Shii; Tan, Kim-Kee; Wong, Meng-Li; Yugavathy, Nava; AbuBakar, Sazaly

    2017-09-01

    To determine the susceptibility status of Aedes albopictus with and without Wolbachia to the four dengue virus serotypes. Two newly colonised colonies of Ae. albopictus from the wild were used for the study. One colony was naturally infected with Wolbachia while in the other Wolbachia was removed by tetracycline treatment. Both colonies were orally infected with dengue virus-infected fresh blood meal. Dengue virus load was measured using quantitative RT-PCR at four-time intervals in the salivary glands, midguts and ovaries. Wolbachia did not significantly affect Malaysian Ae. albopictus dengue infection or the dissemination rate for all four dengue virus serotypes. Malaysian Ae. albopictus had the highest replication kinetics for DENV-1 and the highest salivary gland and midgut infection rate for DENV-4. Wolbachia, which naturally exists in Malaysian Ae. albopictus, does not significantly affect dengue virus replication. Malaysian Ae. albopictus is susceptible to dengue virus infections and capable of transmitting dengue virus, especially DENV-1 and DENV-4. Removal of Wolbachia from Malaysian Ae. albopictus would not reduce their susceptibility status. © 2017 John Wiley & Sons Ltd.

  13. A Role for Human Skin Mast Cells in Dengue Virus Infection and Systemic Spread.

    Science.gov (United States)

    Troupin, Andrea; Shirley, Devon; Londono-Renteria, Berlin; Watson, Alan M; McHale, Cody; Hall, Alex; Hartstone-Rose, Adam; Klimstra, William B; Gomez, Gregorio; Colpitts, Tonya M

    2016-12-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious global human disease and mortality. Skin immune cells are an important component of initial DENV infection and systemic spread. Here, we show that mast cells are a target of DENV in human skin and that DENV infection of skin mast cells induces degranulation and alters cytokine and growth factor expression profiles. Importantly, to our knowledge, we also demonstrate for the first time that DENV localizes within secretory granules in infected skin mast cells. In addition, DENV within extracellular granules was infectious in vitro and in vivo, trafficking through lymph to draining lymph nodes in mice. We demonstrate an important role for human skin mast cells in DENV infection and identify a novel mechanism for systemic spread of DENV infection from the initial peripheral mosquito injection site. Copyright © 2016 by The American Association of Immunologists, Inc.

  14. Patient-based dengue virus surveillance in Aedes aegypti from Recife, Brazil

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    D.R.D. Guedes

    2010-06-01

    Full Text Available Background & objectives: Dengue is currently one of the most important arthropod-borne diseasesand may be caused by four different dengue virus serotypes (DENV-1 to DENV-4, transmittedmainly by Aedes aegypti (Diptera: Culicidae mosquitoes. With the lack of a dengue vaccine,vector control strategies constitute a crucial mode to prevent or reduce disease transmission. Inthis context, DENV detection in natural Ae. aegypti populations may serve as a potential additionaltool for early prediction systems of dengue outbreaks, leading to an intensification of vector controlmeasures, aimed at reducing disease transmission. In Brazil, this type of surveillance has beenperformed sporadically by a few groups and has not been incorporated as a routine activity incontrol programs. This study aimed at detecting DENV in natural Ae. aegypti from Recife,Pernambuco, to check the circulating serotypes and the occurrence of transovarial transmission inlocal mosquito populations.Methods: From January 2005 to June 2006, mosquitoes (adults and eggs were collected in houseswhere people with clinical suspicion of dengue infection lived at. RNA was extracted from pooledmosquitoes and RT-PCR was performed in these samples for detection of the four DENV serotypes.Results & conclusion: Out of 83 pools of adult mosquitoes collected in the field, nine were positivefor DENV: five for DENV-1, two for DENV-2 and two for DENV-3. From 139 pools of adultmosquitoes reared from collected eggs, there were 17 positive pools: three for DENV-1, 10 forDENV-2, and four for DENV-3. These results are discussed in the paper in regard to the localdengue epidemiological data. The conclusions clearly point to the informative power and sensitivityof DENV entomological surveillance and to the importance of including mosquito immature formsin this strategy.

  15. Competitive advantage of a dengue 4 virus when co-infecting the mosquito Aedes aegypti with a dengue 1 virus.

    Science.gov (United States)

    Vazeille, Marie; Gaborit, Pascal; Mousson, Laurence; Girod, Romain; Failloux, Anna-Bella

    2016-07-08

    Dengue viruses (DENV) are comprised in four related serotypes (DENV-1 to 4) and are critically important arboviral pathogens affecting human populations in the tropics. South American countries have seen the reemergence of DENV since the 1970's associated with the progressive re-infestation by the mosquito vector, Aedes aegypti. In French Guiana, DENV is now endemic with the co-circulation of different serotypes resulting in viral epidemics. Between 2009 and 2010, a predominant serotype change occurred from DENV-1 to DENV-4 suggesting a competitive displacement. The aim of the present study was to evaluate the potential role of the mosquito in the selection of the new epidemic serotype. To test this hypothesis of competitive displacement of one serotype by another in the mosquito vector, we performed mono- and co-infections of local Ae. aegypti collected during the inter-epidemic period with both viral autochthonous epidemic serotypes and compared infection, dissemination and transmission rates. We performed oral artificial infections of F1 populations in BSL-3 conditions and analyzed infection, dissemination and transmission rates. When two populations of Ae. aegypti from French Guiana were infected with either serotype, no significant differences in dissemination and transmission were observed between DENV-1 and DENV-4. However, in co-infection experiments, a strong competitive advantage for DENV-4 was seen at the midgut level leading to a much higher dissemination of this serotype. Furthermore only DENV-4 was present in Ae. aegypti saliva and therefore able to be transmitted. In an endemic context, mosquito vectors may be infected by several DENV serotypes. Our results suggest a possible competition between serotypes at the midgut level in co-infected mosquitoes leading to a drastically different transmission potential and, in this case, favoring the competitive displacement of DENV-1 by DENV-4. This phenomenon was observed despite a similar replicative fitness

  16. Molecular epidemiology of dengue virus serotypes 2 and 3 in Paraguay during 2001-2006: the association of viral clade introductions with shifting serotype dominance.

    Science.gov (United States)

    Aquino, Jose D J Diaz; Tang, Wei-Feng; Ishii, Ryoichi; Ono, Tetsuro; Eshita, Yuki; Aono, Hiroshi; Makino, Yoshihiro

    2008-11-01

    To determine the genetic variability of dengue viruses (DENVs) in Paraguay, the complete envelope gene was sequenced for 4 DENV-2 and 22 DENV-3 strains isolated from 2001 to 2006. The sequence data were used in Bayesian phylogenetic analyses, which revealed that Paraguayan DENV-2 strains fell into two distinct clades within the American/Asian genotype, thus suggesting that the introduction of a new DENV-2 clade was likely associated with the shift of dominant serotype from DENV-3 to DENV-2 in 2005 and might have caused an outbreak of DENV-2. This study also indicated that DENV-3 strains fell into genotype III, of which, several 2006 isolates varied from the remaining isolates in their tree locations. The introduction of this new clade was likely associated with the shift of dominant serotype from DENV-2 to DENV-3 in 2006 and might have caused an epidemic of DENV-3. More data are needed to test this hypothesis.

  17. Some epitopes conservation in non structural 3 protein dengue virus serotype 4

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    Tegar A. P. Siregar

    2016-03-01

    Full Text Available AbstrakLatar belakang: Protein Non Struktural 3 (NS3 virus dengue menginduksi respon antibodi netralisasidan respon sel T CD4+ dan CD8+, serta berperan dalam replikasi virus. Protein NS3 memiliki epitopepitopsel T dan B yang terdapat perbedaan kelestarian pada berbagai strain virus dengue serotipe 4(DENV-4. Penelitian ini bertujuan untuk mengetahui kelestarian epitop sel T dan B pada protein NS3DENV-4 strain-strain dunia dan keempat serotipe virus dengue strain Indonesia.Metode: Penelitian ini dilakukan di Departemen Mikrobiologi Fakultas Kedokteran UI sejak Juni 2013 - April2014. Sekuens asam amino NS3 DENV-4 strain 081 didapatkan setelah produk PCR gen NS3 DENV-4 081disekuensing. Epitop-epitop sel T dan sel B protein NS3 DENV-4 081 dianalisis dan dibandingkan dengansekuens asam amino protein NS3 dari 124 strain DENV-4 di dunia dan keempat serotipe DENV strain Indonesia.Strain-strain dunia merupakan strain yang ada di benua Amerika (Venezuela, Colombia, dll dan Asia (Cina,Singapura, dll. Referensi posisi epitop sel T dan B protein NS3 diperoleh dari laporan penelitian terdahulu.Hasil: Delapan epitop sel T dan 2 epitop sel B dari protein NS3 DENV-4 081 ternyata identik dan lestaripada protein NS3 dari 124 strain DENV-4 dunia. Epitop sel B di posisi asam amino 537-544 pada proteinNS3 DENV-4 081 ternyata identik dan lestari dengan epitop sel B protein NS3 dari keempat serotipeDENV strain Indonesia.Kesimpulan: Kelestarian yang luas dari epitop sel T dan B pada hampir seluruh strain DENV-4 dunia danserotipe-serotipe DENV strain Indonesia. (Health Science Journal of Indonesia 2015;6:126-31Kata kunci: virus dengue, protein NS3, epitop sel T, epitop sel B AbstractBackground: Non Structural 3 (NS3 protein of dengue virus (DENV is known to induce antibody, CD4+and CD8+ T cell responses, and playing role in viral replication. NS3 protein has T and B cell epitopes,which has conservation difference between DENV-4 strains. This study aimed to identify

  18. Dengue virus replicates and accumulates in Aedes aegypti salivary glands.

    Science.gov (United States)

    Raquin, Vincent; Lambrechts, Louis

    2017-07-01

    Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Natural strain variation and antibody neutralization of dengue serotype 3 viruses.

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    Wahala M P B Wahala

    2010-03-01

    Full Text Available Dengue viruses (DENVs are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII. It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge "type specific" epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.

  20. Honokiol, a Lignan Biphenol Derived from the Magnolia Tree, Inhibits Dengue Virus Type 2 Infection

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    Chih-Yeu Fang

    2015-09-01

    Full Text Available Dengue is the most widespread arbovirus infection and poses a serious health and economic issue in tropical and subtropical countries. Currently no licensed vaccine or compounds can be used to prevent or manage the severity of dengue virus (DENV infection. Honokiol, a lignan biphenol derived from the Magnolia tree, is commonly used in Eastern medicine. Here we report that honokiol has profound antiviral activity against serotype 2 DENV (DENV-2. In addition to inhibiting the intracellular DENV-2 replicon, honokiol was shown to suppress the replication of DENV-2 in baby hamster kidney (BHK and human hepatocarcinoma Huh7 cells. At the maximum non-toxic dose of honokiol treatment, the production of infectious DENV particles was reduced >90% in BHK and Huh7 cells. The underlying mechanisms revealed that the expression of DENV-2 nonstructural protein NS1/NS3 and its replicating intermediate, double-strand RNA, was dramatically reduced by honokiol treatment. Honokiol has no effect on the expression of DENV putative receptors, but may interfere with the endocytosis of DENV-2 by abrogating the co-localization of DENV envelope glycoprotein and the early endosomes. These results indicate that honokiol inhibits the replication, viral gene expression, and endocytotic process of DENV-2, making it a promising agent for chemotherapy of DENV infection.

  1. Molecular Epidemiology of Dengue Viruses Co-circulating in Upper Myanmar in 2006.

    Science.gov (United States)

    Thant, Kyaw Zin; Tun, Mya Myat Ngwe; Parquet, Maria Del Carmen; Inoue, Shingo; Lwin, Yee Yee; Lin, Sanda; Aye, Kay Thi; Khin, Pe Thet; Myint, Tin; Htwe, Khin; Nabeshima, Takeshi; Morita, Kouichi

    2015-03-01

    To understand the molecular epidemiology of circulating dengue viruses (DENV) in Upper Myanmar, DENV isolation was attempted by inoculating the sera of a panel of 110 serum samples onto a C6/36 mosquito cell line. The samples were collected from dengue (DEN) patients admitted at Mandalay Children's Hospital in 2006. Infected culture fluids were subjected to a RT-PCR to detect the DENV genome. Three DENV strains were isolated. This was the first DENV isolation performed either in Mandalay or in Upper Myanmar. One strain belonged to DENV serotype-3 (DENV-3), and two other strains belonged to DENV serotype-4 (DEN-4). The sequence data for the envelope gene of these strains were used in a phylogenetic comparison of DENV-3 and DENV-4 from various countries. Phylogenetic analyses revealed that this DENV-3 strain was clustered within genotype II, and the two DENV-4 strains were clustered within genotype I in each serotype. The Myanmar strains were closely related to strains from the neighboring countries of Thailand and Bangladesh. These results are important for elucidating the trends of recent and future DEN outbreaks in Myanmar.

  2. KDEL Receptors Assist Dengue Virus Exit from the Endoplasmic Reticulum

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    Ming Yuan Li

    2015-03-01

    Full Text Available Membrane receptors at the surface of target cells are key host factors for virion entry; however, it is unknown whether trafficking and secretion of progeny virus requires host intracellular receptors. In this study, we demonstrate that dengue virus (DENV interacts with KDEL receptors (KDELR, which cycle between the ER and Golgi apparatus, for vesicular transport from ER to Golgi. Depletion of KDELR by siRNA reduced egress of both DENV progeny and recombinant subviral particles (RSPs. Coimmunoprecipitation of KDELR with dengue structural protein prM required three positively charged residues at the N terminus, whose mutation disrupted protein interaction and inhibited RSP transport from the ER to the Golgi. Finally, siRNA depletion of class II Arfs, which results in KDELR accumulation in the Golgi, phenocopied results obtained with mutagenized prME and KDELR knockdown. Our results have uncovered a function for KDELR as an internal receptor involved in DENV trafficking.

  3. Dendritic cells in dengue virus infection: Targets of virus replication and mediators of immunity

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    Michael A. Schmid

    2014-12-01

    Full Text Available Dendritic cells (DCs are sentinels of the immune system and detect pathogens at sites of entry, such as the skin. In addition to the ability of DCs to control infections directly via their innate immune functions, DCs help to prime adaptive B and T cell responses via antigen presentation in lymphoid tissues. Infected Aedes aegypti or Ae. albopictus mosquitoes transmit the four dengue virus (DENV serotypes to humans while probing for small blood vessels in the skin. DENV causes the most prevalent arthropod-borne viral disease in humans, yet no vaccine or specific therapeutic is currently approved. Although primary DENV infection confers life-long protective immunity against re-infection with the same DENV serotype, secondary infection with a different DENV serotype can lead to increased disease severity via cross-reactive T cells or enhancing antibodies. This review summarizes recent findings in humans and animal models about DENV infection of DCs, monocytes and macrophages. We discuss the dual role of DCs as both targets of DENV replication and mediators of innate and adaptive immunity, and summarize immune evasion strategies whereby DENV impairs the function of infected DCs. We suggest that DCs play a key role in priming DENV-specific neutralizing or potentially harmful memory B and T cell responses, and that future DC-directed therapies may help induce protective memory responses and reduce dengue pathogenesis.

  4. Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India

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    Guleria Randeep

    2008-01-01

    Full Text Available Abstract Background Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating during one outbreak. Results Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 69 samples tested for dengue virus RNA, 48 (69.5% were found to be positive. All the four dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 being the predominant serotype. In addition in 9 of 48 (19% dengue virus positive samples, concurrent infection with more than one dengue virus serotype were identified. Conclusion This is the first report in which concurrent infections with different dengue virus serotypes is being reported during an outbreak from India. Delhi is now truly hyperendemic for dengue.

  5. Comparison of Vector Competence of Aedes mediovittatus and Aedes aegypti for Dengue Virus: Implications for Dengue Control in the Caribbean

    Science.gov (United States)

    Poole-Smith, B. Katherine; Hemme, Ryan R.; Delorey, Mark; Felix, Gilberto; Gonzalez, Andrea L.; Amador, Manuel; Hunsperger, Elizabeth A.; Barrera, Roberto

    2015-01-01

    Background Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes. Methods To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1–4 per os at viral titers of 5–6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution. Results Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus. Conclusions This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs. PMID:25658951

  6. Vaccination with dengue virus-like particles induces humoral and cellular immune responses in mice

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    Zhang Quanfu

    2011-06-01

    Full Text Available Abstract Background The incidence of dengue, an infectious disease caused by dengue virus (DENV, has dramatically increased around the world in recent decades and is becoming a severe public health threat. However, there is currently no specific treatment for dengue fever, and licensed vaccine against dengue is not available. Vaccination with virus-like particles (VLPs has shown considerable promise for many viral diseases, but the effect of DENV VLPs to induce specific immune responses has not been adequately investigated. Results By optimizing the expression plasmids, recombinant VLPs of four antigenically different DENV serotypes DENV1-4 were successfully produced in 293T cells. The vaccination effect of dengue VLPs in mice showed that monovalent VLPs of each serotype stimulated specific IgG responses and potent neutralizing antibodies against homotypic virus. Tetravalent VLPs efficiently enhanced specific IgG and neutralizing antibodies against all four serotypes of DENV. Moreover, vaccination with monovalent or tetravalent VLPs resulted in the induction of specific cytotoxic T cell responses. Conclusions Mammalian cell expressed dengue VLPs are capable to induce VLP-specific humoral and cellular immune responses in mice, and being a promising subunit vaccine candidate for prevention of dengue virus infection.

  7. Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells.

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    Hilde M van der Schaar

    2008-12-01

    Full Text Available Dengue virus (DENV is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.

  8. Human Leukocyte Antigen (HLA) Class I Restricted Epitope Discovery in Yellow Fewer and Dengue Viruses: Importance of HLA Binding Strength

    DEFF Research Database (Denmark)

    Lund, Ole; Nascimento, Eduardo J. M.; Maciel, Milton, Jr

    2011-01-01

    Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV...

  9. Identification of a new dengue virus inhibitor that targets the viral NS4B protein and restricts genomic RNA replication

    NARCIS (Netherlands)

    Cleef, K.W.R. van; Overheul, G.J.; Thomassen, M.C.; Kaptein, S.J.; Davidson, A.D.; Jacobs, M.; Neyts, J.; Kuppeveld, F.J.M. van; Rij, R.P. van

    2013-01-01

    Dengue virus (DENV) is an important human arthropod-borne virus with a major impact on public health. Nevertheless, a licensed vaccine or specific treatment is still lacking. We therefore screened the NIH Clinical Collection (NCC), a library of drug-like small molecules, for inhibitors of DENV repli

  10. Susceptibility of Florida Aedes aegypti and Aedes albopictus to dengue viruses from Puerto Rico.

    Science.gov (United States)

    Alto, Barry W; Smartt, Chelsea T; Shin, Dongyoung; Bettinardi, David; Malicoate, Jolene; Anderson, Sheri L; Richards, Stephanie L

    2014-12-01

    Locally acquired dengue cases in the continental U.S. are rare. However, outbreaks of dengue-1 during 2009, 2010, and 2013 in Florida and dengue-1 and -2 in Texas suggest vulnerability to transmission. Travel and commerce between Puerto Rico and the U.S. mainland is common, which may pose a risk for traveler-imported dengue cases. Mosquitoes were collected in Florida and used to evaluate their susceptibility to dengue viruses (DENV) from Puerto Rico. Aedes aegypti and Ae. albopictus were susceptible to virus infection with DENV-1 and -2. No significant differences were observed in rates of midgut infection or dissemination between Ae. aegypti or Ae. albopictus for DENV-1 (6-14%). Aedes aegypti was significantly more susceptible to midgut infection with DENV-2 than Ae. albopictus (Ae. aegypti, ∼28%; Ae. albopictus, ∼9%). The dissemination rate with dengue-2 virus for Ae. aegypti (23%) was greater than Ae. albopictus (0%), suggesting that Ae. albopictus is not likely to be an important transmitter of the DENV-2 isolate from Puerto Rico. These results are discussed in light of Florida's vulnerability to DENV transmission.

  11. Dynamics of cellular immune responses in the acute phase of dengue virus infection.

    Science.gov (United States)

    Yoshida, Tomoyuki; Omatsu, Tsutomu; Saito, Akatsuki; Katakai, Yuko; Iwasaki, Yuki; Kurosawa, Terue; Hamano, Masataka; Higashino, Atsunori; Nakamura, Shinichiro; Takasaki, Tomohiko; Yasutomi, Yasuhiro; Kurane, Ichiro; Akari, Hirofumi

    2013-06-01

    In this study, we examined the dynamics of cellular immune responses in the acute phase of dengue virus (DENV) infection in a marmoset model. Here, we found that DENV infection in marmosets greatly induced responses of CD4/CD8 central memory T and NKT cells. Interestingly, the strength of the immune response was greater in animals infected with a dengue fever strain than in those infected with a dengue hemorrhagic fever strain of DENV. In contrast, when animals were re-challenged with the same DENV strain used for primary infection, the neutralizing antibody induced appeared to play a critical role in sterilizing inhibition against viral replication, resulting in strong but delayed responses of CD4/CD8 central memory T and NKT cells. The results in this study may help to better understand the dynamics of cellular and humoral immune responses in the control of DENV infection.

  12. Molecular surveillance of dengue in Minas Gerais provides insights on dengue virus 1 and 4 circulation in Brazil.

    Science.gov (United States)

    Dutra, Karina Rocha; Drumond, Betânia Paiva; de Rezende, Izabela Maurício; Nogueira, Maurício Lacerda; de Oliveira Lopes, Débora; Calzavara Silva, Carlos Eduardo; Siqueira Ferreira, Jaqueline Maria; Dos Santos, Luciana Lara

    2017-06-01

    Dengue, caused by any of the four types of Dengue virus (DENV) is the most important arbovirus in the world. In this study we performed a molecular surveillance of dengue during the greatest dengue outbreak that took place in Divinópolis, Minas Gerais state, Southeast Brazil, in 2013. Samples from 100 patients with clinical symptoms of dengue were studied and 26 were positive. The capsid/premembrane (CprM) and envelope gene sequences of some samples were amplified and sequenced. Molecular analyses demonstrated that two DENV-1 lineages, belonging to genotype V were introduced and co-circulated in Divinópolis. When compared to each other, those lineages presented high genetic diversity and showed unique amino acids substitutions in the envelope protein, including in domains I, II, and III. DENV-4 strains from Divinópolis clustered within genotype IIb and the most recent common ancestor was probably introduced into the city three years before the 2013 epidemic. Here we demonstrated for the first time the circulation of DENV-4 and the co-circulation of two DENV-1 lineages in Midwest region of Minas Gerais, Brazil. Moreover our analysis indicated the introduction of five DENV-1 lineages, genotype V into Brazil, in different times. J. Med. Virol. 89:966-973, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Dengue Virus Serotypes Circulating in Khyber Pakhtunkhwa Province, Pakistan, 2013-2015.

    Science.gov (United States)

    Suleman, Muhammad; Faryal, Rani; Alam, Muhammad Masroor; Sharif, Salmaan; Shaukat, Shahzad; Aamir, Uzma Bashir; Khurshid, Adnan; Angez, Mehar; Umair, Massab; Sufian, Mian Muhammad; Arshad, Yasir; Zaidi, Syed Sohail Zahoor

    2017-03-01

    From 2013 to 2015, the National Institute of Health, Pakistan, received 1,270 blood samples of suspected dengue cases reported from inpatient and outpatient departments of various hospitals in Khyber Pakhtunkhwa (KPK) province. In this study, we determined the circulating dengue virus (DENV) serotypes using real-time reverse transcriptase (RT)-PCR to understand the serotype-based epidemiology of DENV. All four serotypes (DENV-1 [6%], DENV-2 [33%], DENV-3 [47%], and DENV-4 [0.1%]) were found circulating during the study period. Our findings suggest the need for an active surveillance system coupled with the laboratory diagnosis, especially in the chronic endemic areas of the country. Public awareness programs are needed for effective control and prevention of outbreaks in the future.

  14. Protective Role of Cross-Reactive CD8 T Cells Against Dengue Virus Infection

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    Annie Elong Ngono

    2016-11-01

    Full Text Available Infection with one of the four dengue virus serotypes (DENV1-4 presumably leads to lifelong immunity against the infecting serotype but not against heterotypic reinfection, resulting in a greater risk of developing Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS during secondary infection. Both antibodies and T cell responses have been implicated in DHF/DSS pathogenesis. According to the T cell-based hypothesis termed “original antigenic sin,” secondary DENV infection is dominated by non-protective, cross-reactive T cells that elicit an aberrant immune response. The goal of our study was to compare the roles of serotype-specific and cross-reactive T cells in protection vs. pathogenesis during DENV infection in vivo. Specifically, we utilized IFN-α/βR−/− HLA*B0702 transgenic mice in the context of peptide vaccination with relevant human CD8 T cell epitopes. IFN-α/βR−/− HLA*B0702 transgenic mice were immunized with DENV serotype 2 (DENV2-specific epitopes or variants found in any of the other three serotypes (DENV1, DENV3 or DENV4, followed by challenge with DENV. Although cross-reactive T cell responses were lower than responses elicited by serotype-specific T cells, immunization with either serotype-specific or variant peptide epitopes enhanced viral clearance, demonstrating that both serotype-specific and cross-reactive T cells can contribute to protection in vivo against DENV infection.

  15. Protective Role of Cross-Reactive CD8 T Cells Against Dengue Virus Infection.

    Science.gov (United States)

    Elong Ngono, Annie; Chen, Hui-Wen; Tang, William W; Joo, Yunichel; King, Kevin; Weiskopf, Daniela; Sidney, John; Sette, Alessandro; Shresta, Sujan

    2016-11-01

    Infection with one of the four dengue virus serotypes (DENV1-4) presumably leads to lifelong immunity against the infecting serotype but not against heterotypic reinfection, resulting in a greater risk of developing Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) during secondary infection. Both antibodies and T cell responses have been implicated in DHF/DSS pathogenesis. According to the T cell-based hypothesis termed "original antigenic sin," secondary DENV infection is dominated by non-protective, cross-reactive T cells that elicit an aberrant immune response. The goal of our study was to compare the roles of serotype-specific and cross-reactive T cells in protection vs. pathogenesis during DENV infection in vivo. Specifically, we utilized IFN-α/βR(-/-) HLA*B0702 transgenic mice in the context of peptide vaccination with relevant human CD8 T cell epitopes. IFN-α/βR(-/-) HLA*B0702 transgenic mice were immunized with DENV serotype 2 (DENV2)-specific epitopes or variants found in any of the other three serotypes (DENV1, DENV3 or DENV4), followed by challenge with DENV. Although cross-reactive T cell responses were lower than responses elicited by serotype-specific T cells, immunization with either serotype-specific or variant peptide epitopes enhanced viral clearance, demonstrating that both serotype-specific and cross-reactive T cells can contribute to protection in vivo against DENV infection.

  16. Identification of sequence motifs involved in Dengue virus-host interactions.

    Science.gov (United States)

    Asnet Mary, J; Paramasivan, R; Shenbagarathai, R

    2016-01-01

    Dengue fever is a rapidly spreading mosquito-borne virus infection, which remains a serious global public health problem. As there is no specific treatment or commercial vaccine available for effective control of the disease, the attempts on developing novel control strategies are underway. Viruses utilize the surface receptor proteins of host to enter into the cells. Though various proteins were said to be receptors of Dengue virus (DENV) using Virus Overlay Protein Binding Assay, the precise interaction between DENV and host is not explored. Understanding the structural features of domain III envelope glycoprotein would help in developing efficient antiviral inhibitors. Therefore, an attempt was made to identify the sequence motifs present in domain III envelope glycoprotein of Dengue virus. Computational analysis revealed that the NGR motif is present in the domain III envelope glycoprotein of DENV-1 and DENV-3. Similarly, DENV-1, DENV-2 and DENV-4 were found to contain Yxxphi motif which is a tyrosine-based sorting signal responsible for the interaction with a mu subunit of adaptor protein complex. High-throughput virtual screening resulted in five compounds as lead molecules based on glide score, which ranges from -4.664 to -6.52 kcal/Mol. This computational prediction provides an additional tool for understanding the virus-host interactions and helps to identify potential targets in the host. Further, experimental evidence is warranted to confirm the virus-host interactions and also inhibitory activity of reported lead compounds.

  17. Diversity and composition of dengue virus type 2 in Venezuela.

    Science.gov (United States)

    Huhtamo, E; Comach, G; Sierra, G; Camacho, D E; Sironen, T; Vapalahti, O; Uzcátegui, N Y

    2013-09-01

    Dengue is a mosquito-borne disease caused by four closely related dengue virus (genus Flavivirus)serotypes (DENV-1–4). The clinical outcomes vary from mild febrile illness to life-threatening haemorrhagic manifestations. DENVs are endemic in the tropics and subtropics globally and currently no specific treatment or vaccines are available. In Venezuela, the American-Asian genotype of DENV-2 is the most prevalent and has been associated with severe disease outcomes.We aimed to follow-up the molecular epidemiology of DENV-2 in Venezuela to investigate if the evolution of the virus has remained the same throughout time or if the same dynamics documented in Brazil (hyperendemic co-circulation) also occurred. The results show that whereas the epidemiology of DENV in several endemic areas is characterized by serotype replacements through time, in Venezuela the American-Asian genotype DENV-2 has evolved into several genetic lineages and has remained in hyperendemic co-circulation with the other serotypes.

  18. Detection of Mayaro virus infections during a dengue outbreak in Mato Grosso, Brazil.

    Science.gov (United States)

    Vieira, Carla Julia da Silva Pessoa; Silva, David José Ferreira da; Barreto, Eriana Serpa; Siqueira, Carlos Eduardo Hassegawa; Colombo, Tatiana Elias; Ozanic, Katia; Schmidt, Diane Johnson; Drumond, Betânia Paiva; Mondini, Adriano; Nogueira, Maurício Lacerda; Bronzoni, Roberta Vieira de Morais

    2015-07-01

    Arboviruses are common agents of human febrile illness worldwide. In dengue-endemic areas illness due to other arboviruses have been misdiagnosed as dengue based only on clinical-epidemiological data. In this study we investigated the presence of Brazilian arboviruses in sera of 200 patients presenting acute febrile illness, during a dengue outbreak in Sinop, MT, Brazil. The results showed that 38 samples were positive to Dengue virus (DENV) type 1, two samples to DENV type 4, and six to Mayaro virus. These results indicate that arboviruses others than DENV are circulating in Sinop and the surrounding region, which are going undiagnosed. In addition, molecular and evolutionary analyses indicate that two MAYV genotypes are co-circulating in Mato Grosso, Brazil. Thus, a strong surveillance program must be implemented to evaluate and monitor the distribution and the true importance of non-dengue arboviruses in the etiology of acute febrile illnesses.

  19. Complex modulation of the Aedes aegypti transcriptome in response to dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Mariangela Bonizzoni

    Full Text Available Dengue fever is the most important arboviral disease world-wide, with Aedes aegypti being the major vector. Interactions between the mosquito host and dengue viruses (DENV are complex and vector competence varies among geographically-distinct Ae. aegypti populations. Additionally, dengue is caused by four antigenically-distinct viral serotypes (DENV1-4, each with multiple genotypes. Each virus genotype interacts differently with vertebrate and invertebrate hosts. Analyses of alterations in mosquito transcriptional profiles during DENV infection are expected to provide the basis for identifying networks of genes involved in responses to viruses and contribute to the molecular-genetic understanding of vector competence. In addition, this knowledge is anticipated to support the development of novel disease-control strategies. RNA-seq technology was used to assess genome-wide changes in transcript abundance at 1, 4 and 14 days following DENV2 infection in carcasses, midguts and salivary glands of the Ae. aegypti Chetumal strain. DENV2 affected the expression of 397 Ae. aegypti genes, most of which were down-regulated by viral infection. Differential accumulation of transcripts was mainly tissue- and time-specific. Comparisons of our data with other published reports reveal conservation of functional classes, but limited concordance of specific mosquito genes responsive to DENV2 infection. These results indicate the necessity of additional studies of mosquito-DENV interactions, specifically those focused on recently-derived mosquito strains with multiple dengue virus serotypes and genotypes.

  20. Complex modulation of the Aedes aegypti transcriptome in response to dengue virus infection.

    Science.gov (United States)

    Bonizzoni, Mariangela; Dunn, W Augustine; Campbell, Corey L; Olson, Ken E; Marinotti, Osvaldo; James, Anthony A

    2012-01-01

    Dengue fever is the most important arboviral disease world-wide, with Aedes aegypti being the major vector. Interactions between the mosquito host and dengue viruses (DENV) are complex and vector competence varies among geographically-distinct Ae. aegypti populations. Additionally, dengue is caused by four antigenically-distinct viral serotypes (DENV1-4), each with multiple genotypes. Each virus genotype interacts differently with vertebrate and invertebrate hosts. Analyses of alterations in mosquito transcriptional profiles during DENV infection are expected to provide the basis for identifying networks of genes involved in responses to viruses and contribute to the molecular-genetic understanding of vector competence. In addition, this knowledge is anticipated to support the development of novel disease-control strategies. RNA-seq technology was used to assess genome-wide changes in transcript abundance at 1, 4 and 14 days following DENV2 infection in carcasses, midguts and salivary glands of the Ae. aegypti Chetumal strain. DENV2 affected the expression of 397 Ae. aegypti genes, most of which were down-regulated by viral infection. Differential accumulation of transcripts was mainly tissue- and time-specific. Comparisons of our data with other published reports reveal conservation of functional classes, but limited concordance of specific mosquito genes responsive to DENV2 infection. These results indicate the necessity of additional studies of mosquito-DENV interactions, specifically those focused on recently-derived mosquito strains with multiple dengue virus serotypes and genotypes.

  1. Epidemiological studies on dengue virus type 3 in Playa municipality, Havana, Cuba, 2001-2002.

    Science.gov (United States)

    Guzman, Maria G; Alvarez, Angel; Vazquez, Susana; Alvarez, Mayling; Rosario, Delfina; Pelaez, Otto; Cruz, Guillermo; Rodriguez, Rosmari; Pavon, Alequis; Gonzalez, Annia; Morier, Luis; Ruiz, Dydie; Kouri, Gustavo; Halstead, Scott B

    2012-03-01

    Recognizing the uniqueness of secondary dengue virus (DENV)-1/3 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) cases at an interval of 24 years, we sought to estimate DENV infections as well as the ratios between mild disease and DHF/DSS by DENV infection sequence in Playa District (Havana, Cuba) during the 2001-2002 outbreak of dengue virus type 3 (DENV-3). A retrospective seroepidemiological study was conducted in 2003 in Playa District. Blood samples were collected from a 1% random sample of residents and were studied for the prevalence of dengue neutralizing antibodies. DENV-3 was found to have infected 7.2% (95% confidence interval (95% CI) 6.0-8.4%) of susceptible individuals (the entire cohort), the majority of whom experienced silent infections. Virtually every individual who had a secondary infection in the sequence DENV-1 then DENV-3 became ill, with a ratio of severe to mild cases of 1:35 (95% CI 1:67-1:23). Secondary infections in the sequence DENV-2/3 were less pathogenic than DENV-1/3. Mild disease accompanying secondary DENV2/3 occurred at a ratio of 1:4.49 infections (95% CI 1:5.77-1:3.42) secondary infections. The results obtained highlight the role of the infecting serotype and also the sequence of the viral infection in the clinical outcome of a dengue infection. Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  2. Recent progress in dengue vaccine development

    Institute of Scientific and Technical Information of China (English)

    Jianchun; Wei; Hui; Chen; Jing; An

    2014-01-01

    Dengue virus(DENV) has four distinct serotypes. DENV infection can result in classic dengue fever and life-threatening dengue hemorrhagic fever/dengue shock syndrome. In recent decades, DENV infection has become an important public health concern in epidemic-prone areas. Vaccination is the most effective measure to prevent and control viral infections. However, several challenges impede the development of effective DENV vaccines, such as the lack of suitable animal models and the antibody-dependent enhancement phenomenon. Although no licensed DENV vaccine is available, significant progress has been made. This review summarizes candidate DENV vaccines from recent investigations.

  3. Characterization of dengue virus infections in a sample of patients suggests unique clinical, immunological, and virological profiles that impact on the diagnosis of dengue and dengue hemorrhagic fever.

    Science.gov (United States)

    Senaratne, Thamarasi; Wimalaratne, Harith; Alahakoon, D G S; Gunawardane, Nirmali; Carr, Jillian; Noordeen, Faseeha

    2016-10-01

    Dengue virus (DENV) infections are increasing with respect to incidence and severity in the Central Province of Sri Lanka. The objective of this study was to define the clinical, immunological, and virological profiles of patients admitted to the General Hospital, Kandy with clinically apparent dengue. Demographic, clinical, hematological parameters, liver enzymes (ALT and AST), and blood samples were collected from 292 patients with fever dengue fever/dengue hemorrhagic fever (DF/DHF). Samples were analyzed for, anti-DENV IgM, IgG, and DENV nucleic acid. Myalgia was the commonest complaint by 65% of the patients. Packed cell volume was >45% in 27% of the patients while 42.12% had reduced platelets and 62.67% had reduced white blood cell counts. In contrast to other studies, positive tourniquet test (PTT) and petechiae were not major indicators of DENV infection or severity of the disease. Clinical profiles were significantly different between DF and DHF/DSS and showed many similarities to that reported elsewhere. Altogether, 43 patients (14.73%) were viremic as detected by RT-PCR; 181 patients (62%) were positive for anti-DENV IgM, and 245 (84%) patients were positive for anti-DENV IgG. In combination, anti-DENV IgM and RT-PCR assays detected 224 (77.5%) of DENV infected cases, thus improving the DENV diagnosis rate. Hence, the diagnostic utility of PTT, anti-DENV IgM/IgG serology, or RT-PCR used alone in the early phase of illness is low in Sri Lanka but the diagnostic value can be improved by a combination of serology and RT-PCR. J. Med. Virol. 88:1703-1710, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Natural vertical transmission of dengue viruses in Aedes aegypti in selected sites in Cebu City, Philippines.

    Science.gov (United States)

    Edillo, Frances E; Sarcos, Janet R; Sayson, Stephanie L

    2015-12-01

    We attempted to determine the vertical transmission of dengue virus (DENV) in Aedes aegypti in selected sites in Cebu City, Philippines. Mosquito sub-adults were collected monthly from households and the field during the wet-dry-wet season from November, 2011 to July, 2012 and were laboratory-reared to adults. Viral RNA extracts in mosquitoes were assayed by hemi-nested RT-PCR. Results showed that 62 (36.26%; n=679) out of 171 mosquito pools (n=2,871) were DENV+. The minimum infection rate (MIR) of DENV ranged from 0 in wet months to 48.22/1,000 mosquitoes in April, 2012 (mid-dry). DENVs were detected in larvae, pupae, and male and female adults, with DENV-4, DENV-3, and DENV-1, in that rank of prevalence. DENV-1 co-infected with either DENV-3 or -4 or with both in April, 2012; DENV-3 and -4 were present in both seasons. More DENV+ mosquitoes were collected from households than in field premises (p0.05). By Generalized Linear Mixed models, the type of premises nested in sites and monthly total rainfall were significant predictors of monthly dengue cases (pdengue outbreak.

  5. Antibodies against the Envelope Glycoprotein Promote Infectivity of Immature Dengue Virus Serotype 2

    NARCIS (Netherlands)

    Voorham, Julia M. da Silva; Rodenhuis-Zybert, Izabela A.; Nunez, Nilda Vanesa Ayala; Colpitts, Tonya M.; van der Ende-Metselaar, Heidi; Fikrig, Erol; Diamond, Michael S.; Wilschut, Jan; Smit, Jolanda M.

    2012-01-01

    Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM

  6. Genomic analysis and growth characteristic of dengue viruses from Makassar, Indonesia.

    Science.gov (United States)

    Sasmono, R Tedjo; Wahid, Isra; Trimarsanto, Hidayat; Yohan, Benediktus; Wahyuni, Sitti; Hertanto, Martin; Yusuf, Irawan; Mubin, Halim; Ganda, Idham J; Latief, Rachmat; Bifani, Pablo J; Shi, Pei-Yong; Schreiber, Mark J

    2015-06-01

    Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makassar, we carried out dengue fever surveillance study during 2007-2010. A total of 455 patients were recruited, in which antigen and serological detection revealed the confirmed dengue cases in 43.3% of patients. Molecular detection confirmed the dengue cases in 27.7% of patients, demonstrating that dengue places a significant disease burden on the community. Serotyping revealed that dengue virus serotype 1 (DENV-1) was the most predominant serotype, followed by DENV-2, -3, and -4. To determine the molecular evolution of the viruses, we conducted whole-genome sequencing of 80 isolates. Phylogenetic analysis grouped DENV-2, -3 and -4 to the Cosmopolitan genotype, Genotype I and Genotype II, respectively. Intriguingly, each serotype paints a different picture of evolution and transmission. DENV-1 appears to be undergoing a clade replacement with Genotype IV being supplanted by Genotype I. The Cosmopolitan DENV-2 isolates were found to be regionally endemic and is frequently being exchanged between countries in the region. By contrast, DENV-3 and DENV-4 isolates were related to strains with a long history in Indonesia although the DENV-3 strains appear to have been following a distinct evolutionary path since approximately 1998. To assess whether the various DENV serotypes/genotypes possess different growth characteristics, we performed growth kinetic assays on selected viruses. We observed the relatively higher rate of replication for DENV-1 and -2 compared to DENV-3 and -4. Within the DENV-1, viruses from Genotype I grow faster than that of Genotype IV. This higher replication rate may underlie their ability to replace the

  7. Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

    Institute of Scientific and Technical Information of China (English)

    Piyathida Pongsiri; Kesmanee Praianantathavorn; Apiradee Theamboonlers; Sunchai Payungporn; Yong Poovorawan

    2012-01-01

    ABSTRACT Objective:To develop diagnostic test for detection chikungunya virus (CHIKV and Dengue virus (DENV)infection.Methods:We have performed a rapid, accurate laboratory confirmative method to simultaneously detect, quantify and differentiateCHIKV and DENV infection by single-step multiplex real-timeRT-PCR.Results: The assay’s sensitivity was97.65%, specificity was 92.59% and accuracy was95.82% when compared to conventional RT-PCR. Additionally, there was no cross-reaction betweenCHIKV, DENV, Japanese encephalitis virus, hepatitis C, hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis ofCHIKV andDENV in a single-step reaction.

  8. Complete nucleotide sequence analysis of a Dengue-1 virus isolated on Easter Island, Chile.

    Science.gov (United States)

    Cáceres, C; Yung, V; Araya, P; Tognarelli, J; Villagra, E; Vera, L; Fernández, J

    2008-01-01

    Dengue-1 viruses responsible for the dengue fever outbreak in Easter Island in 2002 were isolated from acute-phase sera of dengue fever patients. In order to analyze the complete genome sequence, we designed primers to amplify contiguous segments across the entire sequence of the viral genome. RT-PCR products obtained were cloned, and complete nucleotide and deduced amino acid sequences were determined. This report constitutes the first complete genetic characterization of a DENV-1 isolate from Chile. Phylogenetic analysis shows that an Easter Island isolate is most closely related to Pacific DENV-1 genotype IV viruses.

  9. Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection-enhancing antibody response.

    Science.gov (United States)

    Sjatha, Fithriyah; Kuwahara, Miwa; Sudiro, T Mirawati; Kameoka, Masanori; Konishi, Eiji

    2014-02-01

    Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) gene-based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.

  10. Potential Antivirals: Natural Products Targeting Replication Enzymes of Dengue and Chikungunya Viruses

    Directory of Open Access Journals (Sweden)

    Ana Flávia Costa da Silveira Oliveira

    2017-03-01

    Full Text Available Dengue virus (DENV and chikungunya virus (CHIKV are reemergent arboviruses that are transmitted by mosquitoes of the Aedes genus. During the last several decades, these viruses have been responsible for millions of cases of infection and thousands of deaths worldwide. Therefore, several investigations were conducted over the past few years to find antiviral compounds for the treatment of DENV and CHIKV infections. One attractive strategy is the screening of compounds that target enzymes involved in the replication of both DENV and CHIKV. In this review, we describe advances in the evaluation of natural products targeting the enzymes involved in the replication of these viruses.

  11. Dengue viruses binding proteins from Aedes aegypti and Aedes polynesiensis salivary glands

    Directory of Open Access Journals (Sweden)

    Cao-Lormeau Van-Mai

    2009-03-01

    Full Text Available Abstract Dengue virus (DENV, the etiological agent of dengue fever, is transmitted to the human host during blood uptake by an infective mosquito. Infection of vector salivary glands and further injection of infectious saliva into the human host are key events of the DENV transmission cycle. However, the molecular mechanisms of DENV entry into the mosquito salivary glands have not been clearly identified. Otherwise, although it was demonstrated for other vector-transmitted pathogens that insect salivary components may interact with host immune agents and impact the establishment of infection, the role of mosquito saliva on DENV infection in human has been only poorly documented. To identify salivary gland molecules which might interact with DENV at these key steps of transmission cycle, we investigated the presence of proteins able to bind DENV in salivary gland extracts (SGE from two mosquito species. Using virus overlay protein binding assay, we detected several proteins able to bind DENV in SGE from Aedes aegypti (L. and Aedes polynesiensis (Marks. The present findings pave the way for the identification of proteins mediating DENV attachment or entry into mosquito salivary glands, and of saliva-secreted proteins those might be bound to the virus at the earliest step of human infection. The present findings might contribute to the identification of new targets for anti-dengue strategies.

  12. Dengue Virus Type 2: Protein Binding and Active Replication in Human Central Nervous System Cells

    Directory of Open Access Journals (Sweden)

    Ma Isabel Salazar

    2013-01-01

    Full Text Available An increased number of dengue cases with neurological complications have been reported in recent years. The lack of reliable animal models for dengue has hindered studies on dengue virus (DENV pathogenesis and cellular tropism in vivo. We further investigate the tropism of DENV for the human central nervous system (CNS, characterizing DENV interactions with cell surface proteins in human CNS cells by virus overlay protein binding assays (VOPBA and coimmunoprecipitations. In VOPBA, three membrane proteins (60, 70, and 130 kDa from the gray matter bound the entire virus particle, whereas only a 70 kDa protein bound in white matter. The coimmunoprecipitation assays revealed three proteins from gray matter consistently binding virus particles, one clearly distinguishable protein (~32 kDa and two less apparent proteins (100 and 130 kDa. Monoclonal anti-NS3 targeted the virus protein in primary cell cultures of human CNS treated with DENV-2, which also stained positive for NeuH, a neuron-specific marker. Thus, our results indicate (1 that DENV-2 exhibited a direct tropism for human neurons and (2 that human neurons sustain an active DENV replication as was demonstrated by the presence of the NS3 viral antigen in primary cultures of these cells treated with DENV-2.

  13. Dengue virus 2 American-Asian genotype identified during the 2006/2007 outbreak in Piauí, Brazil reveals a Caribbean route of introduction and dissemination of dengue virus in Brazil.

    Science.gov (United States)

    Barcelos Figueiredo, Leandra; Sakamoto, Tetsu; Leomil Coelho, Luiz Felipe; de Oliveira Rocha, Eliseu Soares; Gomes Cota, Marcela Menezes; Ferreira, Gustavo Portela; de Oliveira, Jaquelline Germano; Kroon, Erna Geessien

    2014-01-01

    Dengue virus (DENV) is the most widespread arthropod-borne virus, and the number and severity of outbreaks has increased worldwide in recent decades. Dengue is caused by DENV-1, DENV- 2, DENV-3 and DENV-4 which are genetically distant. The species has been subdivided into genotypes based on phylogenetic studies. DENV-2, which was isolated from dengue fever patients during an outbreak in Piaui, Brazil in 2006/2007 was analyzed by sequencing the envelope (E) gene. The results indicated a high similarity among the isolated viruses, as well as to other DENV-2 from Brazil, Central America and South America. A phylogenetic and phylogeographic analysis based on DENV-2E gene sequences revealed that these viruses are grouped together with viruses of the American-Asian genotype in two distinct lineages. Our results demonstrate the co-circulation of two American-Asian genotype lineages in northeast Brazil. Moreover, we reveal that DENV-2 lineage 2 was detected in Piauí before it disseminated to other Brazilian states and South American countries, indicating the existence of a new dissemination route that has not been previously described.

  14. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

    Science.gov (United States)

    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  15. Dengue virus capsid protein binds core histones and inhibits nucleosome formation in human liver cells.

    Directory of Open Access Journals (Sweden)

    Tonya M Colpitts

    Full Text Available Dengue virus (DENV is a member of the Flaviviridae and a globally (reemerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection.

  16. First isolation of dengue virus from Lao PDR in a Chinese traveler

    Science.gov (United States)

    2013-01-01

    Background Epidemic dengue activity has been demonstrated in several southern regions of China, but not in Yunnan province, which borders countries in Southeast Asia where dengue is endemic. Many dengue cases imported from Southeast Asia to Yunnan have been reported, but dengue virus (DENV) has not been isolated from any patients. This study is the first to report the isolation of DENV from a Chinese traveler returning to Yunnan from Lao PDR. Findings A serum sample was collected from a patient presenting with a febrile illness who returned from Lao PDR in 2009 and was used to inoculate Aedes albopictus C6/36 cells for viral isolation. The viral isolate was identified using reverse transcription-polymerase chain reaction, and phylogenetic analyses based on the full E sequence were performed using Clustalx 1.8 software. The analyses detected DENV genome, and thus, a DENV isolate was obtained from the patient’s serum sample. The new DENV isolate was grouped into genotype Asia 1, serotype 2. The viral E protein shared the greatest nucleotide sequence identity (99.6%) with the D2/Thailand/0606aTw strain isolated from Thailand in 2006 and demonstrated 94.3% to 100% identity with the predicted amino acid sequence of other DENV 2 strains. Conclusions Our findings indicate that DENV serotype 2 is circulating in Lao PDR, and surveillance of patients suspected of infection with dengue should be conducted not only by a serological test but also by pathogenic detection methods. PMID:23497045

  17. Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

    Science.gov (United States)

    Jeewandara, Chandima; Adikari, Thiruni N.; Gomes, Laksiri; Fernando, Samitha; Fernando, R. H.; Perera, M. K. T.; Ariyaratne, Dinuka; Kamaladasa, Achala; Salimi, Maryam; Prathapan, Shamini

    2015-01-01

    Background Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood. Methodology/Principal findings Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone. We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus. Conclusions/significance The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials. PMID:25875020

  18. Functionality of dengue virus specific memory T cell responses in individuals who were hospitalized or who had mild or subclinical dengue infection.

    Directory of Open Access Journals (Sweden)

    Chandima Jeewandara

    2015-04-01

    Full Text Available Although antibody responses to dengue virus (DENV in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood.Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338, who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02, those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03 or TNFα alone. We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus.The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.

  19. Pichia pastoris-Expressed Dengue 2 Envelope Forms Virus-Like Particles without Pre-Membrane Protein and Induces High Titer Neutralizing Antibodies

    OpenAIRE

    Shailendra Mani; Lav Tripathi; Rajendra Raut; Poornima Tyagi; Upasana Arora; Tarani Barman; Ruchi Sood; Alka Galav; Wahala Wahala; Aravinda de Silva; Sathyamangalam Swaminathan; Navin Khanna

    2013-01-01

    Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust imm...

  20. Kinetics of the association of dengue virus capsid protein with the granular component of nucleolus.

    Science.gov (United States)

    Tiwary, Ashish Kumar; Cecilia, D

    2017-02-01

    Dengue virus (DENV) replicates in the cytoplasm but translocation of the capsid protein (C) to the nucleoli of infected cells has been shown to facilitate virus multiplication for DENV-2. This study demonstrates that the nucleolar localization of C occurs with all four serotypes of DENV. The interaction of C with the nucleolus was found to be dynamic with a mobile fraction of 66% by FRAP. That the C shuttled between the nucleus and cytoplasm was suggested by FLIP and translation inhibition experiments. Colocalization with B23 indicated that DENV C targeted the granular component (GC) of the nucleolus. Presence of DENV C in the nucleolus affected the recovery kinetics of B23 in infected and transfected cells. Sub-nucleolar localization of DENV C of all serotypes to the GC, its mobility in and out of the nucleolus and its affect on the dynamics of B23 is being shown for the first time. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Towards a Casa Segura: a consumer product study of the effect of insecticide-treated curtains on Aedes aegypti and dengue virus infections in the home.

    Science.gov (United States)

    Loroño-Pino, María Alba; García-Rejón, Julián E; Machain-Williams, Carlos; Gomez-Carro, Salvador; Nuñez-Ayala, Guadalupe; Nájera-Vázquez, Maria del Rosario; Losoya, Arturo; Aguilar, Lyla; Saavedra-Rodriguez, Karla; Lozano-Fuentes, Saul; Beaty, Meaghan K; Black, William C; Keefe, Thomas J; Eisen, Lars; Beaty, Barry J

    2013-08-01

    The home, or domicile, is the principal environment for transmission of dengue virus (DENV) between humans and mosquito vectors. Community-wide distribution of insecticide-treated curtains (ITCs), mimicking vector control program-driven interventions, has shown promise to reduce DENV infections. We conducted a Casa Segura consumer product intervention study in Mérida, Mexico to determine the potential to reduce intradomicillary DENV transmission through ITC use in individual homes. Dengue virus infections in mosquitoes and in humans were reduced in homes with ITCs in one of two study subareas. Overall, ITCs reduced intradomicillary DENV transmission; ITC homes were significantly less likely to experience multiple DENV infections in humans than NTC homes. Dengue virus-infected Aedes aegypti females were reduced within the ITC homes where curtain use was highest. Some homes yielded up to nine infected Ae. aegypti females. This study provides insights regarding best practices for Casa Segura interventions to protect homes from intradomicillary DENV transmission.

  2. Proteomic profile of human monocytic cells infected with dengue virus

    Institute of Scientific and Technical Information of China (English)

    Viviana Martnez-Betancur; Marlen Martnez-Gutierrez

    2016-01-01

    Objective: To identify the changes in the proteome of U937 cells infected with dengue virus (DENV). Methods: In this study, differentiated U937 cultures were infected with two DENV-2 strains, one of which was associated with dengue (DENV-2/NG) and the other one with severe dengue (DENV-2/16681), with the aim of determining the cellular proteomic profiles under different infection conditions. Cellular proteins were extracted and sepa-rated by two-dimensional electrophoresis, and those proteins with differential expression profiles were identified by mass spectrometry. The obtained results were correlated with cellular viability, the number of infectious viral particles, and the viral DNA/protein quantity. Results: In comparison with non-infected cultures, in the cells infected with the DENV-2/NG strain, nine proteins were expressed differentially (five were upregulated and four were downregulated); in those cultures infected with the DENV-2/16681 strain, six proteins were differentially expressed (two were downregulated and four were upregu-lated). The downregulated proteins included fatty acid-binding protein, heterogeneous nuclear ribonucleoprotein 1, protein disulfide isomerase, enolase 1, heat shock 70 kDa protein 9, phosphotyrosyl phosphatase, and annexin IV. The upregulated proteins included heat shock 90 kDa protein AA1, tubulin beta, enolase 1, pyruvate kinase, transaldolase and phospholipase C-alpha. Conclusions: Because the monocyte/macrophage lineage is critical for disease patho-genicity, additional studies on these proteins could provide a better understanding of the cellular response to DENV infection and could help identify new therapeutic targets against infection.

  3. Dengue virus type 3 in Brazil: a phylogenetic perspective

    Directory of Open Access Journals (Sweden)

    Josélio Maria Galvão de Araújo

    2009-05-01

    Full Text Available 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.

  4. Longitudinal Analysis of Natural Killer Cells in Dengue Virus-Infected Patients in Comparison to Chikungunya and Chikungunya/Dengue Virus-Infected Patients.

    Directory of Open Access Journals (Sweden)

    Caroline Petitdemange

    2016-03-01

    Full Text Available Dengue virus (DENV is the most prominent arbovirus worldwide, causing major epidemics in South-East Asia, South America and Africa. In 2010, a major DENV-2 outbreak occurred in Gabon with cases of patients co-infected with chikungunya virus (CHIKV. Although the innate immune response is thought to be of primordial importance in the development and outcome of arbovirus-associated pathologies, our knowledge of the role of natural killer (NK cells during DENV-2 infection is in its infancy.We performed the first extensive comparative longitudinal characterization of NK cells in patients infected by DENV-2, CHIKV or both viruses. Hierarchical clustering and principal component analyses were performed to discriminate between CHIKV and DENV-2 infected patients.We observed that both activation and differentiation of NK cells are induced during the acute phase of infection by DENV-2 and CHIKV. Combinatorial analysis however, revealed that both arboviruses induced two different signatures of NK-cell responses, with CHIKV more associated with terminal differentiation, and DENV-2 with inhibitory KIRs. We show also that intracellular production of interferon-γ (IFN-γ by NK cells is strongly stimulated in acute DENV-2 infection, compared to CHIKV.Although specific differences were observed between CHIKV and DENV-2 infections, the significant remodeling of NK cell populations observed here suggests their potential roles in the control of both infections.

  5. Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses.

    Directory of Open Access Journals (Sweden)

    Stefan W Metz

    2016-10-01

    Full Text Available Dengue virus (DENV is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE, purified and adsorbed to poly (lactic-co-glycolic acid (PLGA nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.

  6. Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

    Science.gov (United States)

    Hoekstra, Gabriel; Yi, Xianwen; Stone, Michelle; Horvath, Katie; Miley, Michael J.; DeSimone, Joseph; Luft, Chris J.; de Silva, Aravinda M.

    2016-01-01

    Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika. PMID:27764114

  7. Antiviral activity of four types of bioflavonoid against dengue virus type-2

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    Zandi Keivan

    2011-12-01

    Full Text Available Abstract Background Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2 in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA and quantitative RT-PCR. Selectivity Index value (SI was determined as the ratio of cytotoxic concentration 50 (CC50 to inhibitory concentration 50 (IC50 for each compound. Results The half maximal inhibitory concentration (IC50 of quercetin against dengue virus was 35.7 μg mL-1 when it was used after virus adsorption to the cells. The IC50 decreased to 28.9 μg mL-1 when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07 and 8.74 μg mL-1, respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited anti-adsorption effects against DENV-2 with IC50 = 168.2 μg mL-1 and its related SI was 1.3. Daidzein showed a weak anti-dengue activity with IC50 = 142.6 μg mL-1 when the DENV-2 infected cells were treated after virus adsorption. The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The findings obtained from Foci Forming Unit Reduction Assay (FFURA were corroborated by findings of the qRT-PCR assays. Quercetin and daidzein (50 μg mL-1 reduced DENV-2 RNA levels by 67% and 25%, respectively. There was no significant inhibition of DENV-2 RNA levels with naringin and hesperetin. Conclusion Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory activities. Other

  8. Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability

    OpenAIRE

    Henry Puerta-Guardo; Dustin R Glasner; Eva Harris

    2016-01-01

    Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV) non-structural protein 1 (NS1) in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate to...

  9. IRAV (FLJ11286), an Interferon-Stimulated Gene with Antiviral Activity against Dengue Virus, Interacts with MOV10

    Science.gov (United States)

    Balinsky, Corey A.; Schmeisser, Hana; Wells, Alexandra I.; Ganesan, Sundar; Jin, Tengchuan; Singh, Kavita

    2016-01-01

    ABSTRACT Dengue virus (DENV) is a member of the genus Flavivirus and can cause severe febrile illness. Here, we show that FLJ11286, which we refer to as IRAV, is induced by DENV in an interferon-dependent manner, displays antiviral activity against DENV, and localizes to the DENV replication complex. IRAV is an RNA binding protein and localizes to cytoplasmic processing bodies (P bodies) in uninfected cells, where it interacts with the MOV10 RISC complex RNA helicase, suggesting a role for IRAV in the processing of viral RNA. After DENV infection, IRAV, along with MOV10 and Xrn1, localizes to the DENV replication complex and associates with DENV proteins. Depletion of IRAV or MOV10 results in an increase in viral RNA. These data serve to characterize an interferon-stimulated gene with antiviral activity against DENV, as well as to propose a mechanism of activity involving the processing of viral RNA. IMPORTANCE Dengue virus, a member of the family Flaviviridae, can result in a life-threatening illness and has a significant impact on global health. Dengue virus has been shown to be particularly sensitive to the effects of type I interferon; however, little is known about the mechanisms by which interferon-stimulated genes function to inhibit viral replication. A better understanding of the interferon-mediated antiviral response to dengue virus may aid in the development of novel therapeutics. Here, we examine the influence of the interferon-stimulated gene IRAV (FLJ11286) on dengue virus replication. We show that IRAV associates with P bodies in uninfected cells and with the dengue virus replication complex after infection. IRAV also interacts with MOV10, depletion of which is associated with increased viral replication. Our results provide insight into a newly identified antiviral gene, as well as broadening our understanding of the innate immune response to dengue virus infection. PMID:27974568

  10. Suppression of chikungunya virus replication and differential innate responses of human peripheral blood mononuclear cells during co-infection with dengue virus

    NARCIS (Netherlands)

    Silva, Mariana Ruiz; Briseno, Jose A. Aguilar; Upasani, Vinit; van der Ende-Metselaar, Heidi; Smit, Jolanda M.; Rodenhuis-Zybert, Izabela A.

    2017-01-01

    Dengue and chikungunya are viral diseases transmitted to humans by infected Aedes spp. mosquitoes. With an estimated 390 million infected people per year dengue virus (DENV) currently causes the most prevalent arboviral disease. During the last decade chikungunya virus (CHIKV) has caused large

  11. Suppression of chikungunya virus replication and differential innate responses of human peripheral blood mononuclear cells during co-infection with dengue virus

    NARCIS (Netherlands)

    Ruiz Silva, Mariana; Aguilar Briseño, José A; Upasani, Vinit; van der Ende-Metselaar, Heidi; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A

    2017-01-01

    Dengue and chikungunya are viral diseases transmitted to humans by infected Aedes spp. mosquitoes. With an estimated 390 million infected people per year dengue virus (DENV) currently causes the most prevalent arboviral disease. During the last decade chikungunya virus (CHIKV) has caused large outbr

  12. Improved Serotype-Specific Dengue Virus Detection in Trinidad and Tobago using a Multiplex, Real-Time RT-PCR

    Science.gov (United States)

    Waggoner, Jesse J.; Sahadeo, Nikita S. D.; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V. F.; Pinsky, Benjamin A.

    2014-01-01

    Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance is limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time RT-PCR detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; p=0.01). PMID:25533614

  13. Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

    OpenAIRE

    Meri Bordignon Nogueira; Vanessa Stella; Juliano Bordignon; Weber Cheli Batista; Luana de Borba; Luis Hildebrando Pereira da Silva; Federico Guillermo Hoffmann; Christian Macagnan Probst; Claudia Nunes Duarte dos Santos

    2008-01-01

    The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast ...

  14. Diagnostic Options and Challenges for Dengue and Chikungunya Viruses

    Directory of Open Access Journals (Sweden)

    Stacey K. Mardekian

    2015-01-01

    Full Text Available Dengue virus (DENV and Chikungunya virus (CHIKV are arboviruses that share the same Aedes mosquito vectors and thus overlap in their endemic areas. These two viruses also cause similar clinical presentations, especially in the initial stages of infection, with neither virus possessing any specific distinguishing clinical features. Because the outcomes and management strategies for these two viruses are vastly different, early and accurate diagnosis is imperative. Diagnosis is also important for surveillance, outbreak control, and research related to vaccine and drug development. Available diagnostic tests are aimed at detection of the virus, its antigenic components, or the host immune antibody response. In this review, we describe the recent progress and continued challenges related to the diagnosis of DENV and CHIKV infections.

  15. Diagnostic Options and Challenges for Dengue and Chikungunya Viruses.

    Science.gov (United States)

    Mardekian, Stacey K; Roberts, Amity L

    2015-01-01

    Dengue virus (DENV) and Chikungunya virus (CHIKV) are arboviruses that share the same Aedes mosquito vectors and thus overlap in their endemic areas. These two viruses also cause similar clinical presentations, especially in the initial stages of infection, with neither virus possessing any specific distinguishing clinical features. Because the outcomes and management strategies for these two viruses are vastly different, early and accurate diagnosis is imperative. Diagnosis is also important for surveillance, outbreak control, and research related to vaccine and drug development. Available diagnostic tests are aimed at detection of the virus, its antigenic components, or the host immune antibody response. In this review, we describe the recent progress and continued challenges related to the diagnosis of DENV and CHIKV infections.

  16. Molecular epidemiology of dengue viruses in southern China from 1978 to 2006

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    Liu Jinhua

    2011-06-01

    Full Text Available Abstract To investigate molecular epidemiology of dengue viruses (DENV in southern China, a total of 14 dengue isolates were collected in southern China during each epidemic year between 1978 and 2006 and their full-length genome sequences were obtained by using RT-PCR method. The E gene sequences from additional 6 dengue fever patients in Guangzhou in 2006 were also obtained by using RT-PCR method. Combined with DENVs sequences published in GenBank, phylogenetic analysis and recombination analysis were performed. One hundred and twenty-five E gene sequences and 60 complete genome sequences published in the GenBank were also involved. Phylogenetic analysis showed that there was a wide genetic diversity of DENVs isolated in southern China. DENV-1 strains exist in almost all of the clades of genotype I and IV except the Asia 1 clade of genotype I; DENV-2 stains are grouped into four of the five genotypes except American genotype. DENV-4 strains are grouped into 2 genotypes (I and II. Phylogenetic analysis also showed that all DENV-4 isolates and two DENV-2 isolates were closely related to the prior isolates from neighboring Southeast Asia countries. The DENV-1 strain isolated during the 2006 epidemic is highly homologous to the strains isolated during the 2001 epidemic. Recombination analysis showed no inter-serotype recombination, but 22 intra-serotype recombination events were found across the 32 complete genomes of all Chinese isolates. The study suggested that dengue fever epidemic in Southern China over the past 30 years presented two important modes, 1 imported-cases-induced endemic prevalence; 2 endogenous epidemic outbreak with natural epidemic focus. Recombination may play an important role in dengue virus evolution and adaptation.

  17. Structural modeling and analysis of dengue-mediated inhibition of interferon signaling pathway.

    NARCIS (Netherlands)

    Aslam, B; Ahmad, J; Ali, a; Paracha, R Z; Tareen, S H K; Khusro, S; Ahmad, T; Muhammad, S a; Niazi6 And V Azevedo, U

    2015-01-01

    Dengue virus (DENV) belongs to the family Flaviviridae and can cause major health problems worldwide, including dengue fever and dengue shock syndrome. DENV replicon in human cells inhibits interferon alpha and beta with the help of its non-structural proteins. Non-structural protein 5 (NS5) of DENV

  18. Development and characterization of a reverse genetic system for studying dengue virus serotype 3 strain variation and neutralization.

    Directory of Open Access Journals (Sweden)

    William B Messer

    Full Text Available Dengue viruses (DENV are enveloped single-stranded positive-sense RNA viruses transmitted by Aedes spp. mosquitoes. There are four genetically distinct serotypes designated DENV-1 through DENV-4, each further subdivided into distinct genotypes. The dengue scientific community has long contended that infection with one serotype confers lifelong protection against subsequent infection with the same serotype, irrespective of virus genotype. However this hypothesis is under increased scrutiny and the role of DENV genotypic variation in protection from repeated infection is less certain. As dengue vaccine trials move increasingly into field-testing, there is an urgent need to develop tools to better define the role of genotypic variation in DENV infection and immunity. To better understand genotypic variation in DENV-3 neutralization and protection, we designed and constructed a panel of isogenic, recombinant DENV-3 infectious clones, each expressing an envelope glycoprotein from a different DENV-3 genotype; Philippines 1982 (genotype I, Thailand 1995 (genotype II, Sri Lanka 1989 and Cuba 2002 (genotype III and Puerto Rico 1977 (genotype IV. We used the panel to explore how natural envelope variation influences DENV-polyclonal serum interactions. When the recombinant viruses were tested in neutralization assays using immune sera from primary DENV infections, neutralization titers varied by as much as ∼19-fold, depending on the expressed envelope glycoprotein. The observed variability in neutralization titers suggests that relatively few residue changes in the E glycoprotein may have significant effects on DENV specific humoral immunity and influence antibody mediated protection or disease enhancement in the setting of both natural infection and vaccination. These genotypic differences are also likely to be important in temporal and spatial microevolution of DENV-3 in the background of heterotypic neutralization. The recombinant and synthetic tools

  19. Infecciones concurrentes por dos serotipos del virus dengue durante un brote en el noroeste de Perú, 2008

    OpenAIRE

    Mamani, Enrique; Laboratorio de Arbovirus, Centro Nacional de Salud Pública, Instituto Nacional de Salud. Lima, Perú. Biólogo. Magister en Microbiología.; Figueroa, Dana; Laboratorio de Arbovirus, Centro Nacional de Salud Pública, Instituto Nacional de Salud. Lima, Perú. Biólogo.; García, María Paquita; Laboratorio de Arbovirus, Centro Nacional de Salud Pública, Instituto Nacional de Salud. Lima, Perú. Tecnóloga Médico.; Garaycochea, María del Carmen; Laboratorio de Arbovirus, Centro Nacional de Salud Pública, Instituto Nacional de Salud. Lima, Perú. Biólogo.; Pozo, Edwar J.; Oficina de Epidemiología, Subregión de Salud Luciano Castilla. Sullana, Perú. Biólogo.

    2010-01-01

    Objetives. To establish the existence of concurrent infections by different dengue virus (DENV) serotypes in an outbreak in the Northwestern in Peru during 2008. Material and methods. 73 serum samples from patients with dengue were analyzed during an outbreak that occurred in Northwestern in Peru between May and June 2008. Molecular biology techniques were used to serotype the DENV, thus, firstly the viral RNA viral was extracted using Viral QIAamp RNA mini kit (Qiagen, Valencia, Californ...

  20. Insights into the molecular evolution of Dengue virus type 4 in Puerto Rico over two decades of emergence.

    Science.gov (United States)

    Martin, Estelle; Chirivella, Maritza; Co, Juliene K G; Santiago, Gilberto A; Gubler, Duane J; Muñoz-Jordán, Jorge L; Bennett, Shannon N

    2016-02-02

    Dengue has emerged globally as a major human health problem since the 1950s and is now the most important arboviral disease of humans, infecting nearly 400 million people annually. While some cases are asymptomatic, others can develop a febrile illness (dengue fever) or even progress to severe and fatal dengue. Dengue is caused by any of 4 closely related but distinct viruses, known as Dengue virus serotype 1 to 4 (DENV-1 to DENV-4) which are maintained in endemic transmission to humans in large urban centers of the tropics by Aedes mosquitoes. Since the early 1960s, Puerto Rico, a major metropolitan center in the Caribbean, has experienced increasingly larger and clinically more severe epidemics following the introduction of all four dengue serotypes. The first dengue hemorrhagic fever epidemic in 1986, and a particularly severe outbreak in 1998 were dominated by novel DENV-4 strains that evolved in Puerto Rico, replacing earlier strains and spreading throughout the region. Sequence characterization of 54 complete DENV-4 genomes and their comparative evolution against 74 previously published viral sequences from the region over several decades shows that DENV-4 strains from these periods were genetically distinct based on unique changes in the envelope and non-structural genes. Their replacement of earlier strains in Puerto Rico progressed rapidly, suggesting that strong natural selection played a role in their fixation. This study confirms that DENVs evolve through rapid lineage turnover driven in part by natural selection and genetic drift.

  1. Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi, India.

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    Nazia Afreen

    2016-03-01

    Full Text Available Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011-2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011-2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.

  2. Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi, India.

    Science.gov (United States)

    Afreen, Nazia; Naqvi, Irshad H; Broor, Shobha; Ahmed, Anwar; Kazim, Syed Naqui; Dohare, Ravins; Kumar, Manoj; Parveen, Shama

    2016-03-01

    Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011-2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011-2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.

  3. Towards a Casa Segura: A Consumer Product Study of the Effect of Insecticide-Treated Curtains on Aedes aegypti and Dengue Virus Infections in the Home

    Science.gov (United States)

    Loroño-Pino, María Alba; García-Rejón, Julián E.; Machain-Williams, Carlos; Gomez-Carro, Salvador; Nuñez-Ayala, Guadalupe; del Rosario Nájera-Vázquez, Maria; Losoya, Arturo; Aguilar, Lyla; Saavedra-Rodriguez, Karla; Lozano-Fuentes, Saul; Beaty, Meaghan K.; Black, William C.; Keefe, Thomas J.; Eisen, Lars; Beaty, Barry J.

    2013-01-01

    The home, or domicile, is the principal environment for transmission of dengue virus (DENV) between humans and mosquito vectors. Community-wide distribution of insecticide-treated curtains (ITCs), mimicking vector control program-driven interventions, has shown promise to reduce DENV infections. We conducted a Casa Segura consumer product intervention study in Mérida, Mexico to determine the potential to reduce intradomicillary DENV transmission through ITC use in individual homes. Dengue virus infections in mosquitoes and in humans were reduced in homes with ITCs in one of two study subareas. Overall, ITCs reduced intradomicillary DENV transmission; ITC homes were significantly less likely to experience multiple DENV infections in humans than NTC homes. Dengue virus–infected Aedes aegypti females were reduced within the ITC homes where curtain use was highest. Some homes yielded up to nine infected Ae. aegypti females. This study provides insights regarding best practices for Casa Segura interventions to protect homes from intradomicillary DENV transmission. PMID:23732254

  4. Dengue virus requires the CC-chemokine receptor CCR5 for replication and infection development.

    Science.gov (United States)

    Marques, Rafael E; Guabiraba, Rodrigo; Del Sarto, Juliana L; Rocha, Rebeca F; Queiroz, Ana Luiza; Cisalpino, Daniel; Marques, Pedro E; Pacca, Carolina C; Fagundes, Caio T; Menezes, Gustavo B; Nogueira, Maurício L; Souza, Danielle G; Teixeira, Mauro M

    2015-08-01

    Dengue is a mosquito-borne disease that affects millions of people worldwide yearly. Currently, there is no vaccine or specific treatment available. Further investigation on dengue pathogenesis is required to better understand the disease and to identify potential therapeutic targets. The chemokine system has been implicated in dengue pathogenesis, although the specific role of chemokines and their receptors remains elusive. Here we describe the role of the CC-chemokine receptor CCR5 in Dengue virus (DENV-2) infection. In vitro experiments showed that CCR5 is a host factor required for DENV-2 replication in human and mouse macrophages. DENV-2 infection induces the expression of CCR5 ligands. Incubation with an antagonist prevents CCR5 activation and reduces DENV-2 positive-stranded (+) RNA inside macrophages. Using an immunocompetent mouse model of DENV-2 infection we found that CCR5(-/-) mice were resistant to lethal infection, presenting at least 100-fold reduction of viral load in target organs and significant reduction in disease severity. This phenotype was reproduced in wild-type mice treated with CCR5-blocking compounds. Therefore, CCR5 is a host factor required for DENV-2 replication and disease development. Targeting CCR5 might represent a therapeutic strategy for dengue fever. These data bring new insights on the association between viral infections and the chemokine receptor CCR5.

  5. Garlic Organosulfur Compounds Reduce Inflammation and Oxidative Stress during Dengue Virus Infection.

    Science.gov (United States)

    Hall, Alex; Troupin, Andrea; Londono-Renteria, Berlin; Colpitts, Tonya M

    2017-06-22

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant global human disease and mortality. One approach to develop treatments for DENV infection and the prevention of severe disease is through investigation of natural medicines. Inflammation plays both beneficial and harmful roles during DENV infection. Studies have proposed that the oxidative stress response may be one mechanism responsible for triggering inflammation during DENV infection. Thus, blocking the oxidative stress response could reduce inflammation and the development of severe disease. Garlic has been shown to both reduce inflammation and affect the oxidative stress response. Here, we show that the garlic active compounds diallyl disulfide (DADS), diallyl sulfide (DAS) and alliin reduced inflammation during DENV infection and show that this reduction is due to the effects on the oxidative stress response. These results suggest that garlic could be used as an alternative treatment for DENV infection and for the prevention of severe disease development.

  6. Discovery of insect and human dengue virus host factors.

    Science.gov (United States)

    Sessions, October M; Barrows, Nicholas J; Souza-Neto, Jayme A; Robinson, Timothy J; Hershey, Christine L; Rodgers, Mary A; Ramirez, Jose L; Dimopoulos, George; Yang, Priscilla L; Pearson, James L; Garcia-Blanco, Mariano A

    2009-04-23

    Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world's population at risk of infection. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat. Given their compact genomes, dengue viruses (DENV-1-4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and alpha-glucosidases), most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host.

  7. Dengue Virus Infection in Africa

    Science.gov (United States)

    Kuritsky, Joel N.; Letson, G. William; Margolis, Harold S.

    2011-01-01

    Reported incidence of dengue has increased worldwide in recent decades, but little is known about its incidence in Africa. During 1960–2010, a total of 22 countries in Africa reported sporadic cases or outbreaks of dengue; 12 other countries in Africa reported dengue only in travelers. The presence of disease and high prevalence of antibody to dengue virus in limited serologic surveys suggest endemic dengue virus infection in all or many parts of Africa. Dengue is likely underrecognized and underreported in Africa because of low awareness by health care providers, other prevalent febrile illnesses, and lack of diagnostic testing and systematic surveillance. Other hypotheses to explain low reported numbers of cases include cross-protection from other endemic flavivirus infections, genetic host factors protecting against infection or disease, and low vector competence and transmission efficiency. Population-based studies of febrile illness are needed to determine the epidemiology and true incidence of dengue in Africa. PMID:21801609

  8. Infection of the central nervous system with dengue virus 3 genotype I causing neurological manifestations in Brazil

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    Danilo Bretas de Oliveira

    2016-02-01

    Full Text Available Abstract: A case of dengue virus 3 (DENV-3 genotype I infection with neurological manifestations occurred in Belo Horizonte, Minas Gerais in October 2012. The serotype was detected by PCR, and the genotype was assessed by sequencing and phylogenetic analysis of the C-prM region. The virus causing neurological manifestations clustered with other sequences of DENV-3 genotype I. Because neurological manifestations of DENV are possibly misdiagnosed in Brazil, this study serves as an alert of the importance of DENV diagnoses in CNS infections.

  9. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes.

    Science.gov (United States)

    Salazar, Ma Isabel; Richardson, Jason H; Sánchez-Vargas, Irma; Olson, Ken E; Beaty, Barry J

    2007-01-30

    To be transmitted by its mosquito vector, dengue virus (DENV) must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP) is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission. After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi). The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands) increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle. Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands) differed in their response to DENV-2 infection.

  10. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes

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    Olson Ken E

    2007-01-01

    Full Text Available Abstract Background To be transmitted by its mosquito vector, dengue virus (DENV must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission. Results After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi. The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle. Conclusion Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands differed in their response to DENV-2 infection.

  11. Emergence of a new lineage of dengue virus type 2 identified in travelers entering Western Australia from Indonesia, 2010-2012.

    Directory of Open Access Journals (Sweden)

    Timo Ernst

    2015-01-01

    Full Text Available Dengue virus (DENV transmission is ubiquitous throughout the tropics. More than 70% of the current global dengue disease burden is borne by people who live in the Asia-Pacific region. We sequenced the E gene of DENV isolated from travellers entering Western Australia between 2010-2012, most of whom visited Indonesia, and identified a diverse array of DENV1-4, including multiple co-circulating viral lineages. Most viruses were closely related to lineages known to have circulated in Indonesia for some time, indicating that this geographic region serves as a major hub for dengue genetic diversity. Most notably, we identified a new lineage of DENV-2 (Cosmopolitan genotype that emerged in Bali in 2011-2012. The spread of this lineage should clearly be monitored. Surveillance of symptomatic returned travellers provides important and timely information on circulating DENV serotypes and genotypes, and can reveal the herald wave of dengue and other emerging infectious diseases.

  12. Detection of Hepatitis C Virus Coinfection in Patients with Dengue Diagnosis

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    Carlos Machain-Williams

    2014-01-01

    Full Text Available Coinfection produced by dengue virus (DENV and hepatitis C virus (HCV is a serious problem of public health in Mexico, as they both circulate in tropical zones and may lead to masking or complicating symptoms. In this research, we detected active coinfected patients by HCV residing in the endemic city of Mérida, Yucatán, Mexico, with positive diagnosis to dengue during the acute phase. We performed a retrospective analysis of 240 serum samples from dengue patients. The IgM-ELISA serological test was used for dengue diagnosis, as well as viral isolation to confirm infection. DENV and HCV were detected by RT-PCR. Thus, 31 (12.9% samples showed DENV-HCV coinfection, but interestingly the highest frequency of coinfection cases was found in male patients presenting hemorrhagic dengue in 19/31 (61.29%, with a predominance of 12 : 7 in males. Firstly, coinfection of DENV-HCV in Mérida, Mexico, was detected in young dengue patients, between 11 and 20 years old (38.7%, followed by those between 21 and 30 years old (32%; only 16.13% were between 0 and 10 years of age. Diagnosis of HCV infection in patients with dengue is highly recommended in order to establish potential risk in clinical manifestations as well as dictate patients' special care.

  13. Laboratory-Based Surveillance and Molecular Characterization of Dengue Viruses in Taiwan, 2014.

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    Chang, Shu-Fen; Yang, Cheng-Fen; Hsu, Tung-Chieh; Su, Chien-Ling; Lin, Chien-Chou; Shu, Pei-Yun

    2016-04-01

    We present the results of a laboratory-based surveillance of dengue in Taiwan in 2014. A total of 240 imported dengue cases were identified. The patients had arrived from 16 countries, and Malaysia, Indonesia, the Philippines, and China were the most frequent importing countries. Phylogenetic analyses showed that genotype I of dengue virus type 1 (DENV-1) and the cosmopolitan genotype of DENV-2 were the predominant DENV strains circulating in southeast Asia. The 2014 dengue epidemic was the largest ever to occur in Taiwan since World War II, and there were 15,492 laboratory-confirmed indigenous dengue cases. Phylogenetic analysis showed that the explosive dengue epidemic in southern Taiwan was caused by a DENV-1 strain of genotype I imported from Indonesia. There were several possible causes of this outbreak, including delayed notification of the outbreak, limited staff and resources for control measures, abnormal weather conditions, and a serious gas pipeline explosion in the dengue hot spot areas in Kaohsiung City. However, the results of this surveillance indicated that both active and passive surveillance systems should be strengthened so appropriate public health measures can be taken promptly to prevent large-scale dengue outbreaks.

  14. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology

    Science.gov (United States)

    SAKUDO, AKIKAZU; VISWAN, ANCHU; CHOU, HAN; SASAKI, TADAHIRO; IKUTA, KAZUYOSHI; NAGATSU, MASAAKI

    2016-01-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito-borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave-excited inductively-coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody-integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV-infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1–4 onto the beads was confirmed using reverse transcription-polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody-integration, represents a significant improvement in the detection of DENV. PMID:27221214

  15. Wolbachia-Based Dengue Virus Inhibition Is Not Tissue-Specific in Aedes aegypti

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    Amuzu, Hilaria E.; McGraw, Elizabeth A.

    2016-01-01

    Background Dengue fever, caused by the dengue virus (DENV), is now the most common arbovirus transmitted disease globally. One novel approach to control DENV is to use the endosymbiotic bacterium, Wolbachia pipientis, to limit DENV replication inside the primary mosquito vector, Aedes aegypti. Wolbachia that is naturally present in a range of insects reduces the capacity for viruses, bacteria, parasites and fungi to replicate inside insects. Wolbachia’s mode of action is not well understood but may involve components of immune activation or competition with pathogens for limited host resources. The strength of Wolbachia-based anti DENV effects appear to correlate with bacterial density in the whole insect and in cell culture. Here we aimed to determine whether particular tissues, especially those with high Wolbachia densities or immune activity, play a greater role in mediating the anti DENV effect. Methodology/findings Ae. aegypti mosquito lines with and without Wolbachia (Wildtype) were orally fed DENV 3 and their viral loads subsequently measured over two time points post infection in the midgut, head, salivary glands, Malpighian tubules, fat body and carcass. We did not find correlations between Wolbachia densities and DENV loads in any tissue, nor with DENV loads in salivary glands, the endpoint of infection. This is in contrast with strong positive correlations between DENV loads in a range of tissues and salivary gland loads for Wildtype mosquitoes. Lastly, there was no evidence of a heightened role for tissues with known immune function including the fat body and the Malpighian tubules in Wolbachia’s limitation of DENV. Conclusion/significance We conclude that the efficacy of DENV blocking in Wolbachia infected mosquitoes is not reliant on any particular tissue. This work therefore suggests that the mechanism of Wolbachia-based antiviral effects is either systemic or acts locally via processes that are fundamental to diverse cell types. We further

  16. Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

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    Perera, Rushika M.; Riley, Catherine; Isaac, Georgis; Hopf- Jannasch, Amber; Moore, Ronald J.; Weitz, Karl K.; Pasa-Tolic, Ljiljana; Metz, Thomas O.; Adamec, Jiri; Kuhn, Richard J.

    2012-03-22

    Dengue virus causes {approx}50-100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

  17. Dengue virus infection perturbs lipid homeostasis in infected mosquito cells.

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    Rushika Perera

    Full Text Available Dengue virus causes ∼50-100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

  18. Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking

    NARCIS (Netherlands)

    van der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaarl, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2007-01-01

    In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain SI on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by

  19. Lineage shift of dengue virus in Eastern India: an increased implication for DHF/DSS.

    Science.gov (United States)

    Shrivastava, A; Soni, M; Shrivastava, S; Sharma, S; Dash, P K; Gopalan, N; Behera, P K; Parida, M M

    2015-06-01

    Dengue fever, a mosquito-borne viral disease, has become a major public health problem with marked expansion in recent decades. Dengue has now become hyperendemic in India with co-circulation of all the four serotypes. Herein, we report an unprecedented outbreak which occurred during August to October 2011 in Odisha, eastern India. This is the first report of a large epidemic in Odisha. Detailed serological and molecular investigation was carried out to identify the aetiology. Almost half of the samples were found to be dengue antigen (NS1) positive. Further molecular assays revealed circulation of mixed dengue serotypes (DENV-2 and DENV-3). Cosmopolitan genotype of DENV-2 and -3 were identified as the aetiology by phylogenetic analysis. Interestingly, a new lineage of DENV-3 within cosmopolitan genotype was incriminated in this outbreak. The emergence of the unprecedented magnitude of the dengue outbreak with the involvement of a novel lineage of DENV in a newer state of India is a major cause for concern. There is an urgent need to monitor phylodynamics of dengue viruses in other endemic areas.

  20. A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate.

    Science.gov (United States)

    Santos, Jefferson J S; Cordeiro, Marli T; Bertani, Giovani R; Marques, Ernesto T A; Gil, Laura H V G

    2014-12-01

    Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.

  1. Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

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    Khunchai, Sasiprapa [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Junking, Mutita [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Suttitheptumrong, Aroonroong; Yasamut, Umpa [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Sawasdee, Nunghathai [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Netsawang, Janjuree [Faculty of Medical Technology, Rangsit University, Bangkok (Thailand); Morchang, Atthapan [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Chaowalit, Prapaipit [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Noisakran, Sansanee [Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); and others

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer For the first time how DENV NS5 increases RANTES production. Black-Right-Pointing-Pointer DENV NS5 physically interacts with human Daxx. Black-Right-Pointing-Pointer Nuclear localization of NS5 is required for Daxx interaction and RANTES production. -- Abstract: Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.

  2. A protective role for dengue virus-specific CD8+ T cells.

    Science.gov (United States)

    Yauch, Lauren E; Zellweger, Raphaël M; Kotturi, Maya F; Qutubuddin, Afrina; Sidney, John; Peters, Bjoern; Prestwood, Tyler R; Sette, Alessandro; Shresta, Sujan

    2009-04-15

    Infection with one of the four serotypes of dengue virus (DENV1-4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8(+) T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8(+) T cell response, whereas viral replication and a robust CD8(+) T cell response were observed after infection of IFN-alpha/betaR(-/-) mice. Depletion of CD8(+) T cells from IFN-alpha/betaR(-/-) mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8(+) T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-alpha/betaR(-/-) mice. DENV-specific CD8(+) T cells produced IFN-gamma, TNF-alpha, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8(+) T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8(+) T cells in the host defense against DENV and demonstrate that the anti-DENV CD8(+) T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

  3. The distinct distribution and phylogenetic characteristics of dengue virus serotypes/genotypes during the 2013 outbreak in Yunnan, China: Phylogenetic characteristics of 2013 dengue outbreak in Yunnan, China.

    Science.gov (United States)

    Wang, Binghui; Yang, Henglin; Feng, Yue; Zhou, Hongning; Dai, Jiejie; Hu, Yunzhang; Zhang, Li; Wang, Yajuan; Baloch, Zulqarnain; Xia, Xueshan

    2016-01-01

    Since 2000, sporadic imported cases of dengue fever were documented almost every year in Yunnan Province, China. Unexpectedly, a large-scale outbreak of dengue virus (DENV) infection occurred from August to December 2013, with 1538 documented cases. In the current study, 81 dengue-positive patient samples were collected from Xishuangbanna, the southernmost prefecture of the Yunnan province, and 23 from Dehong, the westernmost prefecture of the Yunnan province. The full-length envelope genes were amplified and sequenced. Phylogenetic analysis revealed that nine strains (39.1%) and 14 strains (60.9%) from the Dehong prefecture were classified as genotype I of DENV-1 and Asian I genotype of DENV-2, respectively. All strains from Xishuangbanna were identified as genotype II of DENV-3. Bayesian coalescent analysis indicates that the outbreak originated from bordering southeastern Asian countries. These three epidemic genotypes were predicted to originate in Thailand and then migrate into Yunnan through different routes.

  4. Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase.

    Science.gov (United States)

    Byrd, Chelsea M; Grosenbach, Douglas W; Berhanu, Aklile; Dai, Dongcheng; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Yang, Guang; Tyavanagimatt, Shanthakumar; Harver, Chris; Wineinger, Kristin A; Page, Jessica; Stavale, Eric; Stone, Melialani A; Fuller, Kathleen P; Lovejoy, Candace; Leeds, Janet M; Hruby, Dennis E; Jordan, Robert

    2013-04-01

    Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

  5. Chikungunya Virus Infections Among Patients with Dengue-Like Illness at a Tertiary Care Hospital in the Philippines, 2012-2013.

    Science.gov (United States)

    Velasco, John Mark; Valderama, Maria Theresa; Lopez, Maria Nila; Chua, Domingo; Latog, Rene; Roque, Vito; Corpuz, June; Klungthong, Chonticha; Rodpradit, Prinyada; Hussem, Kittinun; Poolpanichupatam, Yongyuth; Macareo, Louis; Fernandez, Stefan; Yoon, In-Kyu

    2015-12-01

    Chikungunya virus (CHIKV) often co-circulates with dengue virus (DENV). A cross-sectional surveillance study was conducted at a tertiary hospital in Manila, Philippines, to describe the prevalence and characteristics of DENV and CHIKV infections among patients seeking care for dengue-like illness. Acute blood samples from patients ≥ 6 months of age clinically diagnosed with dengue from November 2012 to December 2013 underwent reverse transcription polymerase chain reaction (RT-PCR) to detect DENV and CHIKV RNA. A total of 118 patients with clinically diagnosed dengue (age range = 1-89 years, mean = 22 years; male-to-female ratio = 1.51) were tested by DENV RT-PCR; 40 (34%) were DENV PCR-positive (age range = 1-45 years, mean = 17 years). All DENV serotypes were detected: 11 (28%) DENV-1, 6 (15%) DENV-2, 6 (15%) DENV-3, and 17 (42%) DENV-4. Of 112 patients clinically diagnosed with dengue and tested by CHIKV RT-PCR, 11 (10%) were CHIKV PCR-positive (age range = 2-47 years, mean = 20.3 years). No coinfections were detected. Presenting signs/symptoms did not differ between DENV- and CHIKV-positive cases. Sequencing of envelope 1 gene from two CHIKV PCR-positive samples showed Asian genotype. This study highlights the potential for misdiagnosis of medically attended CHIKV infections as DENV infection and the difficulty in clinically differentiating dengue and chikungunya based on presenting signs/symptoms alone. This underscores the necessity for diagnostic laboratory tests to distinguish CHIKV infections in the background of actively co-circulating DENV.

  6. Chikungunya Virus Infections among Patients with Dengue-Like Illness at a Tertiary Care Hospital in the Philippines, 2012–2013

    Science.gov (United States)

    Velasco, John Mark; Valderama, Maria Theresa; Lopez, Maria Nila; Chua, Domingo; Latog, Rene; Roque, Vito; Corpuz, June; Klungthong, Chonticha; Rodpradit, Prinyada; Hussem, Kittinun; Poolpanichupatam, Yongyuth; Macareo, Louis; Fernandez, Stefan; Yoon, In-Kyu

    2015-01-01

    Chikungunya virus (CHIKV) often co-circulates with dengue virus (DENV). A cross-sectional surveillance study was conducted at a tertiary hospital in Manila, Philippines, to describe the prevalence and characteristics of DENV and CHIKV infections among patients seeking care for dengue-like illness. Acute blood samples from patients ≥ 6 months of age clinically diagnosed with dengue from November 2012 to December 2013 underwent reverse transcription polymerase chain reaction (RT-PCR) to detect DENV and CHIKV RNA. A total of 118 patients with clinically diagnosed dengue (age range = 1–89 years, mean = 22 years; male-to-female ratio = 1.51) were tested by DENV RT-PCR; 40 (34%) were DENV PCR-positive (age range = 1–45 years, mean = 17 years). All DENV serotypes were detected: 11 (28%) DENV-1, 6 (15%) DENV-2, 6 (15%) DENV-3, and 17 (42%) DENV-4. Of 112 patients clinically diagnosed with dengue and tested by CHIKV RT-PCR, 11 (10%) were CHIKV PCR-positive (age range = 2–47 years, mean = 20.3 years). No coinfections were detected. Presenting signs/symptoms did not differ between DENV- and CHIKV-positive cases. Sequencing of envelope 1 gene from two CHIKV PCR-positive samples showed Asian genotype. This study highlights the potential for misdiagnosis of medically attended CHIKV infections as DENV infection and the difficulty in clinically differentiating dengue and chikungunya based on presenting signs/symptoms alone. This underscores the necessity for diagnostic laboratory tests to distinguish CHIKV infections in the background of actively co-circulating DENV. PMID:26416109

  7. Membranotropic regions of the dengue virus prM protein.

    Science.gov (United States)

    Nemésio, Henrique; Villalaín, José

    2014-08-19

    The Dengue virus (DENV) prM protein consists of two moieties, the pr and M domains. Apart from preventing the premature fusion activity of the DENV E protein, prM has several other unknown biological roles, displaying both protein-protein and membrane-protein interactions. Although the prM protein is an essential component of the DENV viral cycle, little is known about its biological functions and what regions of this protein are responsible for said functions. By performing an exhaustive study of membrane rupture induced by a prM peptide library on simple and complex model membranes as well as their ability to modulate the phospholipid phase transitions of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol], we identified six membranotropic regions on the prM protein. Apart from the previously identified two transmembrane segments of the protein, one of these regions probably interacts with the fusion E protein and another one, the stem segment, would interact with the membrane modulating its structure. These data will help us understand the molecular mechanism of viral entry and morphogenesis, allow the identification of new targets for the treatment of Dengue virus infection, and make possible the future development of DENV entry inhibitors.

  8. Global phylogeography of Dengue type 1 and 2 viruses reveals the role of India.

    Science.gov (United States)

    Walimbe, Atul M; Lotankar, Mrunalini; Cecilia, D; Cherian, Sarah S

    2014-03-01

    Patterns in virus dispersal and epidemiology of viral diseases can be revealed by phylogeographic studies. Currently knowledge about phylogeography of Dengue virus (DENV) Types 1 and 2 is limited. We carried out the phylogeographic analyses for DENV-1 and DENV-2, by the Bayesian Markov Chain Monte Carlo (MCMC) approach, with emphasis on Indian isolates in relation to the global evolutionary dynamics of the viruses. More than 250 E-gene sequences of each virus, available in GenBank, were used for the analyses. The study was focused on understanding the most likely geographical origin for the major genotypes and sub-lineages of DENV-1/DENV-2 and also the possible pathways in the dispersal of the virus. The results showed that for DENV-1, Southeast Asia was the most likely geographical origin and India was determined to be the ancestral location of the Cosmopolitan genotype circulating in India, Sri Lanka, West and East Africa, Caribbean region, East and Southeast Asia. For DENV-2, the ancestral source could not be precisely inferred. Further, in spite of the earliest isolate from Trinidad-1953 of the American genotype, it was depicted that India may have been the probable ancestor of this genotype. India was also determined to be the ancestral location of a subgroup of the Cosmopolitan genotype. It was noted that DENV-1 and DENV-2 were introduced into India during 1940s and 1910s respectively. Subsequently, dispersal of both the viruses between India and different regions including West, East and Central Africa, Southeast and East Asia and Caribbean was inferred. Overall, the current study provides insight into the spatial as well as temporal dynamics of dengue virus serotypes 1 and 2.

  9. Dengue virus antibody database: Systematically linking serotype-specificity with epitope mapping in dengue virus

    Science.gov (United States)

    Chaudhury, Sidhartha; Gromowski, Gregory D.; Ripoll, Daniel R.; Khavrutskii, Ilja V.; Desai, Valmik; Wallqvist, Anders

    2017-01-01

    Background A majority infections caused by dengue virus (DENV) are asymptomatic, but a higher incidence of severe illness, such as dengue hemorrhagic fever, is associated with secondary infections, suggesting that pre-existing immunity plays a central role in dengue pathogenesis. Primary infections are typically associated with a largely serotype-specific antibody response, while secondary infections show a shift to a broadly cross-reactive antibody response. Methods/Principal findings We hypothesized that the basis for the shift in serotype-specificity between primary and secondary infections can be found in a change in the antibody fine-specificity. To investigate the link between epitope- and serotype-specificity, we assembled the Dengue Virus Antibody Database, an online repository containing over 400 DENV-specific mAbs, each annotated with information on 1) its origin, including the immunogen, host immune history, and selection methods, 2) binding/neutralization data against all four DENV serotypes, and 3) epitope mapping at the domain or residue level to the DENV E protein. We combined epitope mapping and activity information to determine a residue-level index of epitope propensity and cross-reactivity and generated detailed composite epitope maps of primary and secondary antibody responses. We found differing patterns of epitope-specificity between primary and secondary infections, where secondary responses target a distinct subset of epitopes found in the primary response. We found that secondary infections were marked with an enhanced response to cross-reactive epitopes, such as the fusion-loop and E-dimer region, as well as increased cross-reactivity in what are typically more serotype-specific epitope regions, such as the domain I-II interface and domain III. Conclusions/Significance Our results support the theory that pre-existing cross-reactive memory B cells form the basis for the secondary antibody response, resulting in a broadening of the response

  10. The distribution of synonymous codon choice in the translation initiation region of dengue virus.

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    Jian-hua Zhou

    Full Text Available Dengue is the most common arthropod-borne viral (Arboviral illness in humans. The genetic features concerning the codon usage of dengue virus (DENV were analyzed by the relative synonymous codon usage, the effective number of codons and the codon adaptation index. The evolutionary distance between DENV and the natural hosts (Homo sapiens, Pan troglodytes, Aedes albopictus and Aedes aegypti was estimated by a novel formula. Finally, the synonymous codon usage preference for the translation initiation region of this virus was also analyzed. The result indicates that the general trend of the 59 synonymous codon usage of the four genotypes of DENV are similar to each other, and this pattern has no link with the geographic distribution of the virus. The effect of codon usage pattern of Aedes albopictus and Aedes aegypti on the formation of codon usage of DENV is stronger than that of the two primates. Turning to the codon usage preference of the translation initiation region of this virus, some codons pairing to low tRNA copy numbers in the two primates have a stronger tendency to exist in the translation initiation region than those in the open reading frame of DENV. Although DENV, like other RNA viruses, has a high mutation to adapt its hosts, the regulatory features about the synonymous codon usage have been 'branded' on the translation initiation region of this virus in order to hijack the translational mechanisms of the hosts.

  11. El Niño-Southern Oscillation, local weather and occurrences of dengue virus serotypes

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    Huang, Xiaodong; Clements, Archie C. A.; Williams, Gail; Devine, Gregor; Tong, Shilu; Hu, Wenbiao

    2015-11-01

    Severe dengue fever is usually associated with secondary infection by a dengue virus (DENV) serotype (1 to 4) that is different to the serotype of the primary infection. Dengue outbreaks only occur following importations of DENV in Cairns, Australia. However, the majority of imported cases do not result in autochthonous transmission in Cairns. Although DENV transmission is strongly associated with the El Niño-Southern Oscillation (ENSO) climate cycle and local weather conditions, the frequency and potential risk factors of infections with the different DENV serotypes, including whether or not they differ, is unknown. This study used a classification tree model to identify the hierarchical interactions between Southern Oscillation Index (SOI), local weather factors, the presence of imported serotypes and the occurrence of the four autochthonous DENV serotypes from January 2000-December 2009 in Cairns. We found that the 12-week moving average of SOI and the 2-week moving average of maximum temperature were the most important factors influencing the variation in the weekly occurrence of the four DENV serotypes, the likelihoods of the occurrence of the four DENV serotypes may be unequal under the same environmental conditions, and occurrence may be influenced by changes in global and local environmental conditions in Cairns.

  12. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

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    Wei, Y; Wang, S; Wang, X

    2014-01-01

    Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

  13. Phylogenetic reconstruction of dengue virus type 2 in Colombia

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    Méndez Jairo A

    2012-03-01

    Full Text Available Abstract Background Dengue fever is perhaps the most important viral re-emergent disease especially in tropical and sub-tropical countries, affecting about 50 million people around the world yearly. In Colombia, dengue virus was first detected in 1971 and still remains as a major public health issue. Although four viral serotypes have been recurrently identified, dengue virus type 2 (DENV-2 has been involved in the most important outbreaks during the last 20 years, including 2010 when the fatality rate highly increased. As there are no major studies reviewing virus origin and genotype distribution in this country, the present study attempts to reconstruct the phylogenetic history of DENV-2 using a sequence analysis from a 224 bp PCR-amplified product corresponding to the carboxyl terminus of the envelope (E gene from 48 Colombian isolates. Results As expected, the oldest isolates belonged to the American genotype (subtype V, but the strains collected since 1990 represent the American/Asian genotype (subtype IIIb as previously reported in different American countries. Interestingly, the introduction of this genotype coincides with the first report of dengue hemorrhagic fever in Colombia at the end of 1989 and the increase of cases during the next years. Conclusion After replacement of the American genotype, several lineages of American/Asian subtype have rapidly spread all over the country evolving in new clades. Nevertheless, the direct association of these new variants in the raise of lethality rate observed during the last outbreak has to be demonstrated.

  14. Paradoxical role of antibodies in dengue virus infections: considerations for prophylactic vaccine development.

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    Acosta, Eliana G; Bartenschlager, Ralf

    2016-01-01

    Highly effective prophylactic vaccines for flaviviruses including yellow fever virus, tick-borne encephalitis virus and Japanese encephalitis virus are currently in use. However, the development of a dengue virus (DENV) vaccine has been hampered by the requirement of simultaneous protection against four distinct serotypes and the threat that DENV-specific antibodies might either mediate neutralization or, on the contrary, exacerbate disease through the phenomenon of antibody-dependent enhancement (ADE) of infection. Therefore, understanding the cellular, biochemical and molecular basis of antibody-mediated neutralization and ADE are fundamental for the development of a safe DENV vaccine. Here we summarize current structural and mechanistic knowledge underlying these phenomena. We also review recent results demonstrating that the humoral immune response triggered during natural DENV infection is able to generate neutralizing antibodies binding complex quaternary epitopes only present on the surface of intact virions.

  15. Evaluation of commercially available diagnostic tests for the detection of dengue virus NS1 antigen and anti-dengue virus IgM antibody.

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    Elizabeth A Hunsperger

    2014-10-01

    Full Text Available Commercially available diagnostic test kits for detection of dengue virus (DENV non-structural protein 1 (NS1 and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO, the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR and the Pediatric Dengue Vaccine Initiative (PDVI. Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA and rapid diagnostic test (RDT formats were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS and the Centers for Disease Control and Prevention (CDC, and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR. Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60-75% and specificity 71-80%; NS1 RDT sensitivity was 38-71% and specificity 76-80%; the IgM anti-DENV RDTs sensitivity was 30-96%, with a specificity of 86-92%, and IgM anti-DENV ELISA sensitivity was 96-98% and specificity 78-91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88-94%.

  16. Evaluation of Commercially Available Diagnostic Tests for the Detection of Dengue Virus NS1 Antigen and Anti-Dengue Virus IgM Antibody

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    Hunsperger, Elizabeth A.; Yoksan, Sutee; Buchy, Philippe; Nguyen, Vinh Chau; Sekaran, Shamala Devi; Enria, Delia A.; Vazquez, Susana; Cartozian, Elizabeth; Pelegrino, Jose L.; Artsob, Harvey; Guzman, Maria G.; Olliaro, Piero; Zwang, Julien; Guillerm, Martine; Kliks, Susie; Halstead, Scott; Peeling, Rosanna W.; Margolis, Harold S.

    2014-01-01

    Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60–75% and specificity 71–80%; NS1 RDT sensitivity was 38–71% and specificity 76–80%; the IgM anti-DENV RDTs sensitivity was 30–96%, with a specificity of 86–92%, and IgM anti-DENV ELISA sensitivity was 96–98% and specificity 78–91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88–94%. PMID:25330157

  17. Galectin-1 exerts inhibitory effects during DENV-1 infection.

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    Karina Alves Toledo

    Full Text Available Dengue virus (DENV is an enveloped RNA virus that is mosquito-transmitted and can infect a variety of immune and non-immune cells. Response to infection ranges from asymptomatic disease to a severe disorder known as dengue hemorrhagic fever. Despite efforts to control the disease, there are no effective treatments or vaccines. In our search for new antiviral compounds to combat infection by dengue virus type 1 (DENV-1, we investigated the role of galectin-1, a widely-expressed mammalian lectin with functions in cell-pathogen interactions and immunoregulatory properties. We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production. In test of this hypothesis we found that exogenous addition of human recombinant Gal-1 (hrGal-1 inhibits the virus production in the three different cell types. This inhibitory effect was dependent on hrGal-1 dimerization and required its carbohydrate recognition domain. Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3. Interestingly, we found that hrGal-1 directly binds to dengue virus and acts, at least in part, during the early stages of DENV-1 infection, by inhibiting viral adsorption and its internalization to target cells. To test the in vivo role of Gal-1 in DENV infection, Gal-1-deficient-mice were used to demonstrate that the expression of endogenous Galectin-1 contributes to resistance of macrophages to in vitro-infection with DENV-1 and it is also important to physiological susceptibility of mice to in vivo infection with DENV-1. These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.

  18. The kinase inhibitor SFV785 dislocates dengue virus envelope protein from the replication complex and blocks virus assembly.

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    Azlinda Anwar

    Full Text Available Dengue virus (DENV is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785, which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.

  19. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

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    Berlin Londono-Renteria

    2015-10-01

    Full Text Available Dengue virus (DENV is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379, whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

  20. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

    Science.gov (United States)

    Londono-Renteria, Berlin; Troupin, Andrea; Conway, Michael J; Vesely, Diana; Ledizet, Michael; Roundy, Christopher M; Cloherty, Erin; Jameson, Samuel; Vanlandingham, Dana; Higgs, Stephen; Fikrig, Erol; Colpitts, Tonya M

    2015-10-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

  1. Cross-protection induced by Japanese encephalitis vaccines against different genotypes of Dengue viruses in mice.

    Science.gov (United States)

    Li, Jieqiong; Gao, Na; Fan, Dongying; Chen, Hui; Sheng, Ziyang; Fu, Shihong; Liang, Guodong; An, Jing

    2016-01-28

    Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) are closely related mosquito-borne flaviviruses that cause very high global disease burdens. Although cross-reactivity and cross-protection within flaviviruses have been demonstrated, the effect of JEV vaccination on susceptibility to DENV infection has not been well elucidated. In this study, we found that vaccination with the JEV inactivated vaccine (INV) and live attenuated vaccine (LAV) could induce cross-immune responses and cross-protection against DENV1-4 in mice. Despite the theoretical risk of immune enhancement, no increased mortality was observed in our mouse model. Additionally, low but consistently detectable cross-neutralizing antibodies against DENV2 and DENV3 were also observed in the sera of JEV vaccine-immunized human donors. The results suggested that both JEV-LAV and JEV-INV could elicit strong cross-immunity and protection against DENVs, indicating that inoculation with JEV vaccines may influence the distribution of DENVs in co-circulated areas and that the cross-protection induced by JEV vaccines against DENVs might provide important information in terms of DENV prevention.

  2. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology.

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    Sakudo, Akikazu; Viswan, Anchu; Chou, Han; Sasaki, Tadahiro; Ikuta, Kazuyoshi; Nagatsu, Masaaki

    2016-07-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito‑borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave‑excited inductively‑coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody‑integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV‑infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1‑4 onto the beads was confirmed using reverse transcription‑polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody‑integration, represents a significant improvement in the detection of DENV.

  3. The dynamics of dengue virus serotype 3 introduction and dispersion in the state of Bahia, Brazil

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    Paulo Roberto Santana de Melo

    2007-12-01

    Full Text Available By 2002, dengue virus serotype 1 (DENV-1 and DENV-2 had circulated for more than a decade in Brazil. In 2002, the introduction of DENV-3 in the state of Bahia produced a massive epidemic and the first cases of dengue hemorrhagic fever. Based on the standardized frequency, timing and location of viral isolations by the state's Central Laboratory, DENV-3 probably entered Bahia through its capital, Salvador, and then rapidly disseminated to other cities, following the main roads. A linear regression model that included traffic flow, distance from the capital and DENV-1 circulation (r² = 0.24, p = 0.001 supported this hypothesis. This pattern was not seen for serotypes already in circulation and was not seen for DENV-3 in the following year. Human population density was another important factor in the intensity of viral circulation. Neither DENV-1 nor DENV-2 fit this model for 2001 or 2003. Since the vector has limited flight range and vector densities fail to correlate with intensity of viral circulation, this distribution represents the movement of infected people and to some extent mosquitoes. This pattern may mimic person-to-person spread of a new infection.

  4. Morphological studies in a model for dengue-2 virus infection in mice

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    Ortrud Monika Barth

    2006-12-01

    Full Text Available One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2. Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.

  5. Prevalencia de anticuerpos neutralizantes contra los serotipos del virus dengue en universitarios de Tabasco, México Prevalence of neutralizing antibodies to dengue virus serotypes in university students from Tabasco, Mexico

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    Gilma Guadalupe Sánchez-Burgos

    2008-10-01

    Full Text Available OBJETIVO: Determinar la seroprevalencia de anticuerpos neutralizantes de los serotipos del virus dengue en estudiantes universitarios de Tabasco, México, durante los meses de septiembre a noviembre del año 2005. MATERIAL Y MÉTODOS: Se determinó la presencia de IgG contra el virus en el suero de estudiantes que acudieron al centro clínico de la universidad; en los sueros positivos se determinaron los anticuerpos neutralizantes mediante el ensayo de reducción de placa lítica. RESULTADOS: La prevalencia de IgG contra el dengue fue de 9.1%; de esta proporción, los anticuerpos neutralizantes fueron DENV-1 (20%, DENV-2 (100%, DENV-3 (4% y DENV-4 (68%. CONCLUSIONES: Este estudio muestra que el serotipo transmitido con mayor frecuencia en el estado de Tabasco es el DENV-2, aunque no ha sido el aislado con más frecuencia. La elevada prevalencia de anticuerpos neutralizantes contra el DENV-4, al parecer de reacción cruzada, podría explicar la baja circulación de este serotipo en Tabasco.OBJECTIVE: Determine the seroprevalence of neutralizing antibodies to dengue virus in students from the state university of Tabasco, Mexico. MATERIAL AND METHODS: A transversal study was conducted of serum collected from students between September and November, 2005. The sera were screened for anti-dengue IgG and those that had evidence of dengue antibodies were analyzed by a plaque reduction neutralization test. RESULTS: Prevalence of anti-dengue IgG was 9.1%. The frequency of neutralizing antibodies was 100% for DENV-2, 68% for DENV-4, 20% for DENV-1, and 4 % for DENV-3. CONCLUSIONS: We found that in this population, DENV-2 circulates more than DENV-3 despite the fact that DENV-3 is more frequently isolated. Unexpectedly, neutralizing antibodies against DENV-4 were frequently found even though this serotype is almost extinct; thus, it is probable that cross-immunity could suppress DEN-4 transmission, as has been suggested.

  6. Phylogenetic analysis of dengue virus types 1 and 4 circulating in Puerto Rico and Key West, Florida, during 2010 epidemics.

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    Añez, Germán; Heisey, Daniel A R; Espina, Luz M; Stramer, Susan L; Rios, Maria

    2012-09-01

    We describe sequences of six strains of dengue virus (DENV): three DENV-1 isolates and two DENV-4 isolates from Puerto Rico, and a DENV-1 strain from Key West, Florida, obtained from blood donors during 2010 epidemics. Phylogenetic analysis revealed that the Puerto Rico DENV-1 strains constitute a new lineage within genotype V different from those that circulated in Puerto Rico during the past two decades. The newer Puerto Rico DENV-1 strains associated with strains from the Caribbean and South America. The DENV-1 strain from Key West, Florida clustered with a strain isolated from mosquito pools collected in that area and with a number of strains from Nicaragua and Mexico circulating during 2006-2009. The Puerto Rico DENV-4 isolates of genotype II associated with strains that have circulated on the island throughout the 1980s and 1990s and with strains from the Caribbean region and Central America. Introduction and circulation of novel DENV lineages in dengue-endemic regions have the potential to increase the severity of dengue cases.

  7. Zika Virus, Chikungunya Virus, and Dengue Virus in Cerebrospinal Fluid from Adults with Neurological Manifestations, Guayaquil, Ecuador

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    Acevedo, Nathalie; Waggoner, Jesse; Rodriguez, Michelle; Rivera, Lissette; Landivar, José; Pinsky, Benjamin; Zambrano, Hector

    2017-01-01

    Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) have been associated with clinical presentations that involve acute neurological complaints. In the current study, we identified ZIKV, CHIKV, and DENV in cerebrospinal fluid (CSF) samples from patients admitted to the Hospital Luis Vernaza (Guayaquil, Ecuador) to the Emergency Room or the Intensive Care Unit, with neurological symptoms and/or concern for acute arboviral infections. Viral RNA from one or more virus was detected in 12/16 patients. Six patients were diagnosed with meningitis or encephalitis, three with Guillain–Barré Syndrome, and one with CNS vasculitis. Two additional patients had a systemic febrile illness including headache that prompted testing of CSF. Two patients, who were diagnosed with encephalitis and meningoencephalitis, died during their hospitalizations. These cases demonstrate the breadth and significance of neurological manifestations associated with ZIKV, CHIKV, and DENV infections. PMID:28174559

  8. Neutralizing antibody titers against dengue virus correlate with protection from symptomatic infection in a longitudinal cohort.

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    Katzelnick, Leah C; Montoya, Magelda; Gresh, Lionel; Balmaseda, Angel; Harris, Eva

    2016-01-19

    The four dengue virus serotypes (DENV1-4) are mosquito-borne flaviviruses that infect ∼ 390 million people annually; up to 100 million infections are symptomatic, and 500,000 cases progress to severe disease. Exposure to a heterologous DENV serotype, the specific infecting DENV strains, and the interval of time between infections, as well as age, ethnicity, genetic polymorphisms, and comorbidities of the host, are all risk factors for severe dengue. In contrast, neutralizing antibodies (NAbs) are thought to provide long-lived protection against symptomatic infection and severe dengue. The objective of dengue vaccines is to provide balanced protection against all DENV serotypes simultaneously. However, the association between homotypic and heterotypic NAb titers and protection against symptomatic infection remains poorly understood. Here, we demonstrate that the titer of preinfection cross-reactive NAbs correlates with reduced likelihood of symptomatic secondary infection in a longitudinal pediatric dengue cohort in Nicaragua. The protective effect of NAb titers on infection outcome remained significant when controlled for age, number of years between infections, and epidemic force, as well as with relaxed or more stringent criteria for defining inapparent DENV infections. Further, individuals with higher NAb titers immediately after primary infection had delayed symptomatic infections compared with those with lower titers. However, overall NAb titers increased modestly in magnitude and remained serotype cross-reactive in the years between infections, possibly due to reexposure. These findings establish that anti-DENV NAb titers correlate with reduced probability of symptomatic DENV infection and provide insights into longitudinal characteristics of antibody-mediated immunity to DENV in an endemic setting.

  9. Consequences of the expanding global distribution of Aedes albopictus for dengue virus transmission.

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    Louis Lambrechts

    Full Text Available The dramatic global expansion of Aedes albopictus in the last three decades has increased public health concern because it is a potential vector of numerous arthropod-borne viruses (arboviruses, including the most prevalent arboviral pathogen of humans, dengue virus (DENV. Ae. aegypti is considered the primary DENV vector and has repeatedly been incriminated as a driving force in dengue's worldwide emergence. What remains unresolved is the extent to which Ae. albopictus contributes to DENV transmission and whether an improved understanding of its vector status would enhance dengue surveillance and prevention. To assess the relative public health importance of Ae. albopictus for dengue, we carried out two complementary analyses. We reviewed its role in past dengue epidemics and compared its DENV vector competence with that of Ae. aegypti. Observations from "natural experiments" indicate that, despite seemingly favorable conditions, places where Ae. albopictus predominates over Ae. aegypti have never experienced a typical explosive dengue epidemic with severe cases of the disease. Results from a meta-analysis of experimental laboratory studies reveal that although Ae. albopictus is overall more susceptible to DENV midgut infection, rates of virus dissemination from the midgut to other tissues are significantly lower in Ae. albopictus than in Ae. aegypti. For both indices of vector competence, a few generations of mosquito colonization appear to result in a relative increase of Ae. albopictus susceptibility, which may have been a confounding factor in the literature. Our results lead to the conclusion that Ae. albopictus plays a relatively minor role compared to Ae. aegypti in DENV transmission, at least in part due to differences in host preferences and reduced vector competence. Recent examples of rapid arboviral adaptation to alternative mosquito vectors, however, call for cautious extrapolation of our conclusion. Vector status is a dynamic

  10. Dengue virus-infected human dendritic cells reveal hierarchies of naturally expressed novel NS3 CD8 T cell epitopes.

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    Piazza, P; Campbell, D; Marques, E; Hildebrand, W H; Buchli, R; Mailliard, R; Rinaldo, C R

    2014-09-01

    Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8(+) T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75-100%), compared to the others (0-50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

  11. Human Immune Responses to Dengue Virus Infection: lessons learned from prospective cohort studies

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    Timothy P Endy

    2014-04-01

    Full Text Available Dengue virus (DENV continues to spread globally and is a major cause of morbidity and mortality. Currently there is no antiviral treatment to diminish severe illness or a vaccine to induce protection from infection. An effective dengue vaccine that protects against all four DENV serotypes is a high priority for endemic countries and several candidates are in development by various United States Federal Agencies and private pharmaceutical companies. Challenges faced by dengue vaccine developers include creating tetravalent formulations that provide tetravalent protection, the lack of a correlate of protective immunity, a changing viral landscape as DENV evolves, and a complex viral-host pathogenesis that can result in a spectrum of illness from subclinical infection to severe hemorrhagic fever. There have been a number of long-term prospective studies on DENV transmission and dengue severity that have provided invaluable information on DENV epidemiology and pathogenesis of this disease. In this section we will review the critical lessons learned from these studies and their application for dengue vaccine development.

  12. Recombinant vesicular stomatitis virus-based dengue-2 vaccine candidate induces humoral response and protects mice against lethal infection.

    Science.gov (United States)

    Lauretti, Flavio; Chattopadhyay, Anasuya; de Oliveira França, Rafael Freitas; Castro-Jorge, Luiza; Rose, John; Fonseca, Benedito A L da

    2016-09-01

    Dengue is the most important arbovirus disease throughout the world and it is responsible for more than 500,000 dengue hemorrhagic cases and 22,000 deaths every year. One vaccine was recently licensed for human use in Brazil, Mexico and Philippines and although at least seven candidates have been in clinical trials the results of the most developed CYD vaccine have demonstrated immunization problems, such as uneven protection and interference between serotypes. We constructed a vaccine candidate based on vesicular stomatitis virus (VSV) expression of pre-membrane (prM) and envelope (E) proteins of dengue-2 virus (DENV-2) and tested it in mice to evaluate immunogenicity and protection against DENV-2 infection. VSV has been successfully used as vaccine vectors for several viruses to induce strong humoral and cellular immune responses. The VSV-DENV-2 recombinant was constructed by inserting the DENV-2 structural proteins into a VSV plasmid DNA for recombinant VSV-DENV-2 recovery. Infectious recombinant VSV viruses were plaque purified and prM and E expression were confirmed by immunofluorescence and radiolabeling of proteins of infected cells. Forty Balb/C mice were inoculated through subcutaneous (s.c.) route with VSV-DENV-2 vaccine in a two doses schedule 15 d apart and 29 d after first inoculation, sera were collected and the mice were challenged with 50 lethal doses (LD50) of a neurovirulent DENV-2. The VSV-DENV-2 induced anti-DENV-2 antibodies and protected animals in the challenge experiment comparable to DENV-2 immunization control group. We conclude that VSV is a promising platform to test as a DENV vaccine and perhaps against others Flaviviridae.

  13. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway.

    Directory of Open Access Journals (Sweden)

    Irma Sánchez-Vargas

    2009-02-01

    Full Text Available A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi, is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA, which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs. These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2 infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.

  14. Presence of Viral Genome in Urine and Development of Hematuria and Pathological Changes in Kidneys in Common Marmoset (Callithrix jacchus after Inoculation with Dengue Virus

    Directory of Open Access Journals (Sweden)

    Hirofumi Akari

    2013-05-01

    Full Text Available Common marmosets (Callithrix jacchus developed high levels of viremia, clinical signs including fever, weight loss, a decrease in activity and hematuria upon inoculation with dengue virus (DENV. Presence of DENV genome in urine samples and pathological changes in kidneys were examined in the present study. Levels of DENV genome were determined in 228 urine samples from 20 primary DENV-inoculated marmosets and in 56 urine samples from four secondary DENV-inoculated marmosets. DENV genome was detected in 75% (15/20 of marmosets after primary DENV infection. No DENV genome was detected in urine samples from the marmosets with secondary infection with homologous DENV (0%, 0/4. Two marmosets demonstrated hematuria. Pathological analysis of the kidneys demonstrated non-suppressive interstitial nephritis with renal tubular regeneration. DENV antigen-positive cells were detected in kidneys. In human dengue virus infections, some patients present renal symptoms. The results indicate that marmosets recapitulate some aspects of the involvement of kidneys in human DENV infection, and suggest that marmosets are potentially useful for the studies of the pathogenesis of DENV infection, including kidneys.

  15. Occurrence of co-infection with dengue viruses during 2014 in New Delhi, India.

    Science.gov (United States)

    Tazeen, A; Afreen, N; Abdullah, M; Deeba, F; Haider, S H; Kazim, S N; Ali, S; Naqvi, I H; Broor, S; Ahmed, A; Parveen, S

    2017-01-01

    Dengue fever is an arthropod-borne viral infection that has become endemic in several parts of India including Delhi. We studied occurrence of co-infection with dengue viruses during an outbreak in New Delhi, India in 2014. For the present study, blood samples collected from symptomatic patients were analysed by RT-PCR. Eighty percent of the samples were positive for dengue virus. The result showed that DENV-1 (77%) was the predominant serotype followed by DENV-2 (60%). Concurrent infection with more than one serotype was identified in 43% of the positive samples. Phylogenetic analysis clustered DENV-1 strains with the American African and DENV-2 strains in Cosmopolitan genotypes. Four common amino-acid mutations were identified in the envelope gene of DENV-1 sequences (F337I, A369T, V380I and L402F) and one common mutation (N390S) in the DENV-2 sequences. Further analysis revealed purifying selection in both the serotypes. A significant number of patients were co-infected with DENV-1 and DENV-2 serotypes. Although we do not have direct evidence to demonstrate co-evolution of these two stereotypes, nonetheless their simultaneous occurrence does indicate that they are favoured by evolutionary forces. An ongoing surveillance and careful analysis of future outbreaks will strengthen the concept of co-evolution or otherwise. Whether the concurrent dengue viral infection is correlated with disease severity in a given population is another aspect to be pursued. This study is envisaged to be useful for future reference in the context of overall epidemiology.

  16. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.

    Directory of Open Access Journals (Sweden)

    Gilberto A Santiago

    Full Text Available Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV. There are four genetically distinct DENVs (DENV-1-4 that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

  17. Prolonged co-circulation of two distinct Dengue virus Type 3 lineages in the hyperendemic area of Medellin, Colombia.

    Science.gov (United States)

    Ospina, Marta C; Diaz, Francisco J; Osorio, Jorge E

    2010-09-01

    During the past two decades, Dengue virus-3 (DENV-3) has re-emerged in the Western Hemisphere causing significant epidemics of dengue fever (DF) and dengue hemorrhagic fever (DHF). In an effort to understand the molecular evolution of DENV-3 and their relationships to other DENV-3 circulating in the western hemisphere, we conducted a phylogenetic study on DENV-3 isolates made between 2002 and 2007 in the metropolitan area of Medellín, Colombia. An unexpected co-circulation of two different variants of DENV-3 subtype III during at least 5 years in Medellín was found. In addition, a more complete analysis of DENV-3 viruses isolated in other South American regions revealed the existence of three different subtype III lineages, all derived from independent introductions. This study documents significant genetic diversity of circulating viruses within the same subtype and an unusual capacity of the population of this city to support continuous circulation of multiple variants of dengue virus.

  18. 登革病毒包膜E蛋白Ⅲ区%Dengue virus envelope protein domain Ⅲ

    Institute of Scientific and Technical Information of China (English)

    杨杰; 饶贤才

    2011-01-01

    登革病毒包膜E蛋白Ⅲ区(DENV ED3)是登革病毒与敏感细胞受体结合的功能区域,对其结构和功能的研究,将有助于理解登革病毒与其敏感细胞问的相互作用和致病机理,为琶革病蓐受体研究和研发特异性阻断剂控制DENV感染奠定基础.现就DENV ED3的研究进展作一综述.%Dengue virus envelope protein domain Ⅲ (DENV ED3) is the functional area of Dengue virus to combine with sensitive cell receptor. Researching the structure and function of DENV ED3 contributes to the understanding of the interaction of dengue virus with sensitive cells and the viral pathogenesis, which also helps lay foundation for investigating the dengue virus receptors and developing the specific blocking agent to control DENV infection. Here we reviewed the recent advancement in study of DENV ED3.

  19. Phylogeography and molecular epidemiology of an epidemic strain of dengue virus type 1 in Sri Lanka.

    Science.gov (United States)

    Ocwieja, Karen E; Fernando, Anira N; Sherrill-Mix, Scott; Sundararaman, Sesh A; Tennekoon, Rashika N; Tippalagama, Rashmi; Krishnananthasivam, Shivankari; Premawansa, Gayani; Premawansa, Sunil; De Silva, Aruna Dharshan

    2014-08-01

    In 2009, a severe epidemic of dengue disease occurred in Sri Lanka, with higher mortality and morbidity than any previously recorded epidemic in the country. It corresponded to a shift to dengue virus 1 as the major disease-causing serotype in Sri Lanka. Dengue disease reached epidemic levels in the next 3 years. We report phylogenetic evidence that the 2009 epidemic DENV-1 strain continued to circulate within the population and caused severe disease in the epidemic of 2012. Bayesian phylogeographic analyses suggest that the 2009 Sri Lankan epidemic DENV-1 strain may have traveled directly or indirectly from Thailand through China to Sri Lanka, and after spreading within the Sri Lankan population, it traveled to Pakistan and Singapore. Our findings delineate the dissemination route of a virulent DENV-1 strain in Asia. Understanding such routes will be of particular importance to global control efforts.

  20. Wolbachia and dengue virus infection in the mosquito Aedes fluviatilis (Diptera: Culicidae).

    Science.gov (United States)

    Silva, Jéssica Barreto Lopes; Magalhães Alves, Debora; Bottino-Rojas, Vanessa; Pereira, Thiago Nunes; Sorgine, Marcos Henrique Ferreira; Caragata, Eric Pearce; Moreira, Luciano Andrade

    2017-01-01

    Dengue represents a serious threat to human health, with billions of people living at risk of the disease. Wolbachia pipientis is a bacterial endosymbiont common to many insect species. Wolbachia transinfections in mosquito disease vectors have great value for disease control given the bacterium's ability to spread into wild mosquito populations, and to interfere with infections of pathogens, such as dengue virus. Aedes fluviatilis is a mosquito with a widespread distribution in Latin America, but its status as a dengue vector has not been clarified. Ae. fluviatilis is also naturally infected by the wFlu Wolbachia strain, which has been demonstrated to enhance infection with the avian malarial parasite Plasmodium gallinaceum. We performed experimental infections of Ae. fluviatilis with DENV-2 and DENV-3 isolates from Brazil via injection or oral feeding to provide insight into its competence for the virus. We also examined the effect of the native Wolbachia infection on the virus using a mosquito line where the wFlu infection had been cleared by antibiotic treatment. Through RT-qPCR, we observed that Ae. fluviatilis could become infected with both viruses via either method of infection, although at a lower rate than Aedes aegypti, the primary dengue vector. We then detected DENV-2 and DENV-3 in the saliva of injected mosquitoes, and observed that injection of DENV-3-infected saliva produced subsequent infections in naïve Ae. aegypti. However, across our data we observed no difference in prevalence of infection and viral load between Wolbachia-infected and -uninfected mosquitoes, suggesting that there is no effect of wFlu on dengue virus. Our results highlight that Ae. fluviatilis could potentially serve as a dengue vector under the right circumstances, although further testing is required to determine if this occurs in the field.

  1. In silico mutation analysis of non-structural protein-5 (NS5) dengue virus

    Science.gov (United States)

    Puspitasari, R. D.; Tambunan, U. S. F.

    2017-04-01

    Dengue fever is a world disease. It is endemic in more than 100 countries. Information about the effect of mutations in the virus is important in drug design and development. In this research, we studied the effect of mutation on NS5 dengue virus. NS5 is the large protein containing 67% amino acid similarity in DENV 1-4 and has multifunctional enzymatic activities. Dengue virus is an RNA virus that has very high mutation frequency with an average of 100 times higher than DNA mutations, and the accumulation of mutations will be possible to generate the new serotype. In this study, we report that mutation occurs in NS5 of DENV serotype 3, glutamine mutates into methionine at position 10 and threonine mutates into isoleucine at position 55. These residues are part of the domain named S-Adenosyl-L-Methionine-Dependent Methyltransferase (IPR029063).

  2. Emergence of Epidemic Dengue-1 Virus in the Southern Province of Sri Lanka

    Science.gov (United States)

    Nagahawatte, Ajith; Devasiri, Vasantha; Kodikara Arachichi, Wasantha; Strouse, John J.; Sessions, October M.; Kurukulasooriya, Ruvini; Uehara, Anna; Howe, Shiqin; Ong, Xin Mei; Tan, Sharon; Chow, Angelia; Tummalapalli, Praveen; De Silva, Aruna D.; Østbye, Truls; Woods, Christopher W.; Gubler, Duane J.; Reller, Megan E.

    2016-01-01

    Background Dengue is a frequent cause of acute febrile illness with an expanding global distribution. Since the 1960s, dengue in Sri Lanka has been documented primarily along the heavily urbanized western coast with periodic shifting of serotypes. Outbreaks from 2005–2008 were attributed to a new clade of DENV-3 and more recently to a newly introduced genotype of DENV-1. In 2007, we conducted etiologic surveillance of acute febrile illness in the Southern Province and confirmed dengue in only 6.3% of febrile patients, with no cases of DENV-1 identified. To re-evaluate the importance of dengue as an etiology of acute febrile illness in this region, we renewed fever surveillance in the Southern Province to newly identify and characterize dengue. Methodology/Principal Findings A cross-sectional surveillance study was conducted at the largest tertiary care hospital in the Southern Province from 2012–2013. A total of 976 patients hospitalized with acute undifferentiated fever were enrolled, with 64.3% male and 31.4% children. Convalescent blood samples were collected from 877 (89.6%). Dengue virus isolation, dengue RT-PCR, and paired IgG ELISA were performed. Acute dengue was confirmed as the etiology for 388 (39.8%) of 976 hospitalizations, with most cases (291, 75.0%) confirmed virologically and by multiple methods. Among 351 cases of virologically confirmed dengue, 320 (91.2%) were due to DENV-1. Acute dengue was associated with self-reported rural residence, travel, and months having greatest rainfall. Sequencing of selected dengue viruses revealed that sequences were most closely related to those described from China and Southeast Asia, not nearby India. Conclusions/Significance We describe the first epidemic of DENV-1 in the Southern Province of Sri Lanka in a population known to be susceptible to this serotype because of prior study. Dengue accounted for 40% of acute febrile illnesses in the current study. The emergence of DENV-1 as the foremost serotype in

  3. Virus antibody dynamics in primary and secondary dengue infections.

    Science.gov (United States)

    Gujarati, Tanvi P; Ambika, G

    2014-12-01

    Dengue viral infections show unique infection patterns arising from its four serotypes, (DENV-1,2,3,4). Its effects range from simple fever in primary infections to potentially fatal secondary infections. We analytically and numerically analyse virus dynamics and humoral response in a host during primary and secondary dengue infection for long periods using micro-epidemic models. The models presented here incorporate time delays, antibody dependent enhancement, a dynamic switch and a correlation factor between different DENV serotypes. We find that the viral load goes down to undetectable levels within 7-14 days as is observed for dengue infection, in both cases. For primary infection, the stability analysis of steady states shows interesting dependence on the time delay involved in the production of antibodies from plasma cells. We demonstrate the existence of a critical value for the immune response parameter, beyond which the infection gets completely cured. For secondary infections with a different serotype, the homologous antibody production is enhanced due to the influence of heterologous antibodies. The antibody production is also controlled by the correlation factor, which is a measure of similarities between the different DENV serotypes involved. Our results agree with clinically observed humoral responses for primary and secondary infections.

  4. Domain Ⅲ of Dengue Virus Serotype 2 Envelope:Expression at High Levels in Escherichia coli and Competitive Inhibition of Virus Entry

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Obejective The domainⅢof dengue virus type 2 envelope was cloned and expressed in Escherichia coli and the inhibited effects of recombinant protein on virus was detected. Methods In this study, the domainⅢ(DⅢ) protein of the dengue virus type-2 (DENV-2) envelope (E) antigen was expressed in Escherichia coli by fusion with a carrier protein. The protein was puriifed using enzymatic cleavage and afifnity puriifcation. Rabbit immunization and antibody detection was carried out. Inhibition of DENV-2 infection was observed by DENV-2 EDⅢprotein and its immunity rabbits serum. Results The recombinant expression DENV-2 EDⅢ protein plasmid was constructed successfully. After isopropyl thiogalactoside induction, a speciifc soluble 29 kD protein was obtained, and the expression product accounted for 68.87%of the total protein of the cell lysate. Western blot demonstrated the reactivity of the recombinant protein with his-tag and DENV (Ⅰ-Ⅳ) monoclonal antibodies. The protein was puriifed using enzymatic cleavage and affinity purification. The purified recombinant EDⅢ protein inhibited the entry of DENV-2 into BHK-21 cells. DENV-2 plaque neutralization assays were carried out using serially diluted antibodies against EDⅢprotein. At a 1︰16 dilution, the antibodies produced at least 90%neutralization of the DENV-2 virus. Furthermore, the antibodies continued to exhibit high neutralization effects (approximately 80%) until the anti-EDⅢantibody titer reached 1︰1 024. Conclusions DENV-2 EDⅢwas cloned and expressed successfully. DENV-2 EDⅢprotein could be useful in the development of inexpensive dengue vaccine. The data also suggested that DENV-2 employed an attachment molecule or receptor for its entry into C6/36 mosquito cells.

  5. Comparative analysis of full genomic sequences among different genotypes of dengue virus type 3

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    Lin Ting-Hsiang

    2008-05-01

    Full Text Available Abstract Background Although the previous study demonstrated the envelope protein of dengue viruses is under purifying selection pressure, little is known about the genetic differences of full-length viral genomes of DENV-3. In our study, complete genomic sequencing of DENV-3 strains collected from different geographical locations and isolation years were determined and the sequence diversity as well as selection pressure sites in the DENV genome other than within the E gene were also analyzed. Results Using maximum likelihood and Bayesian approaches, our phylogenetic analysis revealed that the Taiwan's indigenous DENV-3 isolated from 1994 and 1998 dengue/DHF epidemics and one 1999 sporadic case were of the three different genotypes – I, II, and III, each associated with DENV-3 circulating in Indonesia, Thailand and Sri Lanka, respectively. Sequence diversity and selection pressure of different genomic regions among DENV-3 different genotypes was further examined to understand the global DENV-3 evolution. The highest nucleotide sequence diversity among the fully sequenced DENV-3 strains was found in the nonstructural protein 2A (mean ± SD: 5.84 ± 0.54 and envelope protein gene regions (mean ± SD: 5.04 ± 0.32. Further analysis found that positive selection pressure of DENV-3 may occur in the non-structural protein 1 gene region and the positive selection site was detected at position 178 of the NS1 gene. Conclusion Our study confirmed that the envelope protein is under purifying selection pressure although it presented higher sequence diversity. The detection of positive selection pressure in the non-structural protein along genotype II indicated that DENV-3 originated from Southeast Asia needs to monitor the emergence of DENV strains with epidemic potential for better epidemic prevention and vaccine development.

  6. Divergence of the dengue virus type 2 Cosmopolitan genotype associated with two predominant serotype shifts between 1 and 2 in Surabaya, Indonesia, 2008-2014.

    Science.gov (United States)

    Kotaki, Tomohiro; Yamanaka, Atsushi; Mulyatno, Kris Cahyo; Churrotin, Siti; Sucipto, Teguh Hari; Labiqah, Amaliah; Ahwanah, Nur Laila Fitriati; Soegijanto, Soegeng; Kameoka, Masanori; Konishi, Eiji

    2016-01-01

    Indonesia is one of the biggest dengue endemic countries, and, thus, is an important place to investigate the evolution of dengue virus (DENV). We have continuously isolated DENV in Surabaya, the second biggest city in Indonesia, since 2008. We previously reported sequential changes in the predominant serotype from DENV type 2 (DENV-2) to DENV type 1 (DENV-1) in November 2008 and from DENV-1 to DENV-2 in July 2013. The predominance of DENV-2 continued in 2014, but not in 2015. We herein phylogenetically investigated DENV-2 transitions in Surabaya between 2008 and 2014 to analyze the divergence and evolution of DENV-2 concomitant with serotype shifts. All DENV-2 isolated in Surabaya were classified into the Cosmopolitan genotype, and further divided into 6 clusters. Clusters 1-3, dominated by Surabaya strains, were defined as the "Surabaya lineage". Clusters 4-6, dominated by strains from Singapore, Malaysia, and many parts of Indonesia, were the "South East Asian lineage". The most recent common ancestor of these strains existed in 1988, coinciding with the time that an Indonesian dengue outbreak took place. Cluster 1 appeared to be unique because no other DENV-2 isolate was included in this cluster. The predominance of DENV-2 in 2008 and 2013-14 were caused by cluster 1, whereas clusters 2 and 3 sporadically emerged in 2011 and 2012. The characteristic amino acids of cluster 1, E-170V and E-282Y, may be responsible for its prevalence in Surabaya. No amino acid difference was observed in the envelope region between strains in 2008 and 2013-14, suggesting that the re-emergence of DENV-2 in Surabaya was due to the loss or decrease of herd immunity in the 5-year period when DENV-2 subsided. The South East Asian lineage primarily emerged in Surabaya in 2014, probably imported from other parts of Indonesia or foreign countries.

  7. Modulation of inflammation and pathology during dengue virus infection by p38 MAPK inhibitor SB203580.

    Science.gov (United States)

    Fu, Yilong; Yip, Andy; Seah, Peck Gee; Blasco, Francesca; Shi, Pei-Yong; Hervé, Maxime

    2014-10-01

    Dengue virus (DENV) infection could lead to dengue fever (DF), dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The disease outcome is controlled by both viral and host factors. Inflammation mediators from DENV-infected cells could contribute to increased vascular permeability, leading to severe DHF/DSS. Therefore, suppression of inflammation could be a potential therapeutic approach for treatment of dengue patients. In this context, p38 MAPK (mitogen-activated protein kinase) is a key enzyme that modulates the initiation of stress and inflammatory responses. Here we show that SB203580, a p38 MAPK inhibitor, suppressed the over production of DENV-induced pro-inflammatory mediators such as TNF-α, IL-8, and RANTES from human PBMCs, monocytic THP-1, and granulocyte KU812 cell lines. Oral administration of SB203580 in DENV-infected AG129 mice prevented hematocrit rise and lymphopenia, limited the development of inflammation and pathology (including intestine leakage), and significantly improved survival. These results, for the first time, have provided experimental evidence to imply that a short term inhibition of p38 MAPK may be beneficial to reduce disease symptoms in dengue patients.

  8. Antibody-dependent enhancement of dengue virus infection is inhibited by SA-17, a doxorubicin derivative

    NARCIS (Netherlands)

    Ayala Nunez, Vanesa; Jarupathirun, Patsaporn; Kaptein, Suzanne; Neyts, Johan; Smit, Jolanda

    2013-01-01

    Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus (DENV)-induced disease during a heterologous re-infection. Despite ADE's clinical impact, only a few antiviral compounds have been assessed for their anti-ADE activity. We reported earlier tha

  9. Functional importance of dengue virus maturation : infectious properties of immature virions

    NARCIS (Netherlands)

    Zybert, Izabela A.; van der Ende-Metselaar, Heidi; Wilschut, Jan; Smit, Jolanda M.

    2008-01-01

    Prior to the release of flavivirus particles from infected cells, the viral surface protein prM is cleaved to M by the cellular enzyme furin. For dengue virus (DENV), this maturation process appears to be very inefficient since a high proportion of progeny virions contain uncleaved prM. Furthermore,

  10. Underrecognized mildly symptomatic viremic dengue virus infections in rural Thai schools and villages

    NARCIS (Netherlands)

    Yoon, I.K.; Rothman, A.L.; Tannitisupawong, D.; Srikiatkhachorn, A.; Jarman, R.G.; Aldstadt, J.; Nisalak, A.; Mammen Jr., M.P.; Thammapalo, S.; Green, S.; Libraty, D.H.; Gibbons, R.V.; Getis, A.; Endy, T.; Jones, J.W.; Koenraadt, C.J.M.; Morrison, A.C.; Fansiri, T.; Pimgate, C.; Scott, T.W.

    2012-01-01

    BACKGROUND: The understanding of dengue virus (DENV) transmission dynamics and the clinical spectrum of infection are critical to informing surveillance and control measures. Geographic cluster studies can elucidate these features in greater detail than cohort studies alone. METHODS: A 4-year longit

  11. Plasma levels of inter-α inhibitor proteins in children with acute dengue virus infection

    NARCIS (Netherlands)

    P. Koraka (Penelope); Y.P. Lim; M.D. Shin (Michael); T.E. Setiati (Tatty); A.T.A. Mairuhu; E.C.M. van Gorp (Eric); A. Soemantri (Augustinus); A.D.M.E. Osterhaus (Albert); B.E.E. Martina (Byron)

    2010-01-01

    textabstractBackground: Inter-α inhibitor proteins (IaIp) belong to a family of protease inhibitors that are involved in the haemostatic and the vascular system. Dengue viruses (DENV) infections are characterized by coagulopathy and increased vascular permeability. In this study we measured the conc

  12. Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2

    Energy Technology Data Exchange (ETDEWEB)

    Dash, Paban Kumar, E-mail: pabandash@rediffmail.com; Sharma, Shashi; Soni, Manisha; Agarwal, Ankita; Parida, Manmohan; Rao, P.V.Lakshmana

    2013-07-05

    Highlights: •Complete genome of Indian DENV-2 was deciphered for the first time in this study. •The recent Indian DENV-2 revealed presence of many unique amino acid residues. •Genotype shift (American to Cosmopolitan) characterizes evolution of DENV-2 in India. •Circulation of a unique clade of DENV-2 in South Asia was identified. -- Abstract: Dengue is the most important arboviral infection of global public health significance. It is now endemic in most parts of the South East Asia including India. Though Dengue virus type 2 (DENV-2) is predominantly associated with major outbreaks in India, complete genome information of Indian DENV-2 is not available. In this study, the full-length genome of five DENV-2 isolates (four from 2001 to 2011 and one from 1960), from different parts of India was determined. The complete genome of the Indian DENV-2 was found to be 10,670 bases long with an open reading frame coding for 3391 amino acids. The recent Indian DENV-2 (2001–2011) revealed a nucleotide sequence identity of around 90% and 97% with an older Indian DENV-2 (1960) and closely related Sri Lankan and Chinese DENV-2 respectively. Presence of unique amino acid residues and non-conservative substitutions in critical amino acid residues of major structural and non-structural proteins was observed in recent Indian DENV-2. Selection pressure analysis revealed positive selection in few amino acid sites of the genes encoding for structural and non-structural proteins. The molecular phylogenetic analysis based on comparison of both complete coding region and envelope protein gene with globally diverse DENV-2 viruses classified the recent Indian isolates into a unique South Asian clade within Cosmopolitan genotype. A shift of genotype from American to Cosmopolitan in 1970s characterized the evolution of DENV-2 in India. Present study is the first report on complete genome characterization of emerging DENV-2 isolates from India and highlights the circulation of a

  13. Suppression of chikungunya virus replication and differential innate responses of human peripheral blood mononuclear cells during co-infection with dengue virus

    OpenAIRE

    Ruiz Silva, Mariana; Aguilar Briseño, José A; Upasani, Vinit; van der Ende-Metselaar, Heidi; Smit, Jolanda M.; Izabela A. Rodenhuis-Zybert

    2017-01-01

    Dengue and chikungunya are viral diseases transmitted to humans by infected Aedes spp. mosquitoes. With an estimated 390 million infected people per year dengue virus (DENV) currently causes the most prevalent arboviral disease. During the last decade chikungunya virus (CHIKV) has caused large outbreaks and has expanded its territory causing millions of cases in Asia, Africa and America. The viruses share a common mosquito vector and during the acute phase cause similar flu-like symptoms that...

  14. Distribution of Dengue Virus Types 1 and 4 in Blood Components from Infected Blood Donors from Puerto Rico.

    Science.gov (United States)

    Añez, Germán; Heisey, Daniel A R; Chancey, Caren; Fares, Rafaelle C G; Espina, Luz M; Souza, Kátia P R; Teixeira-Carvalho, Andréa; Krysztof, David E; Foster, Gregory A; Stramer, Susan L; Rios, Maria

    2016-02-01

    Dengue is a mosquito-borne viral disease caused by the four dengue viruses (DENV-1 to 4) that can also be transmitted by blood transfusion and organ transplantation. The distribution of DENV in the components of blood from infected donors is poorly understood. We used an in-house TaqMan qRT-PCR assay to test residual samples of plasma, cellular components of whole blood (CCWB), serum and clot specimens from the same collection from blood donors who were DENV-RNA-reactive in a parallel blood safety study. To assess whether DENV RNA detected by TaqMan was associated with infectious virus, DENV infectivity in available samples was determined by culture in mosquito cells. DENV RNA was detected by TaqMan in all tested blood components, albeit more consistently in the cellular components; 78.8% of CCWB, 73.3% of clots, 86.7% of sera and 41.8% of plasma samples. DENV-1 was detected in 48 plasma and 97 CCWB samples while DENV-4 was detected in 21 plasma and 31 CCWB samples. In mosquito cell cultures, 29/111 (26.1%) plasma and 32/97 (32.7%) CCWB samples were infectious. A subset of samples from 29 donors was separately analyzed to compare DENV viral loads in the available blood components. DENV viral loads did not differ significantly between components and ranged from 3-8 log10 PCR-detectable units/ml. DENV was present in all tested components from most donors, and viral RNA was not preferentially distributed in any of the tested components. Infectious DENV was also present in similar proportions in cultured plasma, clot and CCWB samples, indicating that these components may serve as a resource when sample sizes are limited. However, these results suggest that the sensitivity of the nucleic acid tests (NAT) for these viruses would not be improved by testing whole blood or components other than plasma.

  15. Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway.

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    Milly M Choy

    2015-11-01

    Full Text Available Dengue virus (DENV relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.

  16. Mouse models to study dengue virus immunology and pathogenesis

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    Raphaël M. Zellweger

    2014-04-01

    Full Text Available The development of a compelling murine model of dengue virus (DENV infection has been challenging, because dengue virus clinical isolates do not readily replicate or cause pathology in immunocompetent mice. However, research using immunocompromised mice and/or mouse-adapted viruses allows to investigate questions that may be impossible to address in human studies. In this review, we discuss the potential strengths and limitations of existing mouse models of dengue disease. Human studies are descriptive by nature; moreover, the strain, time, and sequence of infection are often unknown. In contrast, in mice, the conditions of infection are well defined and a large number of experimental parameters can be varied at will. Therefore, mouse models offer an opportunity to experimentally test hypotheses that are based on epidemiological observations. In particular, gain-of-function or loss-of-function models can be established to assess how different components of the immune system (either alone or in combination contribute to protection or pathogenesis during secondary infections or after vaccination. In addition, mouse models have been used for pre-clinical testing of antiviral drug or for vaccine development studies. Conclusions based on mouse experiments must be extrapolated to DENV infection in humans with caution due to the inherent limitations of animal models. However, research in mouse models is a useful complement to in vitro and epidemiological data, and may delineate new areas that deserve attention during future human studies.

  17. T-cell Responses in Individuals Infected with Zika Virus and in Those Vaccinated Against Dengue Virus

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    Dominic Paquin-Proulx

    2017-06-01

    Full Text Available Background: The outbreak of Zika virus (ZIKV infection in Brazil has raised concerns that infection during pregnancy could cause microcephaly and other severe neurodevelopmental malformations in the fetus. The mechanisms by which ZIKV causes fetal abnormalities are largely unknown. The importance of pre-infection with dengue virus (DENV, or other flaviviruses endemic to Brazil, remains to be investigated. It has been reported that antibodies directed against DENV can increase ZIKV infectivity by antibody dependent enhancement (ADE, suggesting that a history of prior DENV infection might worsen the outcome of ZIKV infection. Methods: We used bioinformatics tools to design 18 peptides from the ZIKV envelope containing predicted HLA-I T-cell epitopes and investigated T-cell cross-reactivity between ZIKV-infected individuals and DENV-vaccinated subjects by IFNg ELISPOT. Results: Three peptides induced IFNg production in both ZIKV-infected subjects and in DENV-vaccinated individuals. Flow cytometry indicated that 1 ZIKV peptide induced a CD4+ T-cell response in DENV-vaccinated subjects. Conclusions: We demonstrated that vaccination against DENV induced a T-cell response against ZIKV and identified one such CD4+ T-cell epitope. The ZIKV-reactive CD4+ T cells induced by DENV vaccination and identified in this study could contribute to the appearance of cross-reactive antibodies mediating ADE.

  18. T-cell Responses in Individuals Infected with Zika Virus and in Those Vaccinated Against Dengue Virus

    Science.gov (United States)

    Paquin-Proulx, Dominic; Leal, Fabio E.; Terrassani Silveira, Cassia G.; Maestri, Alvino; Brockmeyer, Claudia; Kitchen, Shannon M.; Cabido, Vinicius D.; Kallas, Esper G.; Nixon, Douglas F.

    2017-01-01

    Background The outbreak of Zika virus (ZIKV) infection in Brazil has raised concerns that infection during pregnancy could cause microcephaly and other severe neurodevelopmental malformations in the fetus. The mechanisms by which ZIKV causes fetal abnormalities are largely unknown. The importance of pre-infection with dengue virus (DENV), or other flaviviruses endemic to Brazil, remains to be investigated. It has been reported that antibodies directed against DENV can increase ZIKV infectivity by antibody dependent enhancement (ADE), suggesting that a history of prior DENV infection might worsen the outcome of ZIKV infection. Methods We used bioinformatics tools to design 18 peptides from the ZIKV envelope containing predicted HLA-I T-cell epitopes and investigated T-cell cross-reactivity between ZIKV-infected individuals and DENV-vaccinated subjects by IFNγ ELISPOT. Results Three peptides induced IFNγ production in both ZIKV-infected subjects and in DENV-vaccinated individuals. Flow cytometry indicated that 1 ZIKV peptide induced a CD4+ T-cell response in DENV-vaccinated subjects. Conclusions We demonstrated that vaccination against DENV induced a T-cell response against ZIKV and identified one such CD4+ T-cell epitope. The ZIKV-reactive CD4+ T cells induced by DENV vaccination and identified in this study could contribute to the appearance of cross-reactive antibodies mediating ADE. PMID:28835931

  19. Structural features of NS3 of Dengue virus serotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin.

    Science.gov (United States)

    Pan, Ankita; Saw, Wuan Geok; Subramanian Manimekalai, Malathy Sony; Grüber, Ardina; Joon, Shin; Matsui, Tsutomu; Weiss, Thomas M; Grüber, Gerhard

    2017-05-01

    Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174PPAVP179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.

  20. Seroprevalence of antibodies against dengue virus among pregnant women in the Democratic Republic of Sao Tome and Principe.

    Science.gov (United States)

    Yen, Tsai-Ying; Trovoada dos Santos, Maria de Jesus; Tseng, Lien-Feng; Chang, Shu-Feng; Cheng, Chien-Fu; Carvalho, Arlindo Vicente de Assunção; Shu, Pei-Yun; Lien, Jih-Ching; Tsai, Kun-Hsien

    2016-03-01

    Dengue fever has become a worldwide public health concern, threatening an estimated 40% of the world's population. However, most resources and attention are still focused on malaria, while dengue statuses are poorly recognized in many African countries. In this serological survey, dengue virus (DENV) transmission was demonstrated by using serum samples collected from 78 pregnant women in the Democratic Republic of Sao Tome and Principe (DRSTP) during 2003 to 2004. Immunofluorescence assay was performed and 31 samples (39.74%) were found positive for DENV antibodies. Indirect enzyme-linked immunosorbent assay (ELISA) showed that 53 samples (67.95%) were positive for dengue E IgG, and 38 samples (48.72%) were positive for NS1 IgG. A prevalence of 35.90% was therefore determined for dengue IgG by considering samples that yielded positive results by all three tests. Cross-reactions with other flaviviruses were examined by indirect ELISA against Japanese encephalitis virus, West Nile virus, and yellow fever virus. Only one sample exhibited stronger absorbance against Japanese encephalitis virus and West Nile virus. Moreover, one sample was positive for dengue IgM. These results agreed with the previous researches in neighboring countries and suggested DENV exposure. The study contributes to raising public awareness of dengue and supporting future control strategies.

  1. Dengue virus infection elicits highly polarized CX3CR1+ cytotoxic CD4+ T cells associated with protective immunity.

    Science.gov (United States)

    Weiskopf, Daniela; Bangs, Derek J; Sidney, John; Kolla, Ravi V; De Silva, Aruna D; de Silva, Aravinda M; Crotty, Shane; Peters, Bjoern; Sette, Alessandro

    2015-08-04

    Dengue virus (DENV) is a rapidly spreading pathogen with unusual pathogenesis, and correlates of protection from severe dengue disease and vaccine efficacy have not yet been established. Although DENV-specific CD8(+) T-cell responses have been extensively studied, the breadth and specificity of CD4(+) T-cell responses remains to be defined. Here we define HLA-restricted CD4(+) T-cell epitopes resulting from natural infection with dengue virus in a hyperepidemic setting. Ex vivo flow-cytometric analysis of DENV-specific CD4(+) T cells revealed that the virus-specific cells were highly polarized, with a strong bias toward a CX3CR1(+) Eomesodermin(+) perforin(+) granzyme B(+) CD45RA(+) CD4 CTL phenotype. Importantly, these cells correlated with a protective HLA DR allele, and we demonstrate that these cells have direct ex vivo DENV-specific cytolytic activity. We speculate that cytotoxic dengue-specific CD4(+) T cells may play a role in the control of dengue infection in vivo, and this immune correlate may be a key target for dengue virus vaccine development.

  2. Development of viable TAP-tagged dengue virus for investigation of host-virus interactions in viral replication.

    Science.gov (United States)

    Poyomtip, Teera; Hodge, Kenneth; Matangkasombut, Ponpan; Sakuntabhai, Anavaj; Pisitkun, Trairak; Jirawatnotai, Siwanon; Chimnaronk, Sarin

    2016-03-01

    Dengue virus (DENV) is a mosquito-borne flavivirus responsible for life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The viral replication machinery containing the core non-structural protein 5 (NS5) is implicated in severe dengue symptoms but molecular details remain obscure. To date, studies seeking to catalogue and characterize interaction networks between viral NS5 and host proteins have been limited to the yeast two-hybrid system, computational prediction and co-immunoprecipitation (IP) of ectopically expressed NS5. However, these traditional approaches do not reproduce a natural course of infection in which a number of DENV NS proteins colocalize and tightly associate during the replication process. Here, we demonstrate the development of a recombinant DENV that harbours a TAP tag in NS5 to study host-virus interactions in vivo. We show that our engineered DENV was infective in several human cell lines and that the tags were stable over multiple viral passages, suggesting negligible structural and functional disturbance of NS5. We further provide proof-of-concept for the use of rationally tagged virus by revealing a high confidence NS5 interaction network in human hepatic cells. Our analysis uncovered previously unrecognized hnRNP complexes and several low-abundance fatty acid metabolism genes, which have been implicated in the viral life cycle. This study sets a new standard for investigation of host-flavivirus interactions.

  3. Clinical, Virologic, and Epidemiologic Characteristics of Dengue Outbreak, Dar es Salaam, Tanzania, 2014.

    Science.gov (United States)

    Vairo, Francesco; Mboera, Leonard E G; De Nardo, Pasquale; Oriyo, Ndekya M; Meschi, Silvia; Rumisha, Susan F; Colavita, Francesca; Mhina, Athanas; Carletti, Fabrizio; Mwakapeje, Elibariki; Capobianchi, Maria Rosaria; Castilletti, Concetta; Di Caro, Antonino; Nicastri, Emanuele; Malecela, Mwelecele N; Ippolito, Giuseppe

    2016-05-01

    We investigated a dengue outbreak in Dar es Salaam, Tanzania, in 2014, that was caused by dengue virus (DENV) serotype 2. DENV infection was present in 101 (20.9%) of 483 patients. Patient age and location of residence were associated with infection. Seven (4.0%) of 176 patients were co-infected with malaria and DENV.

  4. Detection of dengue virus in platelets isolated from dengue patients.

    Science.gov (United States)

    Noisakran, Sansanee; Gibbons, Robert V; Songprakhon, Pucharee; Jairungsri, Aroonroong; Ajariyakhajorn, Chuanpis; Nisalak, Ananda; Jarman, Richard G; Malasit, Prida; Chokephaibulkit, Kulkanya; Perng, Guey Chuen

    2009-03-01

    Though thrombocytopenia or dysfunction of platelets is common in dengue virus infection, the role of platelets has not been established. We enrolled 33 hospitalized children with serologically confirmed dengue virus infection. Blood specimens were collected during hospitalization. Platelets and plasma were isolated from the whole blood. Detection of dengue virus in plasma and platelets was carried out by RT-PCR with primers that can differentiate different dengue serotypes simultaneously, and by electron transmission microscopy (EM). Dengue viral RNA was detected in the platelets and plasma by conventional RT-PCR. A significantly higher percentage of dengue viral RNA was detected in platelets than in plasma (p = 0.03). Platelets isolated 5 days after onset of fever were most likely positive for viral RNA. Concurrent infection or co-circulation with multiple dengue serotypes was observed in 12% of patients. Infrequently, negative-stranded dengue viral RNA was detected in platelets and in plasma. Importantly, EM confirmed the presence of dengue viral-like particles inside platelets prepared from dengue patients. Our findings suggest the presence of dengue virus in platelets may be associated with the dysfunction of platelets observed in dengue patients.

  5. Recombinant Dengue virus protein NS2B alters membrane permeability in different membrane models

    OpenAIRE

    León-Juárez, Moisés; Martínez-Castillo, Macario; Shrivastava, Gaurav; García-Cordero, Julio; Villegas-Sepulveda, Nicolás; Mondragón-Castelán, Mónica; Mondragón-Flores, Ricardo; Cedillo-Barrón, Leticia

    2016-01-01

    Background One of the main phenomena occurring in cellular membranes during virus infection is a change in membrane permeability. It has been observed that numerous viral proteins can oligomerize and form structures known as viroporins that alter the permeability of membranes. Previous findings have identified such proteins in cells infected with Japanese encephalitis virus (JEV), a member of the same family that Dengue virus (DENV) belongs to (Flaviviridae). In the present work, we investiga...

  6. Unusual dengue virus 3 epidemic in Nicaragua, 2009.

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    Gamaliel Gutierrez

    2011-11-01

    Full Text Available The four dengue virus serotypes (DENV1-4 cause the most prevalent mosquito-borne viral disease affecting humans worldwide. In 2009, Nicaragua experienced the largest dengue epidemic in over a decade, marked by unusual clinical presentation, as observed in two prospective studies of pediatric dengue in Managua. From August 2009-January 2010, 212 dengue cases were confirmed among 396 study participants at the National Pediatric Reference Hospital. In our parallel community-based cohort study, 170 dengue cases were recorded in 2009-10, compared to 13-65 cases in 2004-9. In both studies, significantly more patients experienced "compensated shock" (poor capillary refill plus cold extremities, tachycardia, tachypnea, and/or weak pulse in 2009-10 than in previous years (42.5% [90/212] vs. 24.7% [82/332] in the hospital study (p<0.001 and 17% [29/170] vs. 2.2% [4/181] in the cohort study (p<0.001. Signs of poor peripheral perfusion presented significantly earlier (1-2 days in 2009-10 than in previous years according to Kaplan-Meier survival analysis. In the hospital study, 19.8% of subjects were transferred to intensive care, compared to 7.1% in previous years - similar to the cohort study. DENV-3 predominated in 2008-9, 2009-10, and 2010-11, and full-length sequencing revealed no major genetic changes from 2008-9 to 2010-11. In 2008-9 and 2010-11, typical dengue was observed; only in 2009-10 was unusual presentation noted. Multivariate analysis revealed only "2009-10" as a significant risk factor for Dengue Fever with Compensated Shock. Interestingly, circulation of pandemic influenza A-H1N1 2009 in Managua was shifted such that it overlapped with the dengue epidemic. We hypothesize that prior influenza A H1N1 2009 infection may have modulated subsequent DENV infection, and initial results of an ongoing study suggest increased risk of shock among children with anti-H1N1-2009 antibodies. This study demonstrates that parameters other than serotype, viral

  7. Autoimmunity in dengue pathogenesis

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    Shu-Wen Wan

    2013-01-01

    Full Text Available Dengue is one of the most important vector-borne viral diseases. With climate change and the convenience of travel, dengue is spreading beyond its usual tropical and subtropical boundaries. Infection with dengue virus (DENV causes diseases ranging widely in severity, from self-limited dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome. Vascular leakage, thrombocytopenia, and hemorrhage are the major clinical manifestations associated with severe DENV infection, yet the mechanisms remain unclear. Besides the direct effects of the virus, immunopathogenesis is also involved in the development of dengue disease. Antibody-dependent enhancement increases the efficiency of virus infection and may suppress type I interferon-mediated antiviral responses. Aberrant activation of T cells and overproduction of soluble factors cause an increase in vascular permeability. DENV-induced autoantibodies against endothelial cells, platelets, and coagulatory molecules lead to their abnormal activation or dysfunction. Molecular mimicry between DENV proteins and host proteins may explain the cross-reactivity of DENV-induced autoantibodies. Although no licensed dengue vaccine is yet available, several vaccine candidates are under development. For the development of a safe and effective dengue vaccine, the immunopathogenic complications of dengue disease need to be considered.

  8. Comparative Susceptibility of Aedes albopictus and Aedes aegypti to Dengue Virus Infection After Feeding on Blood of Viremic Humans: Implications for Public Health.

    Science.gov (United States)

    Whitehorn, James; Kien, Duong Thi Hue; Nguyen, Nguyet Minh; Nguyen, Hoa L; Kyrylos, Peter P; Carrington, Lauren B; Tran, Chau Nguyen Bich; Quyen, Nguyen Thanh Ha; Thi, Long Vo; Le Thi, Dui; Truong, Nguyen Thanh; Luong, Tai Thi Hue; Nguyen, Chau Van Vinh; Wills, Bridget; Wolbers, Marcel; Simmons, Cameron P

    2015-10-15

    Aedes albopictus is secondary to Aedes aegypti as a vector of dengue viruses (DENVs) in settings of endemicity, but it plays an important role in areas of dengue emergence. This study compared the susceptibility of these 2 species to DENV infection by performing 232 direct blood-feeding experiments on 118 viremic patients with dengue in Vietnam. Field-derived A. albopictus acquired DENV infections as readily as A. aegypti after blood feeding. Once infected, A. albopictus permitted higher concentrations of DENV RNA to accumulate in abdominal tissues, compared with A. aegypti. However, the odds of A. albopictus having infectious saliva were lower than the odds observed for A. aegypti (odds ratio, 0.70; 95% confidence interval, .52-.93). These results quantitate the susceptibility of A. albopictus to DENV infection and will assist parameterization of models for predicting disease risk in settings where A. albopictus is present.

  9. Induction of neutralizing antibodies against four serotypes of dengue viruses by MixBiEDIII, a tetravalent dengue vaccine.

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    Hui Zhao

    Full Text Available The worldwide expansion of four serotypes of dengue virus (DENV poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII was proposed. Tandem EDIIIs of two serotypes (type 1-2 and type 3-4 of DENV connected by a Gly-Ser linker ((Gly4Ser3 were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.

  10. Neutralizing activities of human immunoglobulin derived from donors in Japan against mosquito-borne flaviviruses, Japanese encephalitis virus, West Nile virus, and dengue virus

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    Yunoki M

    2016-07-01

    Full Text Available Mikihiro Yunoki,1-3 Takeshi Kurosu,2 Ritsuko Kubota Koketsu,2,4 Kazuo Takahashi,5 Yoshinobu Okuno,4 Kazuyoshi Ikuta2,4 1Research and Development Division, Japan Blood Products Organization, Tokyo, 2Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, 3Pathogenic Risk Evaluation, Graduate School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, 4Research and Development Division, The Research Foundation for Microbial Diseases of Osaka University, Kagawa, 5Osaka Prefectural Institute of Public Health, Osaka, Japan Abstract: Japanese encephalitis virus (JEV, West Nile virus (WNV, and dengue virus (DenV are causal agents of Japanese encephalitis, West Nile fever, and dengue fever, respectively. JEV is considered to be indigenized and widespread in Japan, whereas WNV and DenV are not indigenized in Japan. Globulin products seem to reflect the status of the donor population according to antivirus neutralization activity. However, the anti-JEV, -WNV, and -DenV neutralization activities of globulin products derived from donors in Japan have not been clarified. Furthermore, potential candidates for the development of an effective immunotherapeutic drug for encephalitis caused by JEV, WNV, or DenV have also not been identified. Therefore, the aim of this study was to determine the overall status of the donor population in Japan based on globulin products by evaluating anti-JEV, -WNV, and -DenV neutralizing activities of intravenous immunoglobulin. Overall, intravenous immunoglobulin products showed stable neutralizing activity against JEV but showed no or only weak activity against WNV or DenV. These results suggest that the epidemiological level against WNV and DenV in the donor population of Japan is still low, suggesting that these viruses are not yet indigenized. In addition, JEV vaccinations and/or infections in the donor population do not induce a cross-reactive antibody against WNV. Keywords

  11. Fine Scale Spatiotemporal Clustering of Dengue Virus Transmission in Children and Aedes aegypti in Rural Thai Villages

    NARCIS (Netherlands)

    Yoon, I.K.; Getis, A.; Aldstadt, J.; Rothman, A.L.; Tannitisupawong, D.; Koenraadt, C.J.M.; Fansiri, T.; Jones, J.W.; Morrison, A.C.; Jarman, R.G.; Nisalak, A.; Mammen Jr., M.P.; Thammapalo, S.; Srikiatkhachorn, A.; Green, S.; Libraty, D.H.; Gibbons, R.V.; Endy, T.; Pimgate, C.; Scott, T.W.

    2012-01-01

    Background Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that

  12. Fine Scale Spatiotemporal Clustering of Dengue Virus Transmission in Children and Aedes aegypti in Rural Thai Villages

    NARCIS (Netherlands)

    Yoon, I.K.; Getis, A.; Aldstadt, J.; Rothman, A.L.; Tannitisupawong, D.; Koenraadt, C.J.M.; Fansiri, T.; Jones, J.W.; Morrison, A.C.; Jarman, R.G.; Nisalak, A.; Mammen Jr., M.P.; Thammapalo, S.; Srikiatkhachorn, A.; Green, S.; Libraty, D.H.; Gibbons, R.V.; Endy, T.; Pimgate, C.; Scott, T.W.

    2012-01-01

    Background Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that supp

  13. Construction and characterisation of a complete reverse genetics system of dengue virus type 3

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    Jefferson Jose da Silva Santos

    2013-12-01

    Full Text Available Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

  14. Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes

    Science.gov (United States)

    Waman, Vaishali P.; Kolekar, Pandurang; Ramtirthkar, Mukund R.; Kale, Mohan M.

    2016-01-01

    Background Dengue is one of the most common arboviral diseases prevalent worldwide and is caused by Dengue viruses (genus Flavivirus, family Flaviviridae). There are four serotypes of Dengue Virus (DENV-1 to DENV-4), each of which is further subdivided into distinct genotypes. DENV-2 is frequently associated with severe dengue infections and epidemics. DENV-2 consists of six genotypes such as Asian/American, Asian I, Asian II, Cosmopolitan, American and sylvatic. Comparative genomic study was carried out to infer population structure of DENV-2 and to analyze the role of evolutionary and spatiotemporal factors in emergence of diversifying lineages. Methods Complete genome sequences of 990 strains of DENV-2 were analyzed using Bayesian-based population genetics and phylogenetic approaches to infer genetically distinct lineages. The role of spatiotemporal factors, genetic recombination and selection pressure in the evolution of DENV-2 is examined using the sequence-based bioinformatics approaches. Results DENV-2 genetic structure is complex and consists of fifteen subpopulations/lineages. The Asian/American genotype is observed to be diversified into seven lineages. The Asian I, Cosmopolitan and sylvatic genotypes were found to be subdivided into two lineages, each. The populations of American and Asian II genotypes were observed to be homogeneous. Significant evidence of episodic positive selection was observed in all the genes, except NS4A. Positive selection operational on a few codons in envelope gene confers antigenic and lineage diversity in the American strains of Asian/American genotype. Selection on codons of non-structural genes was observed to impact diversification of lineages in Asian I, cosmopolitan and sylvatic genotypes. Evidence of intra/inter-genotype recombination was obtained and the uncertainty in classification of recombinant strains was resolved using the population genetics approach. Discussion Complete genome-based analysis revealed that the

  15. Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes

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    Vaishali P. Waman

    2016-08-01

    Full Text Available Background Dengue is one of the most common arboviral diseases prevalent worldwide and is caused by Dengue viruses (genus Flavivirus, family Flaviviridae. There are four serotypes of Dengue Virus (DENV-1 to DENV-4, each of which is further subdivided into distinct genotypes. DENV-2 is frequently associated with severe dengue infections and epidemics. DENV-2 consists of six genotypes such as Asian/American, Asian I, Asian II, Cosmopolitan, American and sylvatic. Comparative genomic study was carried out to infer population structure of DENV-2 and to analyze the role of evolutionary and spatiotemporal factors in emergence of diversifying lineages. Methods Complete genome sequences of 990 strains of DENV-2 were analyzed using Bayesian-based population genetics and phylogenetic approaches to infer genetically distinct lineages. The role of spatiotemporal factors, genetic recombination and selection pressure in the evolution of DENV-2 is examined using the sequence-based bioinformatics approaches. Results DENV-2 genetic structure is complex and consists of fifteen subpopulations/lineages. The Asian/American genotype is observed to be diversified into seven lineages. The Asian I, Cosmopolitan and sylvatic genotypes were found to be subdivided into two lineages, each. The populations of American and Asian II genotypes were observed to be homogeneous. Significant evidence of episodic positive selection was observed in all the genes, except NS4A. Positive selection operational on a few codons in envelope gene confers antigenic and lineage diversity in the American strains of Asian/American genotype. Selection on codons of non-structural genes was observed to impact diversification of lineages in Asian I, cosmopolitan and sylvatic genotypes. Evidence of intra/inter-genotype recombination was obtained and the uncertainty in classification of recombinant strains was resolved using the population genetics approach. Discussion Complete genome-based analysis

  16. Serotype-specific anti-Dengue virus NS1 mouse antibodies cross-react with prM and are potentially involved in virus production.

    Science.gov (United States)

    Masrinoul, Promsin; Omokoko, Magot Diata; Pambudi, Sabar; Ikuta, Kazuyoshi; Kurosu, Takeshi

    2013-08-01

    Dengue virus (DENV) infection induces a strong B-cell immune response against the viral nonstructural protein 1 (NS1). Anti-NS1 antibodies (Abs) may affect virus production because they coexist with the virus in the patients' blood. The present study examined whether ten mouse monoclonal antibodies (MAbs) raised against NS1 affected production of the DENV-2. Three MAbs, 4C2, 4G11, and 4E5, showed weak neutralizing activity in a focus reduction assay. In addition, two serotype-specific MAbs, 4C2 and 4G11, protected suckling mice from lethal infection with DENV-2. An immunoprecipitation assay with DENV-2 showed that these MAbs, which were specific for the NS1 of DENV-4 and DENV-1, cross-reacted with the DENV-2 pre-membrane (prM) protein, but not with DENV-2 NS1. Interestingly, high concentrations of MAb 4G11 showed antibody-dependent enhancement of DENV-2 infection in human monocyte THP-1 cells. Taken together, these observations suggest that serotype-specific anti-NS1 MAbs are potentially involved in virus production.

  17. Dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera.

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    Ruklanthi de Alwis

    2014-10-01

    Full Text Available Dengue viruses (DENV are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to

  18. Binding of dengue virus particles and dengue proteins onto solid surfaces.

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    Pereira, Edla M A; Dario, Aline F; França, Rafael F O; Fonseca, Benedito A L; Petri, Denise F S

    2010-09-01

    The interaction between dengue virus particles (DENV), sedimentation hemagglutinin particles (SHA), dengue virus envelope protein (Eprot), and solid surfaces was investigated by means of ellipsometry and atomic force microscopy (AFM). The surfaces chosen are bare Si/SiO2 wafers and Si/SiO2 wafers covered with concanavalin A (ConA), jacalin (Jac), polystyrene (PS), or poly(styrene sulfonate) (PSS) films. Adsorption experiments at pH 7.2 and pH 3 onto all surfaces revealed that (i) adsorption of DENV particles took place only onto ConA under pH 7.2, because of specific recognition between glycans on DENV surface and ConA binding site; (ii) DENV particles did not attach to any of the surfaces at pH 3, suggesting the presence of positive charges on DENV surface at this pH, which repel the positively charged lectin surfaces; (iii) SHA particles are positively charged at pH 7.2 and pH 3 because they adhered to negatively charged surfaces at pH 7.2 and repelled positively charged layers at pH 3; and (iv) SHA particles carry polar groups on the surface because they attached to silanol surfaces at pH 3 and avoided hydrophobic PS films at pH 3 and pH 7.2. The adsorption behavior of Eprot at pH 7.2 revealed affinity for ConA>Jac>PSS>PS≈bare Si/SiO2 layers. These findings indicate that selectivity of the Eprot adsorption is higher when it is part of virus structure than when it is free in solution. The correlation between surface energy values determined by means of contact angle measurements and DENV, SHA, or Eprot adsorption behavior was used to understand the intermolecular forces at the interfaces. A direct correlation was not found because the contributions from surface energy were probably surpassed by specific contributions.

  19. Temporal dynamics of the transcriptional response to dengue virus infection in Nicaraguan children.

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    Stephen J Popper

    Full Text Available Dengue is the most prevalent mosquito-borne human illness worldwide. The ability to predict disease severity during the earliest days of the illness is a long-sought, but unachieved goal.We examined human genome-wide transcript abundance patterns in daily peripheral blood mononuclear cell (PBMC samples from 41 children hospitalized with dengue virus (DENV infection in Nicaragua, as well as 8 healthy control subjects. Nine patients had primary dengue fever (DF1, 11 had dengue fever with serologic evidence of prior DENV infection, i.e., secondary dengue fever (DF2, 12 had dengue hemorrhagic fever (DHF, and 9 had dengue shock syndrome (DSS. We identified 2,092 genes for which transcript abundance differed significantly between patients on days 3-6 of fever and healthy subjects (FDR<1%. Prior DENV infection explained the greatest amount of variation in gene expression among patients. The number of differentially expressed genes was greatest on fever day 3 in patients with DF1, while the number in patients with DF2 or DHF/DSS was greatest on day 5. Genes associated with the mitotic cell cycle and B cell differentiation were expressed at higher levels, and genes associated with signal transduction and cell adhesion were expressed at lower levels, in patients versus healthy controls. On fever day 3, a set of interferon-stimulated gene transcripts was less abundant in patients who subsequently developed DSS than in other patient groups (p<0.05, ranksum. Patients who later developed DSS also had higher levels of transcripts on day 3 associated with mitochondrial function (p<0.01, ranksum. These day 3 transcript abundance findings were not evident on subsequent fever days.In conclusion, we identified differences in the timing and magnitude of human gene transcript abundance changes in DENV patients that were associated with serologic evidence of prior infection and with disease severity. Some of these differential features may predict the outcome of DENV

  20. Inactivation of Dengue and Yellow Fever viruses by heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX.

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    Assunção-Miranda, I; Cruz-Oliveira, C; Neris, R L S; Figueiredo, C M; Pereira, L P S; Rodrigues, D; Araujo, D F F; Da Poian, A T; Bozza, M T

    2016-03-01

    To investigate the effect of heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX (CoPPIX and SnPPIX), macrocyclic structures composed by a tetrapyrrole ring with a central metallic ion, on Dengue Virus (DENV) and Yellow Fever Virus (YFV) infection. Treatment of HepG2 cells with heme, CoPPIX and SnPPIX after DENV infection reduced infectious particles without affecting viral RNA contents in infected cells. The reduction of viral load occurs only with the direct contact of DENV with porphyrins, suggesting a direct effect on viral particles. Previously incubation of DENV and YFV with heme, CoPPIX and SnPPIX resulted in viral particles inactivation in a dose-dependent manner. Biliverdin, a noncyclical porphyrin, was unable to inactivate the viruses tested. Infection of HepG2 cells with porphyrin-pretreated DENV2 results in a reduced or abolished viral protein synthesis, RNA replication and cell death. Treatment of HepG2 or THP-1 cell lineage with heme or CoPPIX after DENV infection with a very low MOI resulted in a decreased DENV replication and protection from death. Heme, CoPPIX and SnPPIX possess a marked ability to inactivate DENV and YFV, impairing its ability to infect and induce cytopathic effects on target cells. These results open the possibility of therapeutic application of porphyrins or their use as models to design new antiviral drugs against DENV and YFV. © 2016 The Society for Applied Microbiology.

  1. A Native Wolbachia Endosymbiont Does Not Limit Dengue Virus Infection in the Mosquito Aedes notoscriptus (Diptera: Culicidae).

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    Skelton, Ellie; Rancès, Edwige; Frentiu, Francesca D; Kusmintarsih, Endang Srimurni; Iturbe-Ormaetxe, Iñaki; Caragata, Eric P; Woolfit, Megan; O'Neill, Scott L

    2016-03-01

    The endosymbiotic bacterium Wolbachia pipientis infects many species of insects and has been transinfected into the mosquito Aedes aegypti (L.), the primary vector of dengue virus (DENV). Recently, it has been shown that Wolbachia blocks the replication and transmission of RNA viruses, such as DENV, in a number of mosquito species including Ae. aegypti and Aedes albopictus (Skuse), which is naturally infected with Wolbachia and considered a secondary vector for DENV. The mosquito species Aedes notoscriptus (Skuse) is highly prevalent in Australia, including in areas where DENV outbreaks have been recorded. The mosquito has been implicated in the transmission of Ross River and Barmah Forest viruses, but not DENV. We investigated whether Wolbachia naturally infects this mosquito species and whether it has an impact on the ability of Ae. notoscriptus to transmit DENV. We show, for the first time, that Ae. notoscriptus is naturally infected with a strain of Wolbachia that belongs to supergroup B and is localized only in the ovaries. However, Wolbachia infection in Ae. notoscriptus did not induce resistance to DENV and had no effect on overall DENV infection rate or titer. The presence of a native Wolbachia in Ae. notoscriptus cannot explain why this mosquito is an ineffective vector of DENV.

  2. Noninvasive bioluminescence imaging of dengue virus infection in the brain of A129 mice.

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    Li, Xiao-Feng; Deng, Yong-Qiang; Zhao, Hui; Ye, Qing; Wang, Hong-Jiang; Li, Shi-Hua; Zhu, Shun-Ya; Shi, Pei-Yong; Qin, E-De; Zhang, Bo; Qin, Cheng-Feng

    2013-05-01

    Dengue virus (DENV) infection is one of the most important public health threats globally; however, no vaccines or effective antivirals are currently available. The bioluminescence imaging technique has emerged as a powerful tool for studies on viral pathogenesis in vitro and in vivo. In this study, using a recombinant DENV that stably expressed Renilla luciferase (Rluc-DENV), we used bioluminescence for imaging of DENV infection in the brain of A129 mice that lacked type I interferon receptors. Upon intracranial inoculation with Rluc-DENV, A129 mice developed typical neurological symptoms and rapidly succumbed to viral infection. Real-time bioluminescence intensity analysis revealed the replication kinetics of Rluc-DENV in the brain of A129 mice. Linear regression analyses showed a good correlation between photon flux and viral titers (R(2) = 0.9923). Finally, the bioluminescence model was validated using a known mouse monoclonal antibody, 2A10G6, and the therapeutic effects of this neutralizing antibody were readily monitored by live imaging in the same animal. The noninvasive bioluminescence imaging of DENV infection as described here shows distinct advantages over traditional animal models and provides a powerful tool for potential antiviral or vaccine assays against DENV infection in vivo.

  3. Complete genome of a dengue virus serotype 4 strain from Amazonas, Brazil.

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    Nascimento, Valdinete Alves do; Souza, Victor Costa de; Naveca, Felipe Gomes

    2016-02-01

    Dengue virus (DENV) infections represent a significant concern for public health worldwide, being considered as the most prevalent arthropod-borne virus regarding the number of reported cases. In this study, we report the complete genome sequencing of a DENV serotype 4 isolate, genotype II, obtained in the city of Manaus, directly from the serum sample, applying Ion Torrent sequencing technology. The use of a massive sequencing technology allowed the detection of two variable sites, one in the coding region for the viral envelope protein and the other in the nonstructural 1 coding region within viral populations.

  4. A novel dengue virus serotype 1 vaccine candidate based on Japanese encephalitis virus vaccine strain SA14-14-2 as the backbone.

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    Yang, Huiqiang; Li, Zhushi; Lin, Hua; Wang, Wei; Yang, Jian; Liu, Lina; Zeng, Xianwu; Wu, Yonglin; Yu, Yongxin; Li, Yuhua

    2016-06-01

    To develop a potential dengue vaccine candidate, a full-length cDNA clone of a novel chimeric virus was constructed using recombinant DNA technology, with Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 as the backbone, with its premembrane (prM) and envelope (E) genes substituted by their counterparts from dengue virus type 1 (DENV1). The chimeric virus (JEV/DENV1) was successfully recovered from primary hamster kidney (PHK) cells by transfection with the in vitro transcription products of JEV/DENV1 cDNA and was identified by complete genome sequencing and immunofluorescent staining. No neuroinvasiveness of this chimeric virus was observed in mice inoculated by the subcutaneous route (s.c.) or by the intraperitoneal route (i.p.), while some neurovirulence was displayed in mice that were inoculated directly by the intracerebral route (i.c.). The chimeric virus was able to stimulate high-titer production of antibodies against DENV1 and provided protection against lethal challenge with neuroadapted dengue virus in mice. These results suggest that the chimeric virus is a promising dengue vaccine candidate.

  5. CD4+ T cells are not required for the induction of dengue virus-specific CD8+ T cell or antibody responses but contribute to protection after vaccination.

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    Yauch, Lauren E; Prestwood, Tyler R; May, Monica M; Morar, Malika M; Zellweger, Raphaël M; Peters, Bjoern; Sette, Alessandro; Shresta, Sujan

    2010-11-01

    The contribution of T cells to the host response to dengue virus (DENV) infection is not well understood. We previously demonstrated a protective role for CD8(+) T cells during primary DENV infection using a mouse-passaged DENV strain and IFN-α/βR(-/-) C57BL/6 mice, which are susceptible to DENV infection. In this study, we examine the role of CD4(+) T cells during primary DENV infection. Four I-A(b)-restricted epitopes derived from three of the nonstructural DENV proteins were identified. CD4(+) T cells expanded and were activated after DENV infection, with peak activation occurring on day 7. The DENV-specific CD4(+) T cells expressed intracellular IFN-γ, TNF, IL-2, and CD40L, and killed peptide-pulsed target cells in vivo. Surprisingly, depletion of CD4(+) T cells before DENV infection had no effect on viral loads. Consistent with this observation, CD4(+) T cell depletion did not affect the DENV-specific IgG or IgM Ab titers or their neutralizing activity, or the DENV-specific CD8(+) T cell response. However, immunization with the CD4(+) T cell epitopes before infection resulted in significantly lower viral loads. Thus, we conclude that whereas CD4(+) T cells are not required for controlling primary DENV infection, their induction by immunization can contribute to viral clearance. These findings suggest inducing anti-DENV CD4(+) T cell responses by vaccination may be beneficial.

  6. Spatial and temporal clustering of dengue virus transmission in Thai villages.

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    Mammen P Mammen

    2008-11-01

    Full Text Available Transmission of dengue viruses (DENV, the leading cause of arboviral disease worldwide, is known to vary through time and space, likely owing to a combination of factors related to the human host, virus, mosquito vector, and environment. An improved understanding of variation in transmission patterns is fundamental to conducting surveillance and implementing disease prevention strategies. To test the hypothesis that DENV transmission is spatially and temporally focal, we compared geographic and temporal characteristics within Thai villages where DENV are and are not being actively transmitted.Cluster investigations were conducted within 100 m of homes where febrile index children with (positive clusters and without (negative clusters acute dengue lived during two seasons of peak DENV transmission. Data on human infection and mosquito infection/density were examined to precisely (1 define the spatial and temporal dimensions of DENV transmission, (2 correlate these factors with variation in DENV transmission, and (3 determine the burden of inapparent and symptomatic infections. Among 556 village children enrolled as neighbors of 12 dengue-positive and 22 dengue-negative index cases, all 27 DENV infections (4.9% of enrollees occurred in positive clusters (p < 0.01; attributable risk [AR] = 10.4 per 100; 95% confidence interval 1-19.8 per 100]. In positive clusters, 12.4% of enrollees became infected in a 15-d period and DENV infections were aggregated centrally near homes of index cases. As only 1 of 217 pairs of serologic specimens tested in positive clusters revealed a recent DENV infection that occurred prior to cluster initiation, we attribute the observed DENV transmission subsequent to cluster investigation to recent DENV transmission activity. Of the 1,022 female adult Ae. aegypti collected, all eight (0.8% dengue-infected mosquitoes came from houses in positive clusters; none from control clusters or schools. Distinguishing features between

  7. Clinical outcome and genetic differences within a monophyletic Dengue virus type 2 population.

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    Hapuarachchige Chanditha Hapuarachchi

    Full Text Available The exact mechanisms of interplay between host and viral factors leading to severe dengue are yet to be fully understood. Even though previous studies have implicated specific genetic differences of Dengue virus (DENV in clinical severity and virus attenuation, similar studies with large-scale, whole genome screening of monophyletic virus populations are limited. Therefore, in the present study, we compared 89 whole genomes of DENV-2 cosmopolitan clade III isolates obtained from patients diagnosed with dengue fever (DF, n = 58, dengue hemorrhagic fever (DHF, n = 30 and dengue shock syndrome (DSS, n = 1 in Singapore between July 2010 and January 2013, in order to determine the correlation of observed viral genetic differences with clinical outcomes. Our findings showed no significant difference between the number of primary and secondary infections that progressed to DHF and DSS (p>0.05 in our study cohort. Despite being highly homogenous, study isolates possessed 39 amino acid substitutions of which 10 substitutions were fixed in three main groups of virus isolates. None of those substitutions were specifically associated with DHF and DSS. Notably, two evolutionarily unique virus groups possessing C-P43T+NS1-S103T+NS2A-V83I+NS3-R337K+ NS3-I600T+ NS5-P136S and NS2A-T119N mutations were exclusively found in patients with DF, the benign form of DENV infections. Those mutants were significantly associated with mild disease outcome. These observations indicated that disease progression into DHF and DSS within our patient population was more likely to be due to host than virus factors. We hypothesize that selection for potentially less virulent groups of DENV-2 in our study cohort may be an evolutionary adaptation of viral strains to extend their survival in the human-mosquito transmission cycle.

  8. Dengue-1 virus and vector competence of Aedes aegypti (Diptera: Culicidae) populations from New Caledonia.

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    Calvez, Elodie; Guillaumot, Laurent; Girault, Dominique; Richard, Vaea; O'Connor, Olivia; Paoaafaite, Tuterarii; Teurlai, Magali; Pocquet, Nicolas; Cao-Lormeau, Van-Mai; Dupont-Rouzeyrol, Myrielle

    2017-08-09

    Dengue virus (DENV) is the arbovirus with the highest incidence in New Caledonia and in the South Pacific region. In 2012-2014, a major DENV-1 outbreak occurred in New Caledonia. The only known vector of DENV in New Caledonia is Aedes aegypti but no study has yet evaluated the competence of New Caledonia Ae. aegypti populations to transmit DENV. This study compared the ability of field-collected Ae. aegypti from different locations in New Caledonia to transmit the DENV-1 responsible for the 2012-2014 outbreak. This study also aimed to compare the New Caledonia results with the vector competence of Ae. aegypti from French Polynesia as these two French countries have close links, including arbovirus circulation. Three wild Ae. aegypti populations were collected in New Caledonia and one in French Polynesia. Female mosquitoes were orally exposed to DENV-1 (10(6) FFU/ml). Mosquito bodies (thorax and abdomen), heads and saliva were analyzed to measure infection, dissemination, transmission rates and transmission efficiency, at 7, 14 and 21 days post-infection (dpi), respectively. DENV-1 infection rates were heterogeneous, but dissemination rates were high and homogenous among the three Ae. aegypti populations from New Caledonia. Despite this high DENV-1 dissemination rate, the transmission rate, and therefore the transmission efficiency, observed were low. Aedes aegypti population from New Caledonia was less susceptible to infection and had lower ability to transmit DENV-1 than Ae. aegypti populations from French Polynesia. This study suggests that even if susceptible to infection, the New Caledonian Ae. aegypti populations were moderately competent vectors for DENV-1 strain from the 2012-2014 outbreak. These results strongly suggest that other factors might have contributed to the spread of this DENV-1 strain in New Caledonia and in the Pacific region.

  9. Investigation Into an Outbreak of Dengue-like Illness in Pernambuco, Brazil, Revealed a Cocirculation of Zika, Chikungunya, and Dengue Virus Type 1.

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    Pessôa, Rodrigo; Patriota, João Veras; Lourdes de Souza, Maria de; Felix, Alvina Clara; Mamede, Nubia; Sanabani, Sabri S

    2016-03-01

    In April 2015, an outbreak of dengue-like illness occurred in Tuparetama, a small city in the northeast region of Brazil; this outbreak was characterized by its fast expansion. An investigation was initiated to identify the viral etiologies and advise the health authorities on implementing control measures to contain the outbreak. This is the first report of this outbreak in the northeast, even though a few cases were documented earlier in a neighboring city.Plasma samples were obtained from 77 suspected dengue patients attending the main hospital in the city. Laboratory assays, such as real-time reverse transcription polymerase chain reaction, virus cDNA sequencing, and enzyme-linked immunosorbent assay, were employed to identify the infecting virus and molecular phylogenetic analysis was performed to define the circulating viral genotypes.RNA of Zika virus (ZIKV) and Dengue virus (DENV) or IgM antibodies (Abs) to DENV or chikungunya (CHIKV) were detected in 40 of the 77 plasma samples (51.9%). DENV was found in 9 patients (11.7%), ZIKV was found in 31 patients (40.2%), CHIKV in 1 patient (1.3%), and coinfection of DENV and ZIKV was detected in 2 patients (2.6%). The phylogenetic analysis of 2 available partial DENV and 14 ZIKV sequences revealed the identities of genotype 1 and the Asiatic lineage, respectively.Consistent with recent reports from the same region, our results showed that the ongoing outbreak is caused by ZIKV, DENV, and CHIKV. This emphasizes the need for a routine and differential diagnosis of arboviruses in patients with dengue-like illness. Coordinated efforts are necessary to contain the outbreak. Continued surveillance will be important to assess the effectiveness of current and future prevention strategies.

  10. Correlation between dengue-specific neutralizing antibodies and serum avidity in primary and secondary dengue virus 3 natural infections in humans.

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    Andreas Puschnik

    Full Text Available Although heterotypic secondary infection with dengue virus (DENV is associated with severe disease, the majority of secondary infections are mild or asymptomatic. The mechanisms of antibody-mediated protection are poorly understood. In 2010, 108 DENV3-positive cases were enrolled in a pediatric hospital-based study in Managua, Nicaragua, with 61 primary and 47 secondary infections. We analyzed DENV-specific neutralization titers (NT50, IgM and IgG avidity, and antibody titer in serum samples collected during acute and convalescent phases and 3, 6, and 18 months post-infection. NT50 titers peaked at convalescence and decreased thereafter. IgG avidity to DENV3 significantly increased between convalescent and 3-month time-points in primary DENV infections and between the acute and convalescent phase in secondary DENV infections. While avidity to DENV2, a likely previous infecting serotype, was initially higher than avidity to DENV3 in secondary DENV infections, the opposite relation was observed 3-18 months post-infection. We found significant correlations between IgM avidity and NT50 in acute primary cases and between IgG avidity and NT50 in secondary DENV infections. In summary, our findings indicate that IgM antibodies likely play a role in early control of DENV infections. IgG serum avidity to DENV, analyzed for the first time in longitudinal samples, switches from targeting mainly cross-reactive serotype(s to the current infecting serotype over time. Finally, serum avidity correlates with neutralization capacity.

  11. Thrombocytopenia in Dengue: Interrelationship between Virus and the Imbalance between Coagulation and Fibrinolysis and Inflammatory Mediators

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    Elzinandes Leal de Azeredo

    2015-01-01

    Full Text Available Dengue is an infectious disease caused by dengue virus (DENV. In general, dengue is a self-limiting acute febrile illness followed by a phase of critical defervescence, in which patients may improve or progress to a severe form. Severe illness is characterized by hemodynamic disturbances, increased vascular permeability, hypovolemia, hypotension, and shock. Thrombocytopenia and platelet dysfunction are common in both cases and are related to the clinical outcome. Different mechanisms have been hypothesized to explain DENV-associated thrombocytopenia, including the suppression of bone marrow and the peripheral destruction of platelets. Studies have shown DENV-infected hematopoietic progenitors or bone marrow stromal cells. Moreover, anti-platelet antibodies would be involved in peripheral platelet destruction as platelets interact with endothelial cells, immune cells, and/or DENV. It is not yet clear whether platelets play a role in the viral spread. Here, we focus on the mechanisms of thrombocytopenia and platelet dysfunction in DENV infection. Because platelets participate in the inflammatory and immune response by promoting cytokine, chemokine, and inflammatory mediator secretion, their relevance as “immune-like effector cells” will be discussed. Finally, an implication for platelets in plasma leakage will be also regarded, as thrombocytopenia is associated with clinical outcome and higher mortality.

  12. Thrombocytopenia in Dengue: Interrelationship between Virus and the Imbalance between Coagulation and Fibrinolysis and Inflammatory Mediators.

    Science.gov (United States)

    de Azeredo, Elzinandes Leal; Monteiro, Robson Q; de-Oliveira Pinto, Luzia Maria

    2015-01-01

    Dengue is an infectious disease caused by dengue virus (DENV). In general, dengue is a self-limiting acute febrile illness followed by a phase of critical defervescence, in which patients may improve or progress to a severe form. Severe illness is characterized by hemodynamic disturbances, increased vascular permeability, hypovolemia, hypotension, and shock. Thrombocytopenia and platelet dysfunction are common in both cases and are related to the clinical outcome. Different mechanisms have been hypothesized to explain DENV-associated thrombocytopenia, including the suppression of bone marrow and the peripheral destruction of platelets. Studies have shown DENV-infected hematopoietic progenitors or bone marrow stromal cells. Moreover, anti-platelet antibodies would be involved in peripheral platelet destruction as platelets interact with endothelial cells, immune cells, and/or DENV. It is not yet clear whether platelets play a role in the viral spread. Here, we focus on the mechanisms of thrombocytopenia and platelet dysfunction in DENV infection. Because platelets participate in the inflammatory and immune response by promoting cytokine, chemokine, and inflammatory mediator secretion, their relevance as "immune-like effector cells" will be discussed. Finally, an implication for platelets in plasma leakage will be also regarded, as thrombocytopenia is associated with clinical outcome and higher mortality.

  13. Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes.

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    Ruchi Sood

    2015-12-01

    Full Text Available Dengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need.Using the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of Cissampelos pariera Linn (Cipa extract was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that Cipa extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of Cipa extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week.Our findings above, taken in the context of the human safety of Cipa, based on its use in Indian traditional medicine, warrant further work to explore Cipa as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.

  14. A Sensitive and Selective Label-Free Electrochemical DNA Biosensor for the Detection of Specific Dengue Virus Serotype 3 Sequences

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    Natália Oliveira

    2015-07-01

    Full Text Available Dengue fever is the most prevalent vector-borne disease in the world, with nearly 100 million people infected every year. Early diagnosis and identification of the pathogen are crucial steps for the treatment and for prevention of the disease, mainly in areas where the co-circulation of different serotypes is common, increasing the outcome of dengue hemorrhagic fever (DHF and dengue shock syndrome (DSS. Due to the lack of fast and inexpensive methods available for the identification of dengue serotypes, herein we report the development of an electrochemical DNA biosensor for the detection of sequences of dengue virus serotype 3 (DENV-3. DENV-3 probe was designed using bioinformatics software and differential pulse voltammetry (DPV was used for electrochemical analysis. The results showed that a 22-m sequence was the best DNA probe for the identification of DENV-3. The optimum concentration of the DNA probe immobilized onto the electrode surface is 500 nM and a low detection limit of the system (3.09 nM. Moreover, this system allows selective detection of DENV-3 sequences in buffer and human serum solutions. Therefore, the application of DNA biosensors for diagnostics at the molecular level may contribute to future advances in the implementation of specific, effective and rapid detection methods for the diagnosis dengue viruses.

  15. A Sensitive and Selective Label-Free Electrochemical DNA Biosensor for the Detection of Specific Dengue Virus Serotype 3 Sequences.

    Science.gov (United States)

    Oliveira, Natália; Souza, Elaine; Ferreira, Danielly; Zanforlin, Deborah; Bezerra, Wessulla; Borba, Maria Amélia; Arruda, Mariana; Lopes, Kennya; Nascimento, Gustavo; Martins, Danyelly; Cordeiro, Marli; Lima-Filho, José

    2015-07-01

    Dengue fever is the most prevalent vector-borne disease in the world, with nearly 100 million people infected every year. Early diagnosis and identification of the pathogen are crucial steps for the treatment and for prevention of the disease, mainly in areas where the co-circulation of different serotypes is common, increasing the outcome of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Due to the lack of fast and inexpensive methods available for the identification of dengue serotypes, herein we report the development of an electrochemical DNA biosensor for the detection of sequences of dengue virus serotype 3 (DENV-3). DENV-3 probe was designed using bioinformatics software and differential pulse voltammetry (DPV) was used for electrochemical analysis. The results showed that a 22-m sequence was the best DNA probe for the identification of DENV-3. The optimum concentration of the DNA probe immobilized onto the electrode surface is 500 nM and a low detection limit of the system (3.09 nM). Moreover, this system allows selective detection of DENV-3 sequences in buffer and human serum solutions. Therefore, the application of DNA biosensors for diagnostics at the molecular level may contribute to future advances in the implementation of specific, effective and rapid detection methods for the diagnosis dengue viruses.

  16. Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses.

    Science.gov (United States)

    Cecilia, D; Kakade, M; Alagarasu, K; Patil, J; Salunke, A; Parashar, D; Shah, P S

    2015-01-01

    Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.

  17. CD8+ T cells prevent antigen-induced antibody-dependent enhancement of dengue disease in mice

    OpenAIRE

    Zellweger, Raphaël M.; Eddy, William E.; Tang, William W.; Miller, Robyn; Shresta, Sujan

    2014-01-01

    Dengue virus (DENV) causes pathologies ranging from the febrile illness dengue fever to the potentially lethal severe dengue disease. A major risk factor for developing severe dengue disease is the presence of sub-protective DENV-reactive antibodies from a previous infection (or from an immune mother), which can induce antibody-dependent enhancement of infection (ADE). However, infection in the presence of sub-protective anti-DENV antibodies does not always result in severe disease, suggestin...

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

    Science.gov (United States)

    Castrillón-Betancur, Juan Camilo; Urcuqui-Inchima, Silvio

    2017-01-01

    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. PMID:28327787

  19. CD4(+) and CD8(+) T-cell immunity to Dengue - lessons for the study of Zika virus.

    Science.gov (United States)

    Rivino, Laura; Lim, Mei Qiu

    2017-02-01

    Dengue virus (DENV) and Zika virus (ZIKV) are rapidly emerging mosquito-borne flaviviruses that represent a public health concern. Understanding host protective immunity to these viruses is critical for the design of optimal vaccines. Over a decade of research has highlighted a significant contribution of the T-cell response to both protection and/or disease enhancement during DENV infection, the latter being mainly associated with sub-optimal cross-reactive T-cell responses during secondary infections. Phase IIb/III clinical trials of the first licensed tetravalent dengue vaccine highlight increased vaccine efficacy in dengue-immune as opposed to dengue-naive vaccinees, suggesting a possible immunoprotective role of pre-existing DENV-specific T cells that are boosted upon vaccination. No vaccine is available for ZIKV and little is known about the T-cell response to this virus. ZIKV and DENV are closely related viruses with a sequence identity ranging from 44% and 56% for the structural proteins capsid and envelope to 68% for the more conserved non-structural proteins NS3/NS5, which represent the main targets of the CD4(+) and CD8(+) T-cell response to DENV, respectively. In this review we discuss our current knowledge of T-cell immunity to DENV and what it can teach us for the study of ZIKV. The extent of T-cell cross-reactivity towards ZIKV of pre-existing DENV-specific memory T cells and its potential impact on protective immunity and/or immunopathology will also be discussed.

  20. Cytotoxic CD4 T Cells: Differentiation, Function, and Application to Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    Yuan Tian

    2016-12-01

    Full Text Available Dengue virus (DENV has spread through most tropical and subtropical areas of the world and represents a serious public health problem. The control of DENV infection has not yet been fully successful due to lack of effective therapeutics or vaccines. Nevertheless, a better understanding of the immune responses against DENV infection may reveal new strategies for eliciting and improving antiviral immunity. T cells provide protective immunity against various viral infections by generating effector cells that cooperate to eliminate antigens and memory cells that can survive for long periods with enhanced abilities to control recurring pathogens. Following activation, CD8 T cells can migrate to sites of infection and kill infected cells, whereas CD4 T cells contribute to the elimination of pathogens by trafficking to infected tissues and providing help to innate immune responses, B cells, as well as CD8 T cells. However, it is now evident that CD4 T cells can also perform cytotoxic functions and induce the apoptosis of target cells. Importantly, accumulating studies demonstrate that cytotoxic CD4 T cells develop following DENV infections and may play a crucial role in protecting the host from severe dengue disease. We review our current understanding of the differentiation and function of cytotoxic CD4 T cells, with a focus on DENV infection, and discuss the potential of harnessing these cells for the prevention and treatment of DENV infection and disease.

  1. Cytotoxic CD4 T Cells: Differentiation, Function, and Application to Dengue Virus Infection.

    Science.gov (United States)

    Tian, Yuan; Sette, Alessandro; Weiskopf, Daniela

    2016-01-01

    Dengue virus (DENV) has spread through most tropical and subtropical areas of the world and represents a serious public health problem. The control of DENV infection has not yet been fully successful due to lack of effective therapeutics or vaccines. Nevertheless, a better understanding of the immune responses against DENV infection may reveal new strategies for eliciting and improving antiviral immunity. T cells provide protective immunity against various viral infections by generating effector cells that cooperate to eliminate antigens and memory cells that can survive for long periods with enhanced abilities to control recurring pathogens. Following activation, CD8 T cells can migrate to sites of infection and kill infected cells, whereas CD4 T cells contribute to the elimination of pathogens by trafficking to infected tissues and providing help to innate immune responses, B cells, as well as CD8 T cells. However, it is now evident that CD4 T cells can also perform cytotoxic functions and induce the apoptosis of target cells. Importantly, accumulating studies demonstrate that cytotoxic CD4 T cells develop following DENV infections and may play a crucial role in protecting the host from severe dengue disease. We review our current understanding of the differentiation and function of cytotoxic CD4 T cells, with a focus on DENV infection, and discuss the potential of harnessing these cells for the prevention and treatment of DENV infection and disease.

  2. Downregulation of Aedes aegypti chromodomain helicase DNA binding protein 7/Kismet by Wolbachia and its effect on dengue virus replication

    Science.gov (United States)

    Asad, Sultan; Hall-Mendelin, Sonja; Asgari, Sassan

    2016-01-01

    Dengue virus (DENV) is a mosquito-transmitted virus imposing a significant burden on human health around the world. Since current control strategies are not sufficient, there is an urgent need to find alternative methods to control DENV transmission. It has been demonstrated that introduction of Wolbachia pipientis in Aedes aegypti mosquitoes can impede DENV transmission with the mechanism(s) not fully understood. Recently, a number of studies have found the involvement of chromodomain DNA binding helicases in case of Human Immunodeficiency virus (HIV) and Influenza A virus infection. In this study, we have identified three chromodomain helicase DNA binding protein (CHD) genes in Ae. aegypti and looked at their response in the case of Wolbachia and DENV infections. Foremost amongst them we have found that AeCHD7/Kismet is significantly downregulated in the presence of Wolbachia infection only in female mosquitoes. Furthermore, AeCHD7 levels showed significant increase during DENV infection, and AeCHD7 depletion led to severe reduction in the replication of DENV. Our data have identified AeCHD7 as a novel Ae. aegypti host factor that is important for DENV replication, and Wolbachia downregulates it, which may contribute towards the mechanism(s) of limiting DENV replication. PMID:27827425

  3. Larval Temperature-Food Effects on Adult Mosquito Infection and Vertical Transmission of Dengue-1 Virus.

    Science.gov (United States)

    Buckner, Eva A; Alto, Barry W; Lounibos, L Philip

    2016-01-01

    Temperature-food interactions in the larval environment can affect life history and population growth of container mosquitoes Aedes aegypti (L.) and Aedes albopictus Skuse, the primary vectors of chikungunya and dengue viruses. We used Ae. aegypti, Ae. albopictus, and dengue-1 virus (DENV-1) from Florida to investigate whether larval rearing temperature can alter the effects of larval food levels on Ae. aegypti and Ae. albopictus life history and DENV-1 infection and vertical transmission. Although we found no effect of larval treatments on survivorship to adulthood, DENV-1 titer, or DENV-1 vertical transmission, rates of vertical transmission up to 16-24% were observed in Ae. albopictus and Ae. aegypti, which may contribute to maintenance of this virus in nature. Larval treatments had no effect on number of progeny and DENV-1 infection in Ae. aegypti, but the interaction between temperature and food affected number of progeny and DENV-1 infection of the female Ae. albopictus parent. The cooler temperature (24°C) yielded the most progeny and this effect was accentuated by high food relative to the other conditions. Low and high food led to the highest (∼90%) and lowest (∼65%) parental infection at the cooler temperature, respectively, whereas intermediate infection rates (∼75-80%) were observed for all food conditions at the elevated temperature. These results suggest that temperature and food availability have minimal influence on rate of vertical transmission and a stronger influence on adults of Ae. albopictus than of Ae. aegypti, which could have consequences for dengue virus epidemiology. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  4. Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

    Energy Technology Data Exchange (ETDEWEB)

    Chotiwan, Nunya; Roehrig, John T. [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Schlesinger, Jacob J. [Department of Medicine, University of Rochester, Rochester, NY 14642 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H., E-mail: yxh0@cdc.gov [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2014-05-15

    Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.

  5. Transcriptomic Profiling of Diverse Aedes aegypti Strains Reveals Increased Basal-level Immune Activation in Dengue Virus-refractory Populations and Identifies Novel Virus-vector Molecular Interactions

    Science.gov (United States)

    Sim, Shuzhen; Jupatanakul, Natapong; Ramirez, José L.; Kang, Seokyoung; Romero-Vivas, Claudia M.; Mohammed, Hamish; Dimopoulos, George

    2013-01-01

    Genetic variation among Aedes aegypti populations can greatly influence their vector competence for human pathogens such as the dengue virus (DENV). While intra-species transcriptome differences remain relatively unstudied when compared to coding sequence polymorphisms, they also affect numerous aspects of mosquito biology. Comparative molecular profiling of mosquito strain transcriptomes can therefore provide valuable insight into the regulation of vector competence. We established a panel of A. aegypti strains with varying levels of susceptibility to DENV, comprising both laboratory-maintained strains and field-derived colonies collected from geographically distinct dengue-endemic regions spanning South America, the Caribbean, and Southeast Asia. A comparative genome-wide gene expression microarray-based analysis revealed higher basal levels of numerous immunity-related gene transcripts in DENV-refractory mosquito strains than in susceptible strains, and RNA interference assays further showed different degrees of immune pathway contribution to refractoriness in different strains. By correlating transcript abundance patterns with DENV susceptibility across our panel, we also identified new candidate modulators of DENV infection in the mosquito, and we provide functional evidence for two potential DENV host factors and one potential restriction factor. Our comparative transcriptome dataset thus not only provides valuable information about immune gene regulation and usage in natural refractoriness of mosquito populations to dengue virus but also allows us to identify new molecular interactions between the virus and its mosquito vector. PMID:23861987

  6. Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation.

    Science.gov (United States)

    Uchida, Leo; Urata, Shuzo; Ulanday, Gianne Eduard L; Takamatsu, Yuki; Yasuda, Jiro; Morita, Kouichi; Hayasaka, Daisuke

    2016-02-10

    Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent.

  7. Coinfection with influenza A(H1N1)pdm09 and dengue virus in fatal cases.

    Science.gov (United States)

    Perdigão, Anne Carolinne Bezerra; Ramalho, Izabel Letícia Cavalcante; Guedes, Maria Izabel Florindo; Braga, Deborah Nunes Melo; Cavalcanti, Luciano Pamplona Góes; Melo, Maria Elisabeth Lisboa de; Araújo, Rafael Montenegro de Carvalho; Lima, Elza Gadelha; Silva, Luciene Alexandre Bié da; Araújo, Lia de Carvalho; Araújo, Fernanda Montenegro de Carvalho

    2016-09-01

    We report on four patients with fatal influenza A(H1N1)pdm09 and dengue virus coinfections. Clinical, necropsy and histopathologic findings presented in all cases were characteristic of influenza-dengue coinfections, and all were laboratory-confirmed for both infections. The possibility of influenza and dengue coinfection should be considered in locations where these two viruses' epidemic periods coincide to avoid fatal outcomes. Dengue is a mosquito-borne viral infection caused by one of the four dengue viruses (DENV-1 to 4). Each of these viruses is capable of causing nonspecific febrile illnesses, classic dengue fever and dengue haemorrhagic fever (Gubler 1998). As a result, dengue is often difficult to diagnose clinically, especially because peak dengue season often coincides with that of other common febrile illnesses in tropical regions (Chacon et al. 2015). In April 2009, a new virus, influenza A/H1N1/pandemic (FluA/H1N1/09pdm), caused a severe outbreak in Mexico. The virus quickly spread throughout the world, and in June 2009, the World Health Organization declared a pandemic (WHO 2010). In Brazil, the first laboratory confirmed case of FluA/H1N1/09pdm was in July 2009 (Pires Neto et al. 2013). The state of Ceará, in Northeast Brazil, is a dengue endemic area. In this state, the virus influenza A(H1N1)pdm09 has circulated since 2009, and through the first half of 2012, 11 deaths caused by the virus were confirmed (Pires Neto et al. 2013). The influenza and dengue seasons in Ceará overlap, which led to diagnostic difficulties. We report four cases of laboratory-confirmed coinfection of deadly influenza A(H1N1)pdm09 with DENV, which occurred during the dengue and influenza season in 2012 and 2013 in Ceará.

  8. Epidemiological and molecular characteristics of emergent dengue virus in Yunnan Province near the China-Myanmar-Laos border, 2013-2015.

    Science.gov (United States)

    Hu, Ting-Song; Zhang, Hai-Lin; Feng, Yun; Fan, Jian-Hua; Tang, Tian; Liu, Yong-Hua; Zhang, Liu; Yin, Xiao-Xiong; Chen, Gang; Li, Hua-Chang; Zu, Jin; Li, Hong-Bin; Li, Yuan-Yuan; Yu, Jing; Zhang, Fu-Qiang; Fan, Quan-Shui

    2017-05-08

    Yunnan Province is located in southwestern China and neighbors the Southeast Asian countries, all of which are dengue-endemic areas. In 2000-2013, sporadic imported cases of dengue fever (DF) were reported almost annually in Yunnan Province. During 2013-2015, we confirmed that a large-scale indigenous DF outbreak emerged in cities of Yunnan Province near the China-Myanmar-Laos border. Epidemiological characteristics of DF in Yunnan Province during 2013-2015 were evaluated by retrospective analysis. A total of 232 dengue virus (DENV)-positive sera were randomly collected for sequence analysis of the capsid/premembrane region of DENV from patients with DF in Yunnan Province. The envelope gene of DENV isolates was also amplified and sequenced. Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model. Phylogenetically, all DENV-positive samples could be classified into DENV-1 genotype I and DENV-2 Asian I genotype during 2013-2015 and DENV-4 genotype I in 2015 from Ruili City; and DENV-3 genotype II in 2013 and DENV-2 Cosmopolitan genotype in 2015 from Xishuangbanna Prefecture. Our results indicated that imported DF from patients from Laos and Myanmar was the primary cause of the DF epidemic in Yunnan Province. Additionally, DENV strains of all four serotypes were identified in indigenous cases in Yunnan Province during the same time period, while the dengue epidemic pattern observed in southwestern Yunnan showed characteristics of a hypoendemic nature: circulation of DENV-1 and DENV-2 over consecutive years.

  9. Sialic acid expression in the mosquito Aedes aegypti and its possible role in dengue virus-vector interactions.

    Science.gov (United States)

    Cime-Castillo, Jorge; Delannoy, Philippe; Mendoza-Hernández, Guillermo; Monroy-Martínez, Verónica; Harduin-Lepers, Anne; Lanz-Mendoza, Humberto; Hernández-Hernández, Fidel de la Cruz; Zenteno, Edgar; Cabello-Gutiérrez, Carlos; Ruiz-Ordaz, Blanca H

    2015-01-01

    Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.

  10. Sialic Acid Expression in the Mosquito Aedes aegypti and Its Possible Role in Dengue Virus-Vector Interactions

    Science.gov (United States)

    Cime-Castillo, Jorge; Delannoy, Philippe; Mendoza-Hernández, Guillermo; Monroy-Martínez, Verónica; Lanz-Mendoza, Humberto; Hernández-Hernández, Fidel de la Cruz; Cabello-Gutiérrez, Carlos; Ruiz-Ordaz, Blanca H.

    2015-01-01

    Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission. PMID:25874215

  11. Sialic Acid Expression in the Mosquito Aedes aegypti and Its Possible Role in Dengue Virus-Vector Interactions

    Directory of Open Access Journals (Sweden)

    Jorge Cime-Castillo

    2015-01-01

    Full Text Available Dengue fever (DF is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence. Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.

  12. Outbreak of viral hemorrhagic fever caused by dengue virus type 3 in Al-Mukalla, Yemen

    Science.gov (United States)

    2013-01-01

    Background Investigations were conducted by the authors to explore an outbreak of viral hemorrhagic fever (VHF) reported in 2010 from Al-Mukalla city, the capital of Hadramout in Yemen. Methods From 15–17 June 2010, the outbreak investigation period, specimens were obtained within 7 days after onset of illness of 18 acutely ill patients hospitalized with VHF and 15 household asymptomatic contacts of 6 acute cases. Additionally, 189 stored sera taken from acutely ill patients with suspected VHF hospitalized in the preceding 12 months were obtained from the Ministry of Health of Yemen. Thus, a total of 222 human specimens were collected; 207 specimens from acute cases and 15 specimens from contacts. All samples were tested with RT-PCR for dengue (DENV), Alkhumra (ALKV), Rift Valley Fever (RVFV), Yellow Fever (YFV), and Chikungunya (CHIKV) viruses. Samples were also tested for DENV IgM, IgG, and NS1-antigen. Medical records of patients were reviewed and demographic, clinical, and laboratory data was collected. Results Of 207 patients tested, 181 (87.4%) patients were confirmed to have acute dengue with positive dengue NS1-antigen (97 patients, 46.9%) and/or IgM (163 patients, 78.7%). Of the 181 patients with confirmed dengue, 100 (55.2%) patients were IgG-positive. DENV RNA was detected in 2 (1%) patients with acute symptoms; both samples were molecularly typed as DENV type 3. No other VHF viruses were detected. For the 15 contacts tested, RT-PCR tests for the five viruses were negative, one contact was dengue IgM positive, and another one was dengue IgG positive. Of the 181 confirmed dengue patients, 120 (66.3%) patients were males and the median age was 24 years. The most common manifestations included fever (100%), headache (94.5%), backache (93.4%), malaise (88.4%), arthralgia (85.1%), myalgia (82.3%), bone pain (77.9%), and leukopenia (76.2%). Two (1.1%) patients died. Conclusions DENV-3 was confirmed to be the cause of an outbreak of VHF in Al

  13. Assessing the risk of dengue virus transmission in a non-endemic city surrounded by endemic and hyperendemic areas.

    Science.gov (United States)

    Kwan, Tsz Ho; Lee, Shui Shan; Chan, Denise Pui Chung; Cheung, Manton; Kam, Kai Man

    2017-02-01

    To assess the potential risk of dengue transmission in a non-endemic city using a spatial epidemiological approach. Past dengue exposure of the general population was examined by dengue virus (DENV) IgG testing of archived samples from voluntary blood donors. Vector intensities were determined by local ovitrap index (OI). Analyses were made in the context of population statistics at both the district and sub-district level. The overall prevalence of DENV IgG was low at 2.25%. Positive donors were more likely to be older, non-Chinese, and female. Neither the OI nor the location of residence was associated with DENV serology. The sub-district level OI was clustered, but no correlation could be confirmed with the location of residence of positive blood donors. The cumulative exposure of Hong Kong residents to dengue has so far been low. Coupled with the lack of a spatial relationship between exposed cases and vector intensities, a high risk of local transmission of DENV is not supported. The apparently higher exposure likelihood of females could be explained by past infection in workers from dengue endemic countries, while frequent travel could have exposed older adults to DENV. Continued surveillance, risk assessment, and intensive vector control remain essential to prevent the transformation of a non-endemic to an endemic city. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  14. Changing pattern of dengue virus serotypes circulating during 2008-2012 and reappearance of dengue serotype 3 may cause outbreak in Kolkata, India.

    Science.gov (United States)

    Saha, Kallol; Ghosh, Monika; Firdaus, Rushna; Biswas, Aritra; Seth, Bikash; Bhattacharya, Debojyoti; Mukherjee, Kheya; Sadhukhan, Provash Chandra

    2016-10-01

    Dengue virus infection is a major cause of morbidity within the endemic tropical and subtropical regions of the world. Dengue virus has four distinct serotypes with specific clinical manifestations. In this study, we observed the changing pattern of dengue serotypes, age-wise dengue infection and useful sero-detection methods needed in a dengue endemic region. We identified dengue serotypes during a period of 5 years among patients with dengue symptoms visiting one of the largest tertiary care infectious disease hospitals of eastern India in Kolkata. A total of 433 dengue RNA positive samples were isolated from 712 acute dengue suspected cases. Age wise distribution highlighted the susceptible age group being >21 years (24.02%) followed by 11-15 years (21.71%) and 5-10 years (21.02%) of the total infected population. Higher numbers of infected cases were found within females as they are involved in more indoor works. The period of study experienced two dengue outbreaks one in 2008 and another in 2012. For early dengue detection, NS1 was found to be more confirmatory than IgM ELISA regarding sensitivity and specificity. DENV-1, 2, and 4 serotypes were the common circulating strains from 2008 until 2010, after which DENV-3 serotype infections rise and led to a massive dengue outbreak in Kolkata with increased numbers of DHF and DSS cases in 2012. The finding within our study emphasizes the public health importance of such prospective surveillance programs with respect to the changing dengue viral etiology and serotypes. J. Med. Virol. 88:1697-1702, 2016. © 2016 Wiley Periodicals, Inc.

  15. DENGUE VACCINES.

    Science.gov (United States)

    Thisyakorn, Usa; Thisyakorn, Chule

    2015-01-01

    The uniqueness of the dengue viruses (DENVs) and the spectrum of disease resulting from infection have made dengue vaccine development difficult. Several vaccine candidates are currently being evaluated in clinical studies. The candidate currently at the most advanced clinical development stage, a live-attenuated tetravalent vaccine based on the chimeric yellow fever-dengue virus (CYD-TDV), has progressed to Phase 3 efficacy studies. Several other live-attenuated vaccines, as well as subunit, DNA, and purified inactivated vaccine candidates are at earlier stages of clinical development. Additional technological approaches, such as virus-vectored and Virus-Like Particles (VLP)-based vaccines are under evaluation in preclinical studies.

  16. Displacement of the predominant dengue virus from type 2 to type 1 with a subsequent genotype shift from IV to I in Surabaya, Indonesia 2008-2010.

    Directory of Open Access Journals (Sweden)

    Atsushi Yamanaka

    Full Text Available Indonesia has annually experienced approximately 100,000 reported cases of dengue fever (DF and dengue hemorrhagic fever (DHF in recent years. However, epidemiological surveys of dengue viruses (DENVs have been limited in this country. In Surabaya, the second largest city, a single report indicated that dengue virus type 2 (DENV2 was the predominant circulating virus in 2003-2005. We conducted three surveys in Surabaya during: (i April 2007, (ii June 2008 to April 2009, and (iii September 2009 to December 2010. A total of 231 isolates were obtained from dengue patients and examined by PCR typing. We found that the predominant DENV shifted from type 2 to type 1 between October and November 2008. Another survey using wild-caught mosquitoes in April 2009 confirmed that dengue type 1 virus (DENV1 was the predominant type in Surabaya. Phylogenetic analyses of the nucleotide sequences of the complete envelope gene of DENV1 indicated that all 22 selected isolates in the second survey belonged to genotype IV and all 17 selected isolates in the third survey belonged to genotype I, indicating a genotype shift between April and September 2009. Furthermore, in December 2010, isolates were grouped into a new clade of DENV1 genotype I, suggesting clade shift between September and December 2010. According to statistics reported by the Surabaya Health Office, the proportion of DHF cases among the total number of dengue cases increased about three times after the type shift in 2008. In addition, the subsequent genotype shift in 2009 was associated with the increased number of total dengue cases. This indicates the need for continuous surveillance of circulating viruses to predict the risk of DHF and DF.

  17. Coinfection with influenza A(H1N1pdm09 and dengue virus in fatal cases

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    Anne Carolinne Bezerra Perdigão

    2016-01-01

    Full Text Available Abstract We report on four patients with fatal influenza A(H1N1pdm09 and dengue virus coinfections. Clinical, necropsy and histopathologic findings presented in all cases were characteristic of influenza-dengue coinfections, and all were laboratory-confirmed for both infections. The possibility of influenza and dengue coinfection should be considered in locations where these two viruses’ epidemic periods coincide to avoid fatal outcomes. Dengue is a mosquito-borne viral infection caused by one of the four dengue viruses (DENV-1 to 4. Each of these viruses is capable of causing nonspecific febrile illnesses, classic dengue fever and dengue haemorrhagic fever (Gubler 1998. As a result, dengue is often difficult to diagnose clinically, especially because peak dengue season often coincides with that of other common febrile illnesses in tropical regions (Chacon et al. 2015. In April 2009, a new virus, influenza A/H1N1/pandemic (FluA/H1N1/09pdm, caused a severe outbreak in Mexico. The virus quickly spread throughout the world, and in June 2009, the World Health Organization declared a pandemic (WHO 2010. In Brazil, the first laboratory confirmed case of FluA/H1N1/09pdm was in July 2009 (Pires Neto et al. 2013. The state of Ceará, in Northeast Brazil, is a dengue endemic area. In this state, the virus influenza A(H1N1pdm09 has circulated since 2009, and through the first half of 2012, 11 deaths caused by the virus were confirmed (Pires Neto et al. 2013. The influenza and dengue seasons in Ceará overlap, which led to diagnostic difficulties. We report four cases of laboratory-confirmed coinfection of deadly influenza A(H1N1pdm09 with DENV, which occurred during the dengue and influenza season in 2012 and 2013 in Ceará.

  18. Coinfection with influenza A(H1N1)pdm09 and dengue virus in fatal cases

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    Perdigão, Anne Carolinne Bezerra; Ramalho, Izabel Letícia Cavalcante; Guedes, Maria Izabel Florindo; Braga, Deborah Nunes Melo; Cavalcanti, Luciano Pamplona Góes; de Melo, Maria Elisabeth Lisboa; Araújo, Rafael Montenegro de Carvalho; Lima, Elza Gadelha; da Silva, Luciene Alexandre Bié; Araújo, Lia de Carvalho; Araújo, Fernanda Montenegro de Carvalho

    2016-01-01

    Abstract We report on four patients with fatal influenza A(H1N1)pdm09 and dengue virus coinfections. Clinical, necropsy and histopathologic findings presented in all cases were characteristic of influenza-dengue coinfections, and all were laboratory-confirmed for both infections. The possibility of influenza and dengue coinfection should be considered in locations where these two viruses’ epidemic periods coincide to avoid fatal outcomes. Dengue is a mosquito-borne viral infection caused by one of the four dengue viruses (DENV-1 to 4). Each of these viruses is capable of causing nonspecific febrile illnesses, classic dengue fever and dengue haemorrhagic fever (Gubler 1998). As a result, dengue is often difficult to diagnose clinically, especially because peak dengue season often coincides with that of other common febrile illnesses in tropical regions (Chacon et al. 2015). In April 2009, a new virus, influenza A/H1N1/pandemic (FluA/H1N1/09pdm), caused a severe outbreak in Mexico. The virus quickly spread throughout the world, and in June 2009, the World Health Organization declared a pandemic (WHO 2010). In Brazil, the first laboratory confirmed case of FluA/H1N1/09pdm was in July 2009 (Pires Neto et al. 2013). The state of Ceará, in Northeast Brazil, is a dengue endemic area. In this state, the virus influenza A(H1N1)pdm09 has circulated since 2009, and through the first half of 2012, 11 deaths caused by the virus were confirmed (Pires Neto et al. 2013). The influenza and dengue seasons in Ceará overlap, which led to diagnostic difficulties. We report four cases of laboratory-confirmed coinfection of deadly influenza A(H1N1)pdm09 with DENV, which occurred during the dengue and influenza season in 2012 and 2013 in Ceará. PMID:27598244

  19. Differential targeting of viral components by CD4+ versus CD8+ T lymphocytes in dengue virus infection.

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    Rivino, Laura; Kumaran, Emmanuelle A P; Jovanovic, Vojislav; Nadua, Karen; Teo, En Wei; Pang, Shyue Wei; Teo, Guo Hui; Gan, Victor Chih Hao; Lye, David C; Leo, Yee Sin; Hanson, Brendon J; Smith, Kenneth G C; Bertoletti, Antonio; Kemeny, David M; MacAry, Paul A

    2013-03-01

    Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4(+) and CD8(+) T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4(+) and 21 are CD8(+) T cell epitopes. We observe that whereas CD8(+) T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4(+) epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4(+) T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4(+) and CD8(+) T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.

  20. Development of real time PCR for detection and quantitation of Dengue Viruses

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    Singh A

    2009-01-01

    Full Text Available Abstract Background Dengue virus (DENV, a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease and virus isolation, which is laborious and time consuming. There is need for a rapid, sensitive and high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB probe approach was used to develop an improved real time RT-PCR (qRT-PCR for DENV in this study. Results The 3'UTR of thirteen Indian strains of DENV was sequenced and aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers and probes for the qRT-PCR. A single MGB probe and a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was10 copies of RNA molecules per reaction. The specificity and sensitivity of the assay was 100% when tested with a panel of 39 known positive and negative samples. Viral RNA could be detected and quantitated in infected mouse brain, cell cultures, mosquitoes and clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE, West Nile (WN, Chikungunya (CHK viruses or with Leptospira, Plasmodium vivax, Plasmodium falciparum and Rickettsia positive clinical samples. Conclusion We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The

  1. Seroprevalence of past dengue virus infection among children and adolescents in Singapore.

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    Ang, Li Wei; Cutter, Jeffery; James, Lyn; Goh, Kee Tai

    2015-12-01

    We conducted a pediatric seroprevalence study of dengue virus (DENV) infection in Singapore, a dengue endemic city-state. Residual sera from 1,200 Singapore residents aged 1-17 years seen in two hospitals between 2008 and 2010 were tested for anti-DENV IgG antibodies. The overall seroprevalence was 10.4% (95%CI: 8.7-12.1%). There had been a marked decline in seroprevalence in the 15-19-year age group over the last three decades, while the prevalence in the 1-5-year olds (12.6%) was significantly higher than that of the 1996-1997 pediatric survey (0.8%). The overall dengue seroprevalence in children and adolescents remained low.

  2. Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia.

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    Francesca D Frentiu

    2014-02-01

    Full Text Available INTRODUCTION: Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV, is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. METHODOLOGY/PRINCIPAL FINDINGS: Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

  3. Serum Metabolomics Investigation of Humanized Mouse Model of Dengue Virus Infection.

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    Cui, Liang; Hou, Jue; Fang, Jinling; Lee, Yie Hou; Costa, Vivian Vasconcelos; Wong, Lan Hiong; Chen, Qingfeng; Ooi, Eng Eong; Tannenbaum, Steven R; Chen, Jianzhu; Ong, Choon Nam

    2017-07-15

    Dengue is an acute febrile illness caused by dengue virus (DENV) and a major cause of morbidity and mortality in tropical and subtropical regions of the world. The lack of an appropriate small-animal model of dengue infection has greatly hindered the study of dengue pathogenesis and the development of therapeutics. In this study, we conducted mass spectrometry-based serum metabolic profiling from a model using humanized mice (humice) with DENV serotype 2 infection at 0, 3, 7, 14, and 28 days postinfection (dpi). Forty-eight differential metabolites were identified, including fatty acids, purines and pyrimidines, acylcarnitines, acylglycines, phospholipids, sphingolipids, amino acids and derivatives, free fatty acids, and bile acid. These metabolites showed a reversible-change trend-most were significantly perturbed at 3 or 7 dpi and returned to control levels at 14 or 28 dpi, indicating that the metabolites might serve as prognostic markers of the disease in humice. The major perturbed metabolic pathways included purine and pyrimidine metabolism, fatty acid β-oxidation, phospholipid catabolism, arachidonic acid and linoleic acid metabolism, sphingolipid metabolism, tryptophan metabolism, phenylalanine metabolism, lysine biosynthesis and degradation, and bile acid biosynthesis. Most of these disturbed pathways are similar to our previous metabolomics findings in a longitudinal cohort of adult human dengue patients across different infection stages. Our analyses revealed the commonalities of host responses to DENV infection between humice and humans and suggested that humice could be a useful small-animal model for the study of dengue pathogenesis and the development of dengue therapeutics.IMPORTANCE Dengue virus is the most widespread arbovirus, causing an estimated 390 million dengue infections worldwide every year. There is currently no effective treatment for the disease, and the lack of an appropriate small-animal model of dengue infection has greatly increased

  4. Phylogeography of recently emerged DENV-2 in southern Viet Nam.

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    Maia A Rabaa

    Full Text Available Revealing the dispersal of dengue viruses (DENV in time and space is central to understanding their epidemiology. However, the processes that shape DENV transmission patterns at the scale of local populations are not well understood, particularly the impact of such factors as human population movement and urbanization. Herein, we investigated trends in the spatial dynamics of DENV-2 transmission in the highly endemic setting of southern Viet Nam. Through a phylogeographic analysis of 168 full-length DENV-2 genome sequences obtained from hospitalized dengue cases from 10 provinces in southern Viet Nam, we reveal substantial genetic diversity in both urban and rural areas, with multiple lineages identified in individual provinces within a single season, and indicative of frequent viral migration among communities. Focusing on the recently introduced Asian I genotype, we observed particularly high rates of viral exchange between adjacent geographic areas, and between Ho Chi Minh City, the primary urban center of this region, and populations across southern Viet Nam. Within Ho Chi Minh City, patterns of DENV movement appear consistent with a gravity model of virus dispersal, with viruses traveling across a gradient of population density. Overall, our analysis suggests that Ho Chi Minh City may act as a source population for the dispersal of DENV across southern Viet Nam, and provides further evidence that urban areas of Southeast Asia play a primary role in DENV transmission. However, these data also indicate that more rural areas are also capable of maintaining virus populations and hence fueling DENV evolution over multiple seasons.

  5. Pichia pastoris-expressed Dengue 3 Envelope-based Virus-like Particles Elicit Predominantly Domain III-Focused High Titer Neutralizing Antibodies

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    Lav eTripathi

    2015-09-01

    Full Text Available Dengue poses a serious public health risk to nearly half the global population. It causes ~400 million infections annually and is considered to be one of the fastest spreading vector-borne diseases. Four distinct serotypes of dengue viruses (DENV-1, -2, -3 and -4 cause dengue disease, which may be either mild or extremely severe. Antibody-dependent enhancement (ADE, by pre-existing cross-reactive antibodies, is considered to be the major mechanism underlying severe disease. This mandates that a preventive vaccine must confer simultaneous and durable immunity to each of the four prevalent DENV serotypes. Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs, in the absence of prM, implicated in the elicitation of ADE-mediating antibodies. These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model. The current work is an extension of this approach to develop prM-lacking DENV-3 E VLPs. Our data reveal that P. pastoris-produced DENV-3 E VLPs not only preserve the antigenic integrity of the major neutralizing epitopes, but also elicit potent DENV-3 virus-neutralizing antibodies. Further, these neutralizing antibodies appear to be exclusively directed towards domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential towards heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way towards developing a DENV E-based, inexpensive, safe and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries.

  6. Quantifying the spatial dimension of dengue virus epidemic spread within a tropical urban environment.

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    Gonzalo M Vazquez-Prokopec

    Full Text Available BACKGROUND: Dengue infection spread in naive populations occurs in an explosive and widespread fashion primarily due to the absence of population herd immunity, the population dynamics and dispersal of Ae. aegypti, and the movement of individuals within the urban space. Knowledge on the relative contribution of such factors to the spatial dimension of dengue virus spread has been limited. In the present study we analyzed the spatio-temporal pattern of a large dengue virus-2 (DENV-2 outbreak that affected the Australian city of Cairns (north Queensland in 2003, quantified the relationship between dengue transmission and distance to the epidemic's index case (IC, evaluated the effects of indoor residual spraying (IRS on the odds of dengue infection, and generated recommendations for city-wide dengue surveillance and control. METHODS AND FINDINGS: We retrospectively analyzed data from 383 DENV-2 confirmed cases and 1,163 IRS applications performed during the 25-week epidemic period. Spatial (local k-function, angular wavelets and space-time (Knox test analyses quantified the intensity and directionality of clustering of dengue cases, whereas a semi-parametric Bayesian space-time regression assessed the impact of IRS and spatial autocorrelation in the odds of weekly dengue infection. About 63% of the cases clustered up to 800 m around the IC's house. Most cases were distributed in the NW-SE axis as a consequence of the spatial arrangement of blocks within the city and, possibly, the prevailing winds. Space-time analysis showed that DENV-2 infection spread rapidly, generating 18 clusters (comprising 65% of all cases, and that these clusters varied in extent as a function of their distance to the IC's residence. IRS applications had a significant protective effect in the further occurrence of dengue cases, but only when they reached coverage of 60% or more of the neighboring premises of a house. CONCLUSION: By applying sound statistical analysis to a

  7. Fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health

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    Vasilakis, Nikos; Cardosa, Jane; Hanley, Kathryn A.; Holmes, Edward C.; Weaver, Scott C.

    2012-01-01

    The four dengue virus (DENV) serotypes that circulate among humans emerged independently from ancestral sylvatic progenitors that were present in non–human primates, following the establishment of human populations that were large and dense enough to support continuous inter-human transmission by mosquitoes. This ancestral sylvatic–DENV transmission cycle still exists and is maintained in non-human primates and Aedes mosquitoes in the forests of Southeast Asia and West Africa. Here, we provide an overview of the ecology and molecular evolution of sylvatic DENV and its potential for adaptation to human transmission. We also emphasize how the study of sylvatic DENV will improve our ability to understand, predict and, ideally, avert further DENV emergence. PMID:21666708

  8. Protection against dengue virus infection in mice by administration of antibodies against modified nonstructural protein 1.

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    Shu-Wen Wan

    Full Text Available BACKGROUND: Infection with dengue virus (DENV may cause life-threatening disease with thrombocytopenia and vascular leakage which are related to dysfunction of platelets and endothelial cells. We previously showed that antibodies (Abs against DENV nonstructural protein 1 (NS1 cross-react with human platelets and endothelial cells, leading to functional disturbances. Based on sequence homology analysis, the C-terminal region of DENV NS1 protein contains cross-reactive epitopes. For safety in vaccine development, the cross-reactive epitopes of DENV NS1 protein should be deleted or modified. METHODOLOGY/PRINCIPAL FINDINGS: We tested the protective effects of Abs against full-length DENV NS1, NS1 lacking the C-terminal amino acids (a.a. 271-352 (designated ΔC NS1, and chimeric DJ NS1 consisting of N-terminal DENV NS1 (a.a. 1-270 and C-terminal Japanese encephalitis virus NS1 (a.a. 271-352. The anti-ΔC NS1 and anti-DJ NS1 Abs showed a lower binding activity to endothelial cells and platelets than that of anti-DENV NS1 Abs. Passive immunization with anti-ΔC NS1 and anti-DJ NS1 Abs reduced DENV-induced prolonged mouse tail bleeding time. Treatment with anti-DENV NS1, anti-ΔC NS1 and anti-DJ NS1 Abs reduced local skin hemorrhage, controlled the viral load of DENV infection in vivo, synergized with complement to inhibit viral replication in vitro, as well as abolished DENV-induced macrophage infiltration to the site of skin inoculation. Moreover, active immunization with modified NS1 protein, but not with unmodified DENV NS1 protein, reduced DENV-induced prolonged bleeding time, local skin hemorrhage, and viral load. CONCLUSIONS/SIGNIFICANCE: These results support the idea that modified NS1 proteins may represent an improved strategy for safe and effective vaccine development against DENV infection.

  9. Manipulation of immunodominant dengue virus E protein epitopes reduces potential antibody-dependent enhancement

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    Hughes Holly R

    2012-06-01

    Full Text Available Abstract Background Dengue viruses (DENV are the most important arboviruses of humans and cause significant disease. Infection with DENV elicits antibody responses to the envelope glycoprotein, predominantly against immunodominant, cross-reactive, weakly-neutralizing epitopes. These weakly-neutralizing antibodies are implicated in enhancing infection via Fcγ receptor bearing cells and can lead to increased viral loads that are associated with severe disease. Here we describe results from the development and testing of cross-reactivity reduced DENV-2 DNA vaccine candidates that contain substitutions in immunodominant B cell epitopes of the fusion peptide and domain III of the envelope protein. Results Cross-reactivity reduced and wild-type vaccine candidates were similarly immunogenic in outbred mice and elicited high levels of neutralizing antibody, however mice immunized with cross-reactivity reduced vaccines produced significantly reduced levels of immunodominant cross-reactive antibodies. Sera from mice immunized with wild-type, fusion peptide-, or domain III- substitution containing vaccines enhanced heterologous DENV infection in vitro, unlike sera from mice immunized with a vaccine containing a combination of both fusion peptide and domain III substitutions. Passive transfer of immune sera from mice immunized with fusion peptide and domain III substitutions also reduced the development of severe DENV disease in AG129 mice when compared to mice receiving wild type immune sera. Conclusions Reducing cross-reactivity in the envelope glycoprotein of DENV may be an approach to improve the quality of the anti-DENV immune response.

  10. Development and characterization of mouse monoclonal antibodies against monomeric dengue virus non-structural glycoprotein 1 (NS1).

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    Gelanew, Tesfaye; Poole-Smith, B Katherine; Hunsperger, Elizabeth

    2015-09-15

    Dengue virus (DENV) nonstructural-1 (NS1) glycoprotein is useful for diagnosis of DENV infections in the first 8 days of illness with any of the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). However, NS1 diagnostics are less sensitive for secondary DENV infections so the utility of NS1 diagnostics in dengue endemic countries where there is predominantly secondary infections is being questioned. Heat-mediated immunecomplex dissociation (ICD) prior to testing serum samples can significantly improve NS1 test sensitivity in secondary infections but requires monoclonal antibodies (MAbs) reactive to heat-denatured NS1. In order to incorporate a simple heat-mediated ICD step, a crucial step was to develop new MAbs with high affinity and specificity to heat-denatured DENV NS1 protein. In the present study, six new MAbs were isolated from BALB/c mice immunized with recombinant monomeric NS1 of DENV-1 and DENV-2. Characterization using three different methods: indirect ELISA, fixed cell ELISA and western blot revealed that all six MAbs are serotype-cross-reactive and capable of recognizing dimeric and hexameric isoforms as well as heat-denatured NS1 from all four DENV serotypes. No cross-reactivity to NS1 of West Nile virus and Yellow fever virus was observed on western blot and indirect ELISA. Five of the six MAbs mapped to the DENV NS1 region of 105-119 amino acids. The remaining MAb mapped to DENV NS1 region of 25-39 amino acids. These two NS1 regions were found to be highly conserved among all four DENV serotypes by sequences analysis and database comparison. These MAbs were used to develop an NS1 capture ELISA and tested using a small panel of clinical specimens. The results from the NS1 capture ELISA indicated at least a three-fold increase in NS1 antigen detection in heat-denatured samples compared to untreated specimens. Furthermore, artificial immunecomplexed results also demonstrated the binding efficiency of these MAbs to heat denatured NS1. Taken together

  11. Infection of bone marrow cells by dengue virus in vivo.

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    Noisakran, Sansanee; Onlamoon, Nattawat; Hsiao, Hui-Mien; Clark, Kristina B; Villinger, Francois; Ansari, Aftab A; Perng, Guey Chuen

    2012-03-01

    Abnormal bone marrow (BM) suppression is one of the hallmarks of dengue virus (DENV) infection in patients. Although the etiology remains unclear, direct viral targeting of the BM has been reasoned to be a contributing factor. The present studies were carried out in an effort to determine the potential effect of DENV infection on the cellularity of BM using a previously established nonhuman primate model of DENV-induced coagulopathy. BM aspirates were collected at various times from the infected nonhuman primate and cells were phenotypically defined and isolated using standard flow cytometry (fluorescence-activated cell sorting). These isolated cells were subjected to detection of DENV utilizing quantitative real-time reverse transcription polymerase chain reaction, electron microscopy, and immunostaining techniques. DENV RNA was detectable by quantitative real-time reverse transcription polymerase chain reaction in BM specimens and the presence of DENV-like particles within platelet was confirmed by electron microscopy. Enumeration of BM cells revealed a transient surge in cellularity at day 1, followed by a gradual decline from days 2 to 10 post infection. Detailed phenotypic studies showed similar kinetics in the frequencies of CD41(+)CD61(+) cells, regardless of CD34 and CD45 expression. The CD61(+) cells were not only the predominant cells that stained for DENV antigen but fluorescence-activated cell sorting-assisted isolation of CD61(+) cells from the BM were shown to contain infectious DENV by coculture with Vero cells. These data support the view that intravenous infection of nonhuman primate with DENV leads to direct infection of the BM, which is likely to be a contributing factor for transient cell suppression in the peripheral blood characteristic of acute DENV infection.

  12. Spatiotemporal characterizations of dengue virus in mainland China: insights into the whole genome from 1978 to 2011.

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    Zhang, Hao; Zhang, Yanru; Hamoudi, Rifat; Yan, Guiyun; Chen, Xiaoguang; Zhou, Yuanping

    2014-01-01

    Temporal-Spatial of dengue virus (DENV) analyses have been performed in previous epidemiological studies in mainland China, but few studies have examined the whole genome of the DENV. Herein, 40 whole genome sequences of DENVs isolated from mainland China were downloaded from GenBank. Phylogenetic analyses and evolutionary distances of the dengue serotypes 1 and 2 were calculated using 14 maximum likelihood trees created from individual genes and whole genome. Amino acid variations were also analyzed in the 40 sequences that included dengue serotypes 1, 2, 3 and 4, and they were grouped according to temporal and spatial differences. The results showed that none of the phylogenetic trees created from each individual gene were similar to the trees created using the complete genome and the evolutionary distances were variable with each individual gene. The number of amino acid variations was significantly different (p = 0.015) between DENV-1 and DENV-2 after 2001; seven mutations, the N290D, L402F and A473T mutations in the E gene region and the R101K, G105R, D340E and L349M mutations in the NS1 region of DENV-1, had significant substitutions, compared to the amino acids of DENV-2. Based on the spatial distribution using Guangzhou, including Foshan, as the indigenous area and the other regions as expanding areas, significant differences in the number of amino acid variations in the NS3 (p = 0.03) and NS1 (p = 0.024) regions and the NS2B (p = 0.016) and NS3 (p = 0.042) regions were found in DENV-1 and DENV-2. Recombination analysis showed no inter-serotype recombination events between the DENV-1 and DENV-2, while six and seven breakpoints were found in DENV-1 and DENV-2. Conclusively, the individual genes might not be suitable to analyze the evolution and selection pressure isolated in mainland China; the mutations in the amino acid residues in the E, NS1 and NS3 regions may play important roles in DENV-1 and DENV-2 epidemics.

  13. Spatiotemporal characterizations of dengue virus in mainland China: insights into the whole genome from 1978 to 2011.

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    Hao Zhang

    Full Text Available Temporal-Spatial of dengue virus (DENV analyses have been performed in previous epidemiological studies in mainland China, but few studies have examined the whole genome of the DENV. Herein, 40 whole genome sequences of DENVs isolated from mainland China were downloaded from GenBank. Phylogenetic analyses and evolutionary distances of the dengue serotypes 1 and 2 were calculated using 14 maximum likelihood trees created from individual genes and whole genome. Amino acid variations were also analyzed in the 40 sequences that included dengue serotypes 1, 2, 3 and 4, and they were grouped according to temporal and spatial differences. The results showed that none of the phylogenetic trees created from each individual gene were similar to the trees created using the complete genome and the evolutionary distances were variable with each individual gene. The number of amino acid variations was significantly different (p = 0.015 between DENV-1 and DENV-2 after 2001; seven mutations, the N290D, L402F and A473T mutations in the E gene region and the R101K, G105R, D340E and L349M mutations in the NS1 region of DENV-1, had significant substitutions, compared to the amino acids of DENV-2. Based on the spatial distribution using Guangzhou, including Foshan, as the indigenous area and the other regions as expanding areas, significant differences in the number of amino acid variations in the NS3 (p = 0.03 and NS1 (p = 0.024 regions and the NS2B (p = 0.016 and NS3 (p = 0.042 regions were found in DENV-1 and DENV-2. Recombination analysis showed no inter-serotype recombination events between the DENV-1 and DENV-2, while six and seven breakpoints were found in DENV-1 and DENV-2. Conclusively, the individual genes might not be suitable to analyze the evolution and selection pressure isolated in mainland China; the mutations in the amino acid residues in the E, NS1 and NS3 regions may play important roles in DENV-1 and DENV-2 epidemics.

  14. Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics.

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    Savidis, George; McDougall, William M; Meraner, Paul; Perreira, Jill M; Portmann, Jocelyn M; Trincucci, Gaia; John, Sinu P; Aker, Aaron M; Renzette, Nicholas; Robbins, Douglas R; Guo, Zhiru; Green, Sharone; Kowalik, Timothy F; Brass, Abraham L

    2016-06-28

    The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.

  15. Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics

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    George Savidis

    2016-06-01

    Full Text Available The flaviviruses dengue virus (DENV and Zika virus (ZIKV are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF, heparin sulfation (NDST1 and EXT1, and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC. We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.

  16. Infecciones concurrentes por dos serotipos del virus dengue durante un brote en el noroeste de Perú, 2008 Concurrent infections by two dengue virus serotypes during an outbreak in northwestern Peru, 2008

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    Enrique Mamani

    2010-03-01

    Full Text Available Objetivo. Describir la existencia de infecciones concurrentes por diferentes serotipos del virus dengue (DENV en un brote ocurrido en el noroeste de Perú durante el 2008. Materiales y métodos. Se analizó 73 muestras séricas de pacientes con dengue en un brote en el noroeste de Perú entre mayo y junio de 2008. Para la serotipificación del DENV se utilizó técnicas de biología molecular; así, primero se realizó la extracción del ARN con el kit QIAamp viral RNA Mini, luego se realizó la transcripción inversa y amplificación de los fragmentos de ADNc viral mediante las técnicas de reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR multiplex y de RT-Anidada PCR (RT-Nested PCR, y finalmente de realizó el secuenciamiento genético de los fragmentos de ADNc viral utilizando el kit Big Dye Terminator v.3,1. Resultados. Los 73 casos de dengue presentaron infecciones por diferentes serotipos: 34 (46,6% por DENV-3, 29 (39,7% por DENV-1, 4 (5,5% por DENV-4 y 6 casos (8,2% por DENV-1 y DENV-3. Las manifestaciones clínicas más frecuentes fueron fiebre y cefalea (100%, mialgia (94,5%, dolor ocular (83,6%, artralgia (78,1%, escalofríos (63,0%, nauseas/vómitos (38,4%, prueba de lazo positiva (30,1% y erupción cutánea (20,5%. Los pacientes con infecciones concurrentes presentaron cuadros leves, excepto una paciente que presentó prueba de lazo positivo y sangrado genital. Conclusión. Es el primer reporte de pacientes peruanos con infecciones concurrentes por dos serotipos del DENV sin formas graves de la enfermedad.Objetives. To establish the existence of concurrent infections by different dengue virus (DENV serotypes in an outbreak in the Northwestern in Peru during 2008. Material and methods. 73 serum samples from patients with dengue were analyzed during an outbreak that occurred in Northwestern in Peru between May and June 2008. Molecular biology techniques were used to serotype the DENV, thus, firstly the viral RNA

  17. Dengue-1 Virus Isolation during First Dengue Fever Outbreak on Easter Island, Chile

    OpenAIRE

    Perret, Cecilia; Abarca, Katia; Ovalle, Jimena; Ferrer, Pablo; Godoy, Paula; Olea, Andrea; Aguilera, Ximena; Ferrés, Marcela

    2003-01-01

    Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription–polymerase chain reaction as being dengue type 1.

  18. Dengue-1 virus isolation during first dengue fever outbreak on Easter Island, Chile.

    Science.gov (United States)

    Perret, Cecilia; Abarca, Katia; Ovalle, Jimena; Ferrer, Pablo; Godoy, Paula; Olea, Andrea; Aguilera, Ximena; Ferrés, Marcela

    2003-11-01

    Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription-polymerase chain reaction as being dengue type 1.

  19. Dengue and Zika viruses: lessons learned from the similarities between these Aedes mosquito-vectored arboviruses.

    Science.gov (United States)

    Suwanmanee, San; Luplertlop, Natthanej

    2017-02-01

    The currently spreading arbovirus epidemic is having a severe impact on human health worldwide. The two most common flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), are transmitted through the same viral vector, Aedes spp. mosquitoes. Since the discovery of DENV in 1943, this virus has been reported to cause around 390 million human infections per year, approximately 500,000 of which require hospitalization and over 20,000 of which are lethal. The present DENV epidemic is primarily concentrated in Southeast Asia. ZIKV, which was discovered in 1952, is another important arthropod-borne flavivirus. The neurotropic role of ZIKV has been reported in infected newborns with microcephaly and in adults with Guillain-Barre syndrome. Despite DENV and ZIKV sharing the same viral vector, their complex pathogenic natures are poorly understood, and the infections they cause do not have specific treatments or effective vaccines. Therefore, this review will describe what is currently known about the clinical characteristics, pathogenesis mechanisms, and transmission of these two viruses. Better understanding of the interrelationships between DENV and ZIKV will provide a useful perspective for developing an effective strategy for controlling both viruses in the future.

  20. Phylogeny of Dengue and Chikungunya viruses in Al Hudayda governorate, Yemen.

    Science.gov (United States)

    Ciccozzi, Massimo; Lo Presti, Alessandra; Cella, Eleonora; Giovanetti, Marta; Lai, Alessia; El-Sawaf, Gamal; Faggioni, Giovanni; Vescio, Fenicia; Al Ameri, Ranya; De Santis, Riccardo; Helaly, Ghada; Pomponi, Alice; Metwally, Dalia; Fantini, Massimo; Qadi, Hussein; Zehender, Gianguglielmo; Lista, Florigio; Rezza, Giovanni

    2014-10-01

    Yemen, which is located in the southwestern end of the Arabian Peninsula, is one of countries most affected by recurrent epidemics caused by emerging vector-borne viruses. Dengue virus (DENV) outbreaks have been reported with increasing frequency in several governorates since the year 2000, and the Chikungunya virus (CHIKV) has been also responsible of large outbreaks and it is now a major public health problem in Yemen. We report the results of the phylogenetic analysis of DENV-2 and CHIKV isolates (NS1 and E1 genes, respectively) detected in an outbreak occurred in Al-Hudayda in 2012. Estimates of the introduction date of CHIKV and DENV-2, and the phylogeographic analysis of DENV-2 are also presented. Phylogenetic analysis showed that the Yemen isolates of DENV belonged to the lineage 2 Cosmopolitan subtype, whereas CHIKV isolates from Yemen belonged to the ECSA genotype. All the CHIKV isolates from Yemen were statistically supported and dated back to the year 2010 (95% HPD: 2009-2011); these sequences showed an alanine in the aminoacid position 226 of the E1 protein. Phylogeographic analysis of DENV-2 virus showed that cluster 1, which included Yemen isolates, dated back to 2003 Burkina Faso strains (95% HPD 1999-2007). The Yemen, cluster dated back to 2011 (95% HPD 2009-2012). Our study sheds light on the global spatiotemporal dynamics of DENV-2 and CHIKV in Yemen. This study reinforces both the need to monitor the spread of CHIKV and DENV, and to apply significant measures for vector control.

  1. First evidence of dengue virus infection in wild caught mosquitoes during an outbreak in Assam, Northeast India

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    Prafulla Dutta

    2015-01-01

    Interpretation & conclusion: This is the maiden report of detection of DENV in wild caught Ae. aegypti mosquitoes from Northeastern Region of India. The study also demonstrates the presence of transovarial transmission of dengue virus in this part of country. This information is useful in respect of both entomological as well as epidemiological point of view for taking appropriate vector control measures.

  2. Salidroside exhibits anti-dengue virus activity by upregulating host innate immune factors.

    Science.gov (United States)

    Sharma, Navita; Mishra, K P; Ganju, Lilly

    2016-12-01

    Dengue is an arboviral disease with no effective therapy available. Therefore, there is an urgent need to find a potent antiviral agent against dengue virus (DENV). In the present study, salidroside, a main bioactive compound of Rhodiola rosea, was evaluated for its antiviral potential against DENV serotype-2 infection and its effect on host innate immune factors. Antiviral effects of salidroside were examined in DENV-infected cells by western blotting, flow cytometry and real-time PCR. Its underlying mechanism involved in antiviral action was determined by evaluating expression of host innate immune factors including RIG-I, IRF-3, IRF-7, PKR, P-eIF2α and NF-κB. Salidroside potently inhibited DENV infection by decreasing DENV envelope protein expression more than tenfold. Salidroside exerts its antiviral activity by increasing expression of RNA helicases such as RIG-I, thereby initiating a downstream signaling cascade that induces upregulation of IRF-3 and IRF-7. It prevents viral protein synthesis by increasing the expression of PKR and P-eIF2α while decreasing NF-κB expression. It was also found to induce the expression of IFN-α. In addition, the number of NK cells and CD8(+) T cells were also found to be increased by salidroside treatment in human PBMCs, which are important in limiting DENV replication during early stages of infection. The findings presented here suggest that salidroside exhibits antiviral activity against DENV by inhibiting viral protein synthesis and boosting host immunity by increasing the expression of host innate immune factors and hence could be considered for the development of an effective therapeutic agent against DENV infection.

  3. Memory CD8+ T cells from naturally acquired primary dengue virus infection are highly cross-reactive.

    Science.gov (United States)

    Friberg, Heather; Burns, Lynne; Woda, Marcia; Kalayanarooj, Siripen; Endy, Timothy P; Stephens, Henry A F; Green, Sharone; Rothman, Alan L; Mathew, Anuja

    2011-01-01

    Cross-reactive memory T cells induced by primary infection with one of the four serotypes of dengue virus (DENV) are hypothesized to have an immunopathological function in secondary heterologous DENV infection. To define the T-cell response to heterologous serotypes, we isolated HLA-A(*)1101-restricted epitope-specific CD8(+) T-cell lines from primary DENV-immune donors. Cell lines exhibited marked cross-reactivity toward peptide variants representing the four DENV serotypes in tetramer binding and functional assays. Many clones responded similarly to homologous and heterologous serotypes with striking cross-reactivity between the DENV-1 and DENV-3 epitope variants. In vitro-stimulated T-cell lines consistently revealed a hierarchical induction of MIP-1β>degranulation>tumor necrosis factor α (TNFα)>interferon-γ (IFNγ), which depended on the concentration of agonistic peptide. Phosphoflow assays showed peptide dose-dependent phosphorylation of ERK1/2, which correlated with cytolysis, degranulation, and induction of TNFα and IFNγ, but not MIP-1β production. This is the first study to show significant DENV serotype-cross-reactivity of CD8(+) T cells after naturally acquired primary infection. We also show qualitatively different T-cell receptor signaling after stimulation with homologous and heterologous peptides. Our data support a model whereby the order of sequential DENV infections influences the immune response to secondary heterologous DENV infection, contributing to varying disease outcomes.

  4. A heparin-functionalized carbon nanotube-based affinity biosensor for dengue virus.

    Science.gov (United States)

    Wasik, Daniel; Mulchandani, Ashok; Yates, Marylynn V

    2017-05-15

    Dengue virus is an arthropod-borne virus transmitted primarily by Aedes mosquitos and is major cause of disease in tropical and subtropical regions. Colloquially known as Dengue Fever, infection can cause hemorrhagic disorders and death in humans and non-human primates. We report a novel electronic biosensor based on a single-walled carbon nanotube network chemiresistive transducer that is functionalized with heparin for low-cost, label-free, ultra-sensitive, and rapid detection of whole dengue virus (DENV). Heparin, an analog of the heparan sulfate proteoglycans that are receptors for dengue virus during infection of Vero cells and hepatocytes, was used for the first time in a biosensor as a biorecognition element instead of traditional antibody. Detection of DENV in viral culture supernatant has similar sensitivity as the corresponding viral titer in phosphate buffer despite the presence of growth media and Vero cell lysate. The biosensor demonstrated sensitivity within the clinically relevant range for humans and infected Aedes aegypti. It has potential application in clinical diagnosis and can improve point-of-care diagnostics of dengue infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Lower activation-induced T-cell apoptosis is related to the pathological immune response in secondary infection with hetero-serotype dengue virus.

    Science.gov (United States)

    Yang, Wang; Yan, Huacheng; Ma, Yuling; Yu, Tiantian; Guo, Hongxia; Kuang, Yuchan; Ren, Ruiwen; Li, Jintao

    2016-03-01

    The available evidence suggests that dengue virus-specific T lymphocytes and cytokine storm play a pivotal role in the immunopathogenesis of plasma leakage. Investigations are underway to identify the immune profiles associated with increased or decreased risk for severe disease. In this study, CD14+ cells from the peripheral blood mononuclear cells (PBMCs) of patients who recovered from DENV-1 infection were infected with DENV-1 or DENV-2 and co-cultured with memory T cells. We found that secondary infection with DENV-2 suppresses the cell reproductive capacity but forms more cell clones and more functional cells to produce more proinflammatory factors (IFN-γ, TNF-α, IL-6, IL-8, IL-12 and IL-17) and less regulatory cytokines (IL-10, TGF-β) which results in higher viral replication compared to secondary infection with DENV-1. Memory dengue virus-specific T cells which are induced in a primary dengue virus infection are reactivated by the heterologous serotype of dengue virus and antigen-presenting cells (APCs) during a secondary infection. Dramatically, less apoptosis and more continuous activation of T cells in secondary infection with hetero-serotype DENV were observed. This discovery which has not been reported previously may be the reasonable and vital interpretation for the cytokine storm and severe symptoms observed in secondary infection with DENV. In summary, secondary infection with hetero-serotype DENV elicits the relatively pathological immune response while secondary infection with homologous-serotype DENV induces the relatively protective immune response by activation-induced cell death (AICD) of T cells.

  6. Modeling the impact on virus transmission of Wolbachia-mediated blocking of dengue virus infection of Aedes aegypti.

    Science.gov (United States)

    Ferguson, Neil M; Kien, Duong Thi Hue; Clapham, Hannah; Aguas, Ricardo; Trung, Vu Tuan; Chau, Tran Nguyen Bich; Popovici, Jean; Ryan, Peter A; O'Neill, Scott L; McGraw, Elizabeth A; Long, Vo Thi; Dui, Le Thi; Nguyen, Hoa L; Chau, Nguyen Van Vinh; Wills, Bridget; Simmons, Cameron P

    2015-03-18

    Dengue is the most common arboviral infection of humans and is a public health burden in more than 100 countries. Aedes aegypti mosquitoes stably infected with strains of the intracellular bacterium Wolbachia are resistant to dengue virus (DENV) infection and are being tested in field trials. To mimic field conditions, we experimentally assessed the vector competence of A. aegypti carrying the Wolbachia strains wMel and wMelPop after challenge with viremic blood from dengue patients. We found that wMelPop conferred strong resistance to DENV infection of mosquito abdomen tissue and largely prevented disseminated infection. wMel conferred less resistance to infection of mosquito abdomen tissue, but it did reduce the prevalence of mosquitoes with infectious saliva. A mathematical model of DENV transmission incorporating the dynamics of viral infection in humans and mosquitoes was fitted to the data collected. Model predictions suggested that wMel would reduce the basic reproduction number, R0, of DENV transmission by 66 to 75%. Our results suggest that establishment of wMelPop-infected A. aegypti at a high frequency in a dengue-endemic setting would result in the complete abatement of DENV transmission. Establishment of wMel-infected A. aegypti is also predicted to have a substantial effect on transmission that would be sufficient to eliminate dengue in low or moderate transmission settings but may be insufficient to achieve complete control in settings where R0 is high. These findings develop a framework for selecting Wolbachia strains for field releases and for calculating their likely impact.

  7. Phylogeny of dengue virus type 3 circulating in Colombia between 2001 and 2007.

    Science.gov (United States)

    Villabona-Arenas, Christian Julián; Miranda-Esquivel, Daniel Rafael; Jimenez, Raquel Elvira Ocazionez

    2009-10-01

    Dengue virus type 3 (DENV-3) re-appeared in Colombia in 2001 after 23 years of apparent absence, in the state of Santander in the North-eastern region near to Venezuelan border. In 2002, the virus was isolated in the state of Valle del Cauca in the South-east region near to Ecuadorian/Peruvian border, and in the state of Antioquia in the North-east region near to Panama border. To gain insight into the molecular epidemiology of DENV-3 in Colombia, we sequenced the complete E gene of 21 isolates sampled in the period 2001-2007. Phylogenetic analyses revealed that Colombian strains seem to have been introduced from Venezuela, Ecuador and Peru, but not from Brazil, Argentina, Paraguay or Central America countries. This study also confirms previous report showing that Colombian isolates is closely related to DENV-3 genotype III.

  8. An Epitope-Substituted DNA Vaccine Improves Safety and Immunogenicity against Dengue Virus Type 2.

    Directory of Open Access Journals (Sweden)

    Chung-Tao Tang

    Full Text Available Dengue virus (DENV, a global disease, is divided into four serotypes (DENV1-4. Cross-reactive and non-neutralizing antibodies against envelope (E protein of DENV bind to the Fcγ receptors (FcγR of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE. Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.

  9. Molecular characterisation of dengue virus type 1 reveals lineage replacement during circulation in Brazilian territory

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    Adriana Ribeiro Carneiro

    2012-09-01

    Full Text Available Dengue fever is the most important arbovirus infection found in tropical regions around the world. Dispersal of the vector and an increase in migratory flow between countries have led to large epidemics and severe clinical outcomes, such as dengue haemorrhagic fever and dengue shock syndrome. This study analysed the genetic variability of the dengue virus serotype 1 (DENV-1 in Brazil with regard to the full-length structural genes C/prM/M/E among 34 strains isolated during epidemics that occurred in the country between 1994-2011. Virus phylogeny and time of divergence were also evaluated with only the E gene of the strains isolated from 1994-2008. An analysis of amino acid differences between these strains and the French Guiana strain (FGA/89 revealed the presence of important nonsynonymous substitutions in the amino acid sequences, including residues E297 (Met→Thr and E338 (Ser→Leu. A phylogenetic analysis of E proteins comparing the studied isolates and other strains selected from the GenBank database showed that the Brazilian DENV-1 strains since 1982 belonged to genotype V. This analysis also showed that different introductions of strains from the 1990s represented lineage replacement, with the identification of three lineages that cluster all isolates from the Americas. An analysis of the divergence time of DENV-1 indicated that the lineage circulating in Brazil emerged from an ancestral lineage that originated approximately 44.35 years ago.

  10. Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability.

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    Henry Puerta-Guardo

    2016-07-01

    Full Text Available Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV non-structural protein 1 (NS1 in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate toll-like receptor 4 signaling in primary human myeloid cells, leading to secretion of pro-inflammatory cytokines and vascular leakage. However, distinct endothelial cell-intrinsic mechanisms of NS1-induced hyperpermeability remained to be defined. The endothelial glycocalyx layer (EGL is a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium that plays a key role in regulating endothelial barrier function. Here, we demonstrate that DENV NS1 disrupts the EGL on human pulmonary microvascular endothelial cells, inducing degradation of sialic acid and shedding of heparan sulfate proteoglycans. This effect is mediated by NS1-induced expression of sialidases and heparanase, respectively. NS1 also activates cathepsin L, a lysosomal cysteine proteinase, in endothelial cells, which activates heparanase via enzymatic cleavage. Specific inhibitors of sialidases, heparanase, and cathepsin L prevent DENV NS1-induced EGL disruption and endothelial hyperpermeability. All of these effects are specific to NS1 from DENV1-4 and are not induced by NS1 from West Nile virus, a related flavivirus. Together, our data suggest an important role for EGL disruption in DENV NS1-mediated endothelial dysfunction during severe dengue disease.

  11. Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability

    Science.gov (United States)

    Puerta-Guardo, Henry; Glasner, Dustin R.; Harris, Eva

    2016-01-01

    Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV) non-structural protein 1 (NS1) in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate toll-like receptor 4 signaling in primary human myeloid cells, leading to secretion of pro-inflammatory cytokines and vascular leakage. However, distinct endothelial cell-intrinsic mechanisms of NS1-induced hyperpermeability remained to be defined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium that plays a key role in regulating endothelial barrier function. Here, we demonstrate that DENV NS1 disrupts the EGL on human pulmonary microvascular endothelial cells, inducing degradation of sialic acid and shedding of heparan sulfate proteoglycans. This effect is mediated by NS1-induced expression of sialidases and heparanase, respectively. NS1 also activates cathepsin L, a lysosomal cysteine proteinase, in endothelial cells, which activates heparanase via enzymatic cleavage. Specific inhibitors of sialidases, heparanase, and cathepsin L prevent DENV NS1-induced EGL disruption and endothelial hyperpermeability. All of these effects are specific to NS1 from DENV1-4 and are not induced by NS1 from West Nile virus, a related flavivirus. Together, our data suggest an important role for EGL disruption in DENV NS1-mediated endothelial dysfunction during severe dengue disease. PMID:27416066

  12. The Effect of Temperature on Wolbachia-Mediated Dengue Virus Blocking in Aedes aegypti.

    Science.gov (United States)

    Ye, Yixin H; Carrasco, Alison M; Dong, Yi; Sgrò, Carla M; McGraw, Elizabeth A

    2016-04-01

    Dengue fever, caused by dengue virus (DENV), is endemic in more than 100 countries. The lack of effective treatment of patients and the suboptimal efficacies of the tetravalent vaccine in trials highlight the urgent need to develop alternative strategies to lessen the burden of dengue fever.Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits the replication of the DENV in the mosquito vector,Aedes aegypti However, several recent studies have demonstrated the sensitivity of pathogens, vectors, and their symbionts to temperature. To understand how the tripartite interactions between the mosquito, DENV, and Wolbachia may change under different temperature regimes, we assessed the vector competence and transmission potential of DENV-infected mosquitoes reared at a common laboratory setting of a constant 25°C and at two diurnal temperature settings with mean of 25°C and 28°C and a fluctuating range of 8°C (±4°C). Temperature significantly affected DENV infection rate in the mosquitoes. Furthermore, temperature significantly influenced the proportion of mosquitoes that achieved transmission potential as measured by the presence of virus in the saliva. Regardless of the temperature regimes,Wolbachia significantly and efficiently reduced the proportion of mosquitoes achieving infection and transmission potential across all the temperature regimes studied. This work reinforces the robustness of the Wolbachia biocontrol strategy to field conditions in Cairns, Australia, and suggests that similar studies are required for local mosquito genotypes and field relevant temperatures for emerging field release sites globally.

  13. Cells in Dengue Virus Infection In Vivo

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    Sansanee Noisakran

    2010-01-01

    Full Text Available Dengue has been recognized as one of the most important vector-borne emerging infectious diseases globally. Though dengue normally causes a self-limiting infection, some patients may develop a life-threatening illness, dengue hemorrhagic fever (DHF/dengue shock syndrome (DSS. The reason why DHF/DSS occurs in certain individuals is unclear. Studies in the endemic regions suggest that the preexisting antibodies are a risk factor for DHF/DSS. Viremia and thrombocytopenia are the key clinical features of dengue virus infection in patients. The amounts of virus circulating in patients are highly correlated with severe dengue disease, DHF/DSS. Also, the disturbance, mainly a transient depression, of hematological cells is a critical clinical finding in acute dengue patients. However, the cells responsible for the dengue viremia are unresolved in spite of the intensive efforts been made. Dengue virus appears to replicate and proliferate in many adapted cell lines, but these in vitro properties are extremely difficult to be reproduced in primary cells or in vivo. This paper summarizes reports on the permissive cells in vitro and in vivo and suggests a hematological cell lineage for dengue virus infection in vivo, with the hope that a new focus will shed light on further understanding of the complexities of dengue disease.

  14. Help Control Mosquitoes that Spread Dengue, Chikungunya, and Zika Viruses

    Science.gov (United States)

    Help Control Mosquitoes that Spread Dengue, Chikungunya, and Zika Viruses B Z Z Z Z . Aside from ... or Aedes albopictus ) can spread dengue, chikungunya, or Zika viruses. People become infected with dengue, chikungunya, or ...

  15. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine

    OpenAIRE

    Babu Ramanathan; Chit Laa Poh; Kristin Kirk; William John Hannan McBride; John Aaskov; Lara Grollo

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction app...

  16. Characterization of RyDEN (C19orf66 as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

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    Youichi Suzuki

    2016-01-01

    Full Text Available Dengue virus (DENV is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN. Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A-binding protein cytoplasmic 1 (PABPC1, and La motif-related protein 1 (LARP1. Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

  17. Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

    Science.gov (United States)

    Suzuki, Youichi; Chin, Wei-Xin; Han, Qi'En; Ichiyama, Koji; Lee, Ching Hua; Eyo, Zhi Wen; Ebina, Hirotaka; Takahashi, Hirotaka; Takahashi, Chikako; Tan, Beng Hui; Hishiki, Takayuki; Ohba, Kenji; Matsuyama, Toshifumi; Koyanagi, Yoshio; Tan, Yee-Joo; Sawasaki, Tatsuya; Chu, Justin Jang Hann; Vasudevan, Subhash G; Sano, Kouichi; Yamamoto, Naoki

    2016-01-01

    Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

  18. Comparative Evaluation of Permissiveness to Dengue Virus Serotype 2 Infection in Primary Rodent Macrophages

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    Jeanette Prada-Arismendy

    2012-01-01

    Full Text Available Infection with dengue virus presents a broad clinical spectrum, which can range from asymptomatic cases to severe cases that are characterised by haemorrhagic syndrome and/or shock. The reason for such variability remains unknown. This work evaluated the in vitro permissiveness of mouse, rat, hamster and guinea pig macrophages to infection by dengue virus 2 (DENV2. The results established that macrophages derived from the BALB/c mouse strain showed higher permissiveness to DENV2 infection than macrophages from other rodent species, although all rodent species studied had the C820T mutation in the oligoadenylate synthetase 1b gene, indicating no relationship to the different in vitro susceptibilities of mouse cells at this locus. Other molecular mechanisms related to flavivirus susceptibility remain to be explored.

  19. Multiploid CD61+ cells are the pre-dominant cell lineage infected during acute dengue virus infection in bone marrow.

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    Kristina B Clark

    Full Text Available Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM. Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM and humans were utilized to identify the cell lineage(s that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.

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

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    de Souza, Renato Pereira; Rocco, Iray M.; Maeda, Adriana Y.; Spenassatto, Carine; Bisordi, Ivani; Suzuki, Akemi; Silveira, Vivian R.; Silva, Sarai J. S.; Azevedo, Roberta M.; Tolentino, Fernanda M.; Assis, Jaqueline C.; Bassi, Margarida G.; Dambrós, Bibiana P.; Tumioto, Gabriela L.; Gregianini, Tatiana S.; Souza, Luiza Terezinha M.; Timenetsky, Maria do Carmo S. T.; Santos, Cecília L. S.

    2011-01-01

    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. PMID:22216365

  1. Seroprevalence of Infections with Dengue, Rift Valley Fever and Chikungunya Viruses in Kenya, 2007.

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    Caroline Ochieng

    Full Text Available Arthropod-borne viruses are a major constituent of emerging infectious diseases worldwide, but limited data are available on the prevalence, distribution, and risk factors for transmission in Kenya and East Africa. In this study, we used 1,091 HIV-negative blood specimens from the 2007 Kenya AIDS Indicator Survey (KAIS 2007 to test for the presence of IgG antibodies to dengue virus (DENV, chikungunya virus (CHIKV and Rift Valley fever virus (RVFV.The KAIS 2007 was a national population-based survey conducted by the Government of Kenya to provide comprehensive information needed to address the HIV/AIDS epidemic. Antibody testing for arboviruses was performed on stored blood specimens from KAIS 2007 through a two-step sandwich IgG ELISA using either commercially available kits or CDC-developed assays. Out of the 1,091 samples tested, 210 (19.2% were positive for IgG antibodies against at least one of the three arboviruses. DENV was the most common of the three viruses tested (12.5% positive, followed by RVFV and CHIKV (4.5% and 0.97%, respectively. For DENV and RVFV, the participant's province of residence was significantly associated (P≤.01 with seropositivity. Seroprevalence of DENV and RVFV increased with age, while there was no correlation between province of residence/age and seropositivity for CHIKV. Females had twelve times higher odds of exposure to CHIK as opposed to DENV and RVFV where both males and females had the same odds of exposure. Lack of education was significantly associated with a higher odds of previous infection with either DENV or RVFV (p <0.01. These data show that a number of people are at risk of arbovirus infections depending on their geographic location in Kenya and transmission of these pathogens is greater than previously appreciated. This poses a public health risk, especially for DENV.

  2. A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease.

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    Yang, Chi-Chen; Hu, Han-Shu; Wu, Ren-Huang; Wu, Szu-Huei; Lee, Shiow-Ju; Jiaang, Weir-Torn; Chern, Jyh-Haur; Huang, Zhi-Shun; Wu, Huey-Nan; Chang, Chung-Ming; Yueh, Andrew

    2014-01-01

    Dengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC50) of 1.03 ± 0.09 μM. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future.

  3. Primary dengue virus infections induce differential cytokine production in Mexican patients

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    Sergio Isaac de la Cruz Hernández

    2016-03-01

    Full Text Available Severe dengue pathogenesis is not fully understood, but high levels of proinflammatory cytokines have been associated with dengue disease severity. In this study, the cytokine levels in 171 sera from Mexican patients with primary dengue fever (DF and dengue haemorrhagic fever (DHF from dengue virus (DENV 1 (n = 116 or 2 (n = 55 were compared. DF and DHF were defined according to the patient’s clinical condition, the primary infections as indicated by IgG enzymatic immunoassay negative results, and the infecting serotype as assessed by real-time reverse transcription-polymerase chain reaction. Samples were analysed for circulating levels of interleukin (IL-12p70, interferon (IFN-γ, tumour necrosis factor (TNF-α, IL-6, and IL-8 using a commercial cytometric bead array. Significantly higher IFN-γ levels were found in patients with DHF than those with DF. However, significantly higher IL-12p70, TNF-α, and IL-6 levels were associated with DHF only in patients who were infected with DENV2 but not with DENV1. Moreover, patients with DF who were infected with DENV1 showed higher levels of IL-12p70, TNF-α, and IL-6 than patients with DHF early after-fever onset. The IL-8 levels were similar in all cases regardless of the clinical condition or infection serotype. These results suggest that the association between high proinflammatory cytokine levels and dengue disease severity does not always stand, and it once again highlights the complex nature of DHF pathogenesis.

  4. Increasing airline travel may facilitate co-circulation of multiple dengue virus serotypes in Asia.

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    Tian, Huaiyu; Sun, Zhe; Faria, Nuno Rodrigues; Yang, Jing; Cazelles, Bernard; Huang, Shanqian; Xu, Bo; Yang, Qiqi; Pybus, Oliver G; Xu, Bing

    2017-08-01

    The incidence of dengue has grown dramatically in recent decades worldwide, especially in Southeast Asia and the Americas with substantial transmission in 2014-2015. Yet the mechanisms underlying the spatio-temporal circulation of dengue virus (DENV) serotypes at large geographical scales remain elusive. Here we investigate the co-circulation in Asia of DENV serotypes 1-3 from 1956 to 2015, using a statistical framework that jointly estimates migration history and quantifies potential predictors of viral spatial diffusion, including socio-economic, air transportation and maritime mobility data. We find that the spread of DENV-1, -2 and -3 lineages in Asia is significantly associated with air traffic. Our analyses suggest the network centrality of air traffic hubs such as Thailand and India contribute to seeding dengue epidemics, whilst China, Cambodia, Indonesia, and Singapore may establish viral diffusion links with multiple countries in Asia. Phylogeographic reconstructions help to explain how growing air transportation networks could influence the dynamics of DENV circulation.

  5. Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice

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    Campos Marco A

    2011-03-01

    Full Text Available Abstract Background Dengue, one of the most important arboviral diseases of humans, may cause severe systemic disease. Although dengue virus (DENV has been considered to be a non-neurotropic virus, dengue infection has been associated recently with a series of neurological syndromes, including encephalitis. In this work, we evaluated behavioral changes and inflammatory parameters in C57BL/6 mice infected with non-adapted dengue virus 3 (DENV-3 genotype I. Methods C57BL/6 mice received 4 × 103 PFU of DENV-3 by an intracranial route. We evaluated the trafficking of leukocytes in brain microvasculature using intravital microscopy, and evaluated chemokine and cytokine profiling by an ELISA test at 3 and 6 days post infection (p.i.. Furthermore, we determined myeloperoxidase activity and immune cell populations, and also performed histopathological analysis and immunostaining for the virus in brain tissue. Results All animals developed signs of encephalitis and died by day 8 p.i. Motor behavior and muscle tone and strength parameters declined at day 7 p.i. We observed increased leukocyte rolling and adhesion in brain microvasculature of infected mice at days 3 and 6 p.i. The infection was followed by significant increases in IFN-γ, TNF-α, CCL2, CCL5, CXCL1, and CXCL2. Histological analysis showed evidence of meningoencephalitis and reactive gliosis. Increased numbers of neutrophils, CD4+ and CD8+ T cells were detected in brain of infected animals, notably at day 6 p.i. Cells immunoreactive for anti-NS-3 were visualized throughout the brain. Conclusion Intracerebral infection with non-adapted DENV-3 induces encephalitis and behavioral changes that precede lethality in mice.

  6. Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice

    Science.gov (United States)

    2011-01-01

    Background Dengue, one of the most important arboviral diseases of humans, may cause severe systemic disease. Although dengue virus (DENV) has been considered to be a non-neurotropic virus, dengue infection has been associated recently with a series of neurological syndromes, including encephalitis. In this work, we evaluated behavioral changes and inflammatory parameters in C57BL/6 mice infected with non-adapted dengue virus 3 (DENV-3) genotype I. Methods C57BL/6 mice received 4 × 103 PFU of DENV-3 by an intracranial route. We evaluated the trafficking of leukocytes in brain microvasculature using intravital microscopy, and evaluated chemokine and cytokine profiling by an ELISA test at 3 and 6 days post infection (p.i.). Furthermore, we determined myeloperoxidase activity and immune cell populations, and also performed histopathological analysis and immunostaining for the virus in brain tissue. Results All animals developed signs of encephalitis and died by day 8 p.i. Motor behavior and muscle tone and strength parameters declined at day 7 p.i. We observed increased leukocyte rolling and adhesion in brain microvasculature of infected mice at days 3 and 6 p.i. The infection was followed by significant increases in IFN-γ, TNF-α, CCL2, CCL5, CXCL1, and CXCL2. Histological analysis showed evidence of meningoencephalitis and reactive gliosis. Increased numbers of neutrophils, CD4+ and CD8+ T cells were detected in brain of infected animals, notably at day 6 p.i. Cells immunoreactive for anti-NS-3 were visualized throughout the brain. Conclusion Intracerebral infection with non-adapted DENV-3 induces encephalitis and behavioral changes that precede lethality in mice. PMID:21388530

  7. High rates of co-infection of Dengue and Chikungunya virus in Odisha and Maharashtra, India during 2013.

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    Saswat, Tanuja; Kumar, Abhishek; Kumar, Sameer; Mamidi, Prabhudutta; Muduli, Sagarika; Debata, Nagen Kumar; Pal, Niladri Shekhar; Pratheek, B M; Chattopadhyay, Subhasis; Chattopadhyay, Soma

    2015-10-01

    Dengue viral (DENV) infection is endemic in different parts of India and because of similar primary signs and symptoms, Chikungunya virus (CHIKV) is mostly undiagnosed. Hence, we investigated 204 suspected Dengue cases in a hospital based cross-sectional study in Odisha, India in 2013. It was observed that 50 samples were positive for DENV only, 28 were positive for CHIKV only and interestingly, 28 patients were co-infected with both DENV and CHIKV. Additionally, a total of 18 confirmed Dengue samples from Maharashtra, India were screened for CHIKV and out of those, 15 were co-infected. All CHIKV strains were of East Central South African (ECSA) type and serotype 2 (genotype IV) was predominant in the DENV samples. Additionally, Dengue serotype 1 and 3 were also detected during this time. Further, sequence analysis of E1 gene of CHIKV strains revealed that two substitution mutations (M269V and D284E) were observed in almost 50% strains and they were from co-infected patients. Similarly, sequence analysis of C-prM gene showed the presence of five substitution mutations, (G70S, L72F, N90S, S93N and I150L) in all serotype 1 and two consistent mutations (A101V and V112A) in serotype 2 Dengue samples. Together, it appears that a significantly high number of dengue patients (43, 44.8%) were co-infected with DENV and CHIKV during this study. This emphasizes the need of a routine diagnosis of CHIKV along with DENV for febrile patients. This will be useful in early and proper recognition of infecting pathogen to study the correlation of clinical symptoms with single or co-infection which will ultimately help to implement proper patient care in future.

  8. Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting.

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    Naze, F; Le Roux, K; Schuffenecker, I; Zeller, H; Staikowsky, F; Grivard, P; Michault, A; Laurent, P

    2009-12-01

    Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.

  9. Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection.

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    Liu, Shuyan; Chen, Lingming; Zeng, Ying; Si, Lulu; Guo, Xiaolan; Zhou, Junmei; Fang, Danyun; Zeng, Gucheng; Jiang, Lifang

    2016-09-01

    Dengue virus (DENV) remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B (GrzB), may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a*, miR-30e, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer (NK) cells and CD8(+) T cells. Further investigation indicated that NK cells, but not CD8(+) T cells, were the major sources of GrzB, and miR-378, but not miR-27a* or miR-30e, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.

  10. Human Immune Responses to Dengue Viruses.

    Science.gov (United States)

    1986-07-01

    between the PBL of non- inmune donors and dengue virus-infected cells, which results in IFN production. We use dengue virus-infected B lymphoblastoid cell...or, J.J., M.W. Brandriss, and E.E. Walsh. 1985. Protection against 17D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies

  11. Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection

    Science.gov (United States)

    Pinto, Amelia K.; Brien, James D.; Lam, Chia-Ying Kao; Johnson, Syd; Chiang, Cindy; Hiscott, John; Sarathy, Vanessa V.; Barrett, Alan D.; Shresta, Sujan

    2015-01-01

    ABSTRACT With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre+ Ifnarflox/flox [denoted as Ifnarf/f herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre+ Ifnarf/f mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre+ Ifnarf/f mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents. PMID:26374123

  12. An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8(+) T cells and antibody when expressed from modified vaccinia Ankara.

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    Quinan, Bárbara R; Flesch, Inge E A; Pinho, Tânia M G; Coelho, Fabiana M; Tscharke, David C; da Fonseca, Flávio G

    2014-05-23

    Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8(+) T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8(+) T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8(+) T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8(+) T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8(+) T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.

  13. Molecular surveillance of Dengue in Sukabumi, West Java province, Indonesia.

    Science.gov (United States)

    Nusa, Roy; Prasetyowati, Heni; Meutiawati, Febrina; Yohan, Benediktus; Trimarsanto, Hidayat; Setianingsih, Tri Yuli; Sasmono, R Tedjo

    2014-06-11

    Dengue is endemic and affects people in all Indonesian provinces. Increasing dengue cases have been observed every year in Sukabumi in West Java province. Despite the endemicity, limited data is available on the genetic of dengue viruses (DENV) circulating in the country. To understand the dynamics of dengue disease, we performed molecular and serological surveillance of dengue in Sukabumi. A total of 113 patients were recruited for this study. Serological data were obtained using anti-dengue IgM and IgG tests plus dengue NS1 antigen detection. Dengue detection and serotyping were performed using real-time RT-PCR. Viruses were isolated and the envelope genes were sequenced. Phylogenetic and evolutionary analyses were performed to determine the genotype of the viruses and their evolutionary rates. Real-time RT-PCR detected DENV in 25 (22%) of 113 samples. Serotyping revealed the predominance of DENV-2 (16 isolates, 64%), followed by DENV-1 (5 isolates, 20%), and DENV-4 (4 isolates, 16%). No DENV-3 was detected in the samples. Co-circulation of genotype I and IV of DENV-1 was observed. The DENV-2 isolates all belonged to the Cosmopolitan genotype, while DENV-4 isolates were grouped into genotype II. Overall, their evolutionary rates were similar to DENV from other countries. We revealed the distribution of DENV serotypes and genotypes in Sukabumi. Compared to data obtained from other cities in Indonesia, we observed the differing predominance of DENV serotypes but similar genotype distribution, where the infecting viruses were closely related with Indonesian endemic viruses isolated previously.

  14. 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

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    Mariana Gandini

    2011-08-01

    Full Text Available 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.

  15. 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.

    Science.gov (United States)

    Gandini, Mariana; Reis, Sonia Regina Nogueira Ignacio; Torrentes-Carvalho, Amanda; Azeredo, Elzinandes Leal; Freire, Marcos da Silva; Galler, Ricardo; Kubelka, Claire Fernandes

    2011-08-01

    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.

  16. Induction of virus-neutralizing antibodies and T cell responses by dengue virus type 1 virus-like particles prepared from Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    TANG Yun-xia; JIANG Li-fang; ZHOU Jun-mei; YIN Yue; YANG Xiao-meng; LIU Wen-quan; FANG Dan-yun

    2012-01-01

    Background Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections.The development of safe and effective vaccines has been hampered by the requirement of conferring complete protection against all four dengue serotypes and the lack of a convenient animal model.Virus-like particles (VLPs) have emerged as a promising subunit vaccine candidate.One strategy of vaccine development is to produce a tetravalent dengue subunit vaccine by mixing recombinant VLPs,corresponding to all four dengue virus serotypes.Towards this end,this study aimed to establish a Pichia pastoris (P.pastoris) expression system for production of dengue virus type 1 (DENV-1) VLPs and evaluate the humoral and cellular immune response of this particle in mice.Methods A recombinant yeast P.pastoris clone containing prM and E genes of DENV-1 was constructed and DENV-1 VLPs expressed by this clone were analyzed by sucrose density gradient centrifugation,Western blotting,and transmission electron microscope.Groups of mice were immunized by these particles plus adjuvant formulations,then mice were tested by ELISA and neutralization assay for humoral immune response,and by lymphocyte proliferation and cytokine production assays for a cellular immune response.Results Our data demonstrated that recombinant DENV-1 VLPs consisting of prM and E protein were successfully expressed in the yeast P.pastoris.Sera of VLPs immunized mice were shown to contain a high-titer of antibodies and the neutralization assay suggested that those antibodies neutralized virus infection in vitro.Data from the T lymphocyte proliferation assay showed proliferation of T cell,and ELISA found elevated secretion levels of interferon IFN-y and IL-4.Conclusions P.pastoris-expressed DENV-1 VLPs can induce virus neutralizing antibodies and T cell responses in immunized mice.Using P.pastoris to produce VLPs offers a promising and economic strategy for dengue virus

  17. RNAi:antiviral therapy against dengue virus

    Institute of Scientific and Technical Information of China (English)

    Sobia Idrees; Usman A Ashfaq

    2013-01-01

    Dengue virus infection has become a global threat affecting around 100 countries in the world. Currently, there is no licensed antiviral agent available against dengue. Thus, there is a strong need to develop therapeutic strategies that can tackle this life threatening disease. RNA interference is an important and effective gene silencing process which degrades targeted RNA by a sequence specific process. Several studies have been conducted during the last decade to evaluate the efficiency of siRNA in inhibiting dengue virus replication. This review summarizes siRNAs as a therapeutic approach against dengue virus serotypes and concludes that siRNAs against virus and host genes can be next generation treatment of dengue virus infection.

  18. [Phylogenetic analysis of envelope gene of dengue virus serotype 2 in Guangzhou, 2001-2015].

    Science.gov (United States)

    Liu, Y; Jiang, L Y; Luo, L; Cao, Y M; Jing, Q L; Yang, Z C

    2017-01-10

    Objective: To investigate the molecular characteristics of dengue virus serotype 2 (DENV2) in Guangzhou during 2001-2015, and analyze the E gene of the strains isolated, the phylogenetic tree and molecular clock were constructed to know about the evolution of the strains. Methods: The serum samples of the patients were detected by real time PCR, and positive samples were used to isolate dengue virus by using C6/36 cells. The E gene of the isolated strains were sequenced. The phylogenetic tree was constructed by using software Mega 4.0, and the molecular clock was drawn by using software BEASTv1.8.2. Results: Twenty-six dengue virus strains were isolated between 2001 and 2015. They were all clustered into 2 genotypes, i.e. cosmopolitan genotype and Asian genotype Ⅰ. The strains isolated in Guangzhou shared high homology with Southeast Asian strains. The cosmopolitan genotype was divided into 2 sub-genotype at about 46 and 35 years ago. The substitution rate of dengue virus serotype 2 in Guangzhou was 7.1 × 10(-4) per year per site. Conclusions: There were close relationship between the Guangzhou strains and Southeast Asian strains. Guangzhou was at high risk of imported dengue fever, outbreak of dengue hemorrhagic fever and dengue shock syndrome. There might be two ways of introduction of cosmopolitan genotype. The substitution rate of the strains in Guangzhou was similar to that in the neighbor countries.

  19. Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus

    Science.gov (United States)

    Ocampo, Clara B.; Caicedo, Paola A.; Jaramillo, Gloria; Ursic Bedoya, Raul; Baron, Olga; Serrato, Idalba M.; Cooper, Dawn M.; Lowenberger, Carl

    2013-01-01

    Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain. PMID:23593426

  20. Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome.

    Science.gov (United States)

    Nawtaisong, Pruksa; Keith, James; Fraser, Tresa; Balaraman, Velmurugan; Kolokoltsov, Andrey; Davey, Robert A; Higgs, Stephen; Mohammed, Ahmed; Rongsriyam, Yupha; Komalamisra, Narumon; Fraser, Malcolm J

    2009-06-04

    Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNA(val) promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

  1. Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome

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    Mohammed Ahmed

    2009-06-01

    Full Text Available Abstract Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNAval promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

  2. Differential expression of apoptosis related genes in selected strains of Aedes aegypti with different susceptibilities to dengue virus.

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    Clara B Ocampo

    Full Text Available Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2 and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1 and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain.

  3. Differential expression of apoptosis related genes in selected strains of Aedes aegypti with different susceptibilities to dengue virus.

    Science.gov (United States)

    Ocampo, Clara B; Caicedo, Paola A; Jaramillo, Gloria; Ursic Bedoya, Raul; Baron, Olga; Serrato, Idalba M; Cooper, Dawn M; Lowenberger, Carl

    2013-01-01

    Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain.

  4. Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus

    Science.gov (United States)

    Jupatanakul, Natapong; Sim, Shuzhen; Angleró-Rodríguez, Yesseinia I.; Souza-Neto, Jayme; Das, Suchismita; Poti, Kristin E.; Rossi, Shannan L.; Bergren, Nicholas; Vasilakis, Nikos

    2017-01-01

    We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role. PMID:28081143

  5. Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus.

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    Natapong Jupatanakul

    2017-01-01

    Full Text Available We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg promoter. Transgene expression inhibits infection with several dengue virus (DENV serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV or chikungunya virus (CHIKV infection, thereby indicating a possible specialization of the pathway's antiviral role.

  6. Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus.

    Science.gov (United States)

    Jupatanakul, Natapong; Sim, Shuzhen; Angleró-Rodríguez, Yesseinia I; Souza-Neto, Jayme; Das, Suchismita; Poti, Kristin E; Rossi, Shannan L; Bergren, Nicholas; Vasilakis, Nikos; Dimopoulos, George

    2017-01-01

    We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway's antiviral role.

  7. Dengue virus transovarial transmission by Aedes aegypti

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    Monica Dwi Hartanti

    2016-02-01

    Full Text Available Dengue is a disease that is caused by dengue virus and transmitted to humans through the bite of infected Aedes mosquitoes, especially Aedes aegypti. The disease is hyper-endemic in Southeast Asia, where a more severe form, dengue hemorrhagic fever (DHF and dengue shock syndrome (DSS, is a major public health concern. The purpose of the present study was to find evidence of dengue virus transovarial transmision in local vectors in Jakarta. Fifteen Aedes larvae were collected in 2009 from two areas in Tebet subdistrict in South Jakarta, namely one area with the highest and one with the lowest DHF prevalence. All mosquitoes were reared inside two cages in the laboratory, eight mosquitoes in one cage and seven mosquitoes in another cage and given only sucrose solution as their food. The results showed that 20% of the mosquitoes were positive for dengue virus. Dengue virus detection with an immunohistochemical method demonstrated the occurrence of transovarial transmission in local DHF vectors in Tebet subdistrict. Transovarial dengue infection in Ae.aegypti larvae appeared to maintain or enhance epidemics. Further research is needed to investigate the relation of dengue virus transovarial transmission with DHF endemicity in Jakarta.

  8. Dengue virus transovarial transmission by Aedes aegypti

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    Monica Dwi Hartanti

    2010-08-01

    Full Text Available Dengue is a disease that is caused by dengue virus and transmitted to humans through the bite of infected Aedes mosquitoes, especially Aedes aegypti. The disease is hyper-endemic in Southeast Asia, where a more severe form, dengue hemorrhagic fever (DHF and dengue shock syndrome (DSS, is a major public health concern. The purpose of the present study was to find evidence of dengue virus transovarial transmision in local vectors in Jakarta. Fifteen Aedes larvae were collected in 2009 from two areas in Tebet subdistrict in South Jakarta, namely one area with the highest and one with the lowest DHF prevalence. All mosquitoes were reared inside two cages in the laboratory, eight mosquitoes in one cage and seven mosquitoes in another cage and given only sucrose solution as their food. The results showed that 20% of the mosquitoes were positive for dengue virus. Dengue virus detection with an immunohistochemical method demonstrated the occurrence of transovarial transmission in local DHF vectors in Tebet subdistrict. Transovarial dengue infection in Ae.aegypti larvae appeared to maintain or enhance epidemics. Further research is needed to investigate the relation of dengue virus transovarial transmission with DHF endemicity in Jakarta.

  9. Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico

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    Cisneros Alejandro

    2009-12-01

    Full Text Available Abstract Background Dengue (DEN is a serious cause of mortality and morbidity in the world including Mexico, where the infection is endemic. One of the states with the highest rate of dengue cases is Oaxaca. The cause of DEN is a positive-sense RNA virus, the dengue virus (DENV that evolves rapidly increasing its variability due to the absence of a repair mechanism that leads to approximately one mutational event per genome replication; which results in enhancement of viral adaptation, including the escape from host immune responses. Additionally, recombination may play a role in driving the evolution of DENV, which may potentially affect virulence and cause host tropism changes. Recombination in DENV has not been described in Mexican strains, neither has been described the relevance in virus evolution in an endemic state such as Oaxaca where the four serotypes of DENV are circulating. Results To study whether there are isolates from Oaxaca having recombination, we obtained the sequence of 6 different isolates of DENV-2 Asian/American genotype from the outbreak 2005-6, one clone of the C(91-prM-E-NS1(2400 structural genes, and 10 clones of the E gene from the isolate MEX_OAX_1656_05. Evidence of recombination was found by using different methods along with two softwares: RDP3 and GARD. The Oaxaca MEX_OAX_1656_05 and MEX_OAX_1038_05 isolates sequenced in this study were recombinant viruses that incorporate the genome sequence from the Cosmopolitan genotype. Furthermore, the clone of the E gene namely MEX_OAX_165607_05 from this study was also recombinant, incorporating genome sequence from the American genotype. Conclusions This is the first report of recombination in DENV-2 in Mexico. Given such a recombinant activity new genomic combinations were produced, this could play a significant role in the DENV evolution and must be considered as a potentially important mechanism generating genetic variation in this virus with serious implications for

  10. Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    Science.gov (United States)

    Papa, Michelle Premazzi; de Morais, Ana Theresa Silveira; Peçanha, Ligia Maria Torres; de Arruda, Luciana Barros

    2015-01-01

    Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients. PMID:26656738

  11. Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies.

    Science.gov (United States)

    Mani, Shailendra; Tripathi, Lav; Raut, Rajendra; Tyagi, Poornima; Arora, Upasana; Barman, Tarani; Sood, Ruchi; Galav, Alka; Wahala, Wahala; de Silva, Aravinda; Swaminathan, Sathyamangalam; Khanna, Navin

    2013-01-01

    Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E) protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+)-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (pdeveloping non-replicating, safe, efficacious and affordable

  12. Differences in type I interferon signaling antagonism by dengue viruses in human and non-human primate cell lines.

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    Freddy A Medina

    2015-03-01

    Full Text Available BACKGROUND/OBJECTIVES: In vitro studies have shown that dengue virus (DENV can thwart the actions of interferon (IFN-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling. METHODOLOGY/PRINCIPAL FINDINGS: In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1. Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells. CONCLUSIONS: The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific

  13. Identification of cytotoxic T lymphocyte epitopes in dengue virus serotype 1.

    Science.gov (United States)

    Duan, Zhiliang; Guo, Jianglong; Huang, Xi; Liu, Huifang; Chen, Xinyu; Jiang, Minghua; Wen, Jinsheng

    2015-07-01

    Dengue virus (DENV) has a serious and growing impact on global health and the exact role of DENV-specific CD8(+) T-cells in DENV infection is still uncertain. In the present study, SYFPEITHI algorithm was used to screen the amino acid sequence of Dengue virus serotype 1 (DENV-1) for potential epitopes, and seven putative HLA-A*1101-restricted and five putative HLA-A*2402-restricted epitopes conserved in hundreds of DENV-1 strains were synthesized. The binding affinity of these epitope candidates to corresponding HLA molecules was evaluated using competitive peptide-binding assay. The immunogenicity and specificity of peptides were further tested in HLA-A*1101 transgenic mice, HLA-A*2402 transgenic mice and peripheral blood mononuclear cells (PBMCs) of patients infected with DENV-1. Percentage inhibition (PI) values calculated in competitive peptide-binding assay showed that six peptides (E39-47 PTLDIELLK, NS5(505-513) GVEGEGLHK, NS2b(15-23) SILLSSLLK, NS5(561-569) ALLATSIFK, NS3(99-107) AVEPGKNPK, and NS4b(159-167) VVYDAKFEK) could bind to HLA-A*1101 molecule with high affinity and five peptides (NS3472-480 QYIYMGQPL, NS4a40-48 AYRHAMEEL, NS5(880-888) DYMTSMKRF, NS3(548-556) SYKVASEGF, and NS3(22-30) IYRILQRGL) have a high affinity for HLA-A*2402 molecule. Enzyme-linked immunospot (ELISPOT) results indicated that these high-affinity peptides were recognized by splenocytes of DENV-1-infected transgenic mice and high-affinity peptide-immunized transgenic mice displayed high levels of peptide-specific IFN-γ-secreting cells. In addition, both peptide-pulsed splenocytes and DENV-1-infected splenic monocytes were efficiently killed by these peptide-specific cytotoxic T lymphocytes. Finally, except NS2b(15-23), 10 high-affinity peptides were recognized by PBMCs of patients infected with DENV-1. These identified epitopes would contribute to the understanding of the function of DENV-specific CD8(+) T-cells.

  14. Immunofluorescence assay method to detect dengue virus in Paniai-Papua

    Science.gov (United States)

    Sucipto, Teguh Hari; Ahwanah, Nur Laila Fitriati; Churrotin, Siti; Matake, Norifumi; Kotaki, Tomohiro; Soegijanto, Soegeng

    2016-03-01

    The dengue viruses (DENV), which include in the family Flaviviridae and the genus Flavivirus, was endemic in tropical areas and had been transmitted to humans by Aedes aegypti. An increasing number of immigrants from endemic areas to the non-endemic areas have emphasized the need for a simple and reliable test for the diagnosis of dengue virus infection. The purpose of this study was to detect the dengue virus by immunofluorescence assay (IFA) in the general population at Paniai-Papua. The results obtained from this study had showed a significantly better discrimination for DENV specific IgG antibodies. A total of 158 samples, 116 samples were IgG antibodies positive and 42 samples were negative. The conclusion of this study, Papua is not only a malaria endemic area, but also dengue virus infections were detected by IFA method. Therefore, the IFA can be used as an important diagnostic tool, which is a quick and an easy way to test samples from immigrants who come to the non-endemic areas.

  15. A Highly Sensitive Diagnostic System for Detecting Dengue Viruses Using the Interaction between a Sulfated Sugar Chain and a Virion.

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    Budi Saksono

    Full Text Available We propose a novel method of detecting trace amounts of dengue virus (DENVs from serum. Our method is based on the interaction between a sulfated sugar chain and a DENV surface glycoprotein. After capturing DENV with the sulfated sugar chain-immobilized gold nanoparticles (SGNPs, the resulting complex is precipitated and viral RNA content is measured using the reverse-transcription quantitative polymerase chain reaction SYBR Green I (RT-qPCR-Syb method. Sugar chains that bind to DENVs were identified using the array-type sugar chain immobilized chip (Sugar Chip and surface plasmon resonance (SPR imaging. Heparin and low-molecular-weight dextran sulfate were identified as binding partners, and immobilized on gold nanoparticles to prepare 3 types of SGNPs. The capacity of these SGNPs to capture and concentrate trace amounts of DENVs was evaluated in vitro. The SGNP with greatest sensitivity was tested using clinical samples in Indonesia in 2013-2014. As a result, the novel method was able to detect low concentrations of DENVs using only 6 μL of serum, with similar sensitivity to that of a Qiagen RNA extraction kit using 140 μL of serum. In addition, this method allows for multiplex-like identification of serotypes of DENVs. This feature is important for good healthcare management of DENV infection in order to safely diagnose the dangerous, highly contagious disease quickly, with high sensitivity.

  16. Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

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    Chu Justin

    2010-02-01

    Full Text Available Abstract Background Dengue virus (DENV is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined. Results In this study, we have used a targeted small interfering RNA (siRNA library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study. Conclusions Together, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti

  17. Antibodies against the Envelope Glycoprotein Promote Infectivity of Immature Dengue Virus Serotype 2

    Science.gov (United States)

    da Silva Voorham, Júlia M.; Rodenhuis-Zybert, Izabela A.; Ayala Nuñez, Nilda Vanesa; Colpitts, Tonya M.; van der Ende-Metselaar, Heidi; Fikrig, Erol; Diamond, Michael S.; Wilschut, Jan; Smit, Jolanda M.

    2012-01-01

    Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles. PMID:22431958

  18. Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2.

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    Júlia M da Silva Voorham

    Full Text Available Cross-reactive dengue virus (DENV antibodies directed against the envelope (E and precursor membrane (prM proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

  19. Selective susceptibility of human skin antigen presenting cells to productive dengue virus infection.

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    Daniela Cerny

    2014-12-01

    Full Text Available Dengue is a growing global concern with 390 million people infected each year. Dengue virus (DENV is transmitted by mosquitoes, thus host cells in the skin are the first point of contact with the virus. Human skin contains several populations of antigen-presenting cells which could drive the immune response to DENV in vivo: epidermal Langerhans cells (LCs, three populations of dermal dendritic cells (DCs, and macrophages. Using samples of normal human skin we detected productive infection of CD14(+ and CD1c(+ DCs, LCs and dermal macrophages, which was independent of DC-SIGN expression. LCs produced the highest viral titers and were less sensitive to IFN-β. Nanostring gene expression data showed significant up-regulation of IFN-β, STAT-1 and CCL5 upon viral exposure in susceptible DC populations. In mice infected intra-dermally with DENV we detected parallel populations of infected DCs originating from the dermis and migrating to the skin-draining lymph nodes. Therefore dermal DCs may simultaneously facilitate systemic spread of DENV and initiate the adaptive anti-viral immune response.

  20. Dengue fever among Israeli expatriates in Delhi, 2015: implications for dengue incidence in Delhi, India.

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    Neuberger, Ami; Turgeman, Avigail; Lustig, Yaniv; Schwartz, Eli

    2016-03-01

    We present the data of 13 dengue cases diagnosed between 1 August and 15 September 2015 among 240 Israeli expatriates residing in Delhi. Attack rates were similar between adults (6/128, 4.7%) and children (7/112, 6.3%). dengue virus (DENV-2) was identified in two and DENV-1 in one dengue-seropositive sample. Another febrile patient was diagnosed with chikungunya virus infection. The reported incidence of dengue fever among people living in Delhi was lower than 0.1% as of September 2015. Based on our results, we hypothesize that the incidence of dengue fever in Delhi is grossly underestimated.

  1. Global temperature constraints on Aedes aegypti and Ae. albopictus persistence and competence for dengue virus transmission

    Science.gov (United States)

    2014-01-01

    Background Dengue is a disease that has undergone significant expansion over the past hundred years. Understanding what factors limit the distribution of transmission can be used to predict current and future limits to further dengue expansion. While not the only factor, temperature plays an important role in defining these limits. Previous attempts to analyse the effect of temperature on the geographic distribution of dengue have not considered its dynamic intra-annual and diurnal change and its cumulative effects on mosquito and virus populations. Methods Here we expand an existing modelling framework with new temperature-based relationships to model an index proportional to the basic reproductive number of the dengue virus. This model framework is combined with high spatial and temporal resolution global temperature data to model the effects of temperature on Aedes aegypti and Ae. albopictus persistence and competence for dengue virus transmission. Results Our model predicted areas where temperature is not expected to permit transmission and/or Aedes persistence throughout the year. By reanalysing existing experimental data our analysis indicates that Ae. albopictus, often considered a minor vector of dengue, has comparable rates of virus dissemination to its primary vector, Ae. aegypti, and when the longer lifespan of Ae. albopictus is considered its competence for dengue virus transmission far exceeds that of Ae. aegypti. Conclusions These results can be used to analyse the effects of temperature and other contributing factors on the expansion of dengue or its Aedes vectors. Our finding that Ae. albopictus has a greater capacity for dengue transmission than Ae. aegypti is contrary to current explanations for the comparative rarity of dengue transmission in established Ae. albopictus populations. This suggests that the limited capacity of Ae. albopictus to transmit DENV is more dependent on its ecology than vector competence. The recommendations, which we

  2. PEMERIKSAAN VIRUS DENGUE-3 PADA NYAMUK Aedes aegypti YANG DIINFEKSI SECARA INTRATHORAKAL DENGAN TEKNIK IMUNOSITOKIMIA MENGGUNAKAN ANTIBODI DSSE10

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    Dyah Widiastuti

    2013-09-01

    Full Text Available ABSTRACTDengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infectedAedes mosquitoes. The most important vector of Dengue virus is the mosquito Ae.aegypti, which should be the main targetof surveillance and control activities. Virologic surveillance for dengue viruses in its vector has been used as an earlywarning system to predict outbreaks. Detection of Dengue virus antigen in mosquito head squash usingimmunocytochemical streptavidin biotin peroxidase complex (SBPC assay is an alternative method for dengue vectorsurveillance. The study aimed to develope immunocytochemical SBPC assay to detect Dengue virus infection in headsquash of Ae.aegypti. The study design was experimental. Artificially-infected adult Ae. aegypti mosquitoes of DENV 3were used as infectious samples and non-infected adult Ae. aegypti mosquitoes were used as normal ones. Theimmunocytochemical SBPC assay using monoclonal antibody DSSE10 then was applied in mosquito head squash todetect Dengue virus antigen. The results were analyzed by descriptive analysis. The immunocytochemical SBPC assaycan detect Dengue virus antigen in mosquito head squash at day 2 postinfection. There are some false positive resultsfound in immunocytochemical SBPC assay.Key Word: Dengue, immunocytochemistry, DSSE10

  3. PEMERIKSAAN VIRUS DENGUE-3 PADA NYAMUK Aedes aegypti YANG DIINFEKSI SECARA INTRATHORAKAL DENGAN TEKNIK IMUNOSITOKIMIA MENGGUNAKAN ANTIBODI DSSE10

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    Dyah Widiastuti

    2013-09-01

    Full Text Available ABSTRACTDengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infectedAedes mosquitoes. The most important vector of Dengue virus is the mosquito Ae.aegypti, which should be the main targetof surveillance and control activities. Virologic surveillance for dengue viruses in its vector has been used as an earlywarning system to predict outbreaks. Detection of Dengue virus antigen in mosquito head squash usingimmunocytochemical streptavidin biotin peroxidase complex (SBPC assay is an alternative method for dengue vectorsurveillance. The study aimed to develope immunocytochemical SBPC assay to detect Dengue virus infection in headsquash of Ae.aegypti. The study design was experimental. Artificially-infected adult Ae. aegypti mosquitoes of DENV 3were used as infectious samples and non-infected adult Ae. aegypti mosquitoes were used as normal ones. Theimmunocytochemical SBPC assay using monoclonal antibody DSSE10 then was applied in mosquito head squash todetect Dengue virus antigen. The results were analyzed by descriptive analysis. The immunocytochemical SBPC assaycan detect Dengue virus antigen in mosquito head squash at day 2 postinfection. There are some false positive resultsfound in immunocytochemical SBPC assay.Key Word: Dengue, immunocytochemistry, DSSE10

  4. Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti.

    Science.gov (United States)

    Richardson, Jason; Molina-Cruz, Alvaro; Salazar, Ma Isabel; Black, William

    2006-01-01

    Dengue virus-2 (DENV-2) RNA was quantified from the midgut and legs of individual Aedes aegypti at each of 14 days postinfectious blood meal (dpi) in a DENV-2 susceptible strain from Chetumal, Mexico. A SYBR Green I based strand-specific, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed. The lower detection and quantitation limits were 20 and 200 copies per reaction, respectively. Amounts of positive and negative strand viral RNA strands were correlated. Numbers of plaque-forming units (PFU) were correlated with DENV-2 RNA copy number in both C6/36 cell cultures and mosquitoes. PFU were consistently lower than RNA copy number by 2-3 log(10). Midgut levels of DENV-2 RNA peaked 8 dpi and fluctuated erratically between 6 and 9 dpi. Copies of DENV-2 RNA varied significantly among infected mosquitoes at each time point. Quantitative real-time RT-PCR is a convenient and reliable method that provides new insights into virus-vector interactions.

  5. Nucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particles.

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    Balinsky, Corey A; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C; Zoon, Kathryn C

    2013-12-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics.

  6. The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis.

    Science.gov (United States)

    Clyde, Karen; Barrera, Julio; Harris, Eva

    2008-09-30

    Dengue virus (DENV) and West Nile virus (WNV) are members of the Flavivirus genus of positive-strand RNA viruses. RNA sequences and structures, primarily in the untranslated regions, have been shown to modulate flaviviral gene expression and genome replication. Previously, we demonstrated that a structure in the DENV coding region (cHP) enhances translation start codon selection and is required for viral replication. Here we further characterize the role of the cHP in the DENV life cycle. We demonstrate that the cHP is required for efficient viral RNA synthesis in a sequence-independent manner. Viruses with a disrupted cHP are rescued by a spontaneous compensatory mutation that restabilizes the structure. Furthermore, the cHP, which is predicted to be conserved among arthropod-borne flaviviruses, is required for WNV replication. We propose that the cHP is a multifunctional determinant of flavivirus replication, functioning in both translation and RNA synthesis.

  7. DENV inhibits type I IFN production in infected cells by cleaving human STING.

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    Sebastian Aguirre

    Full Text Available Dengue virus (DENV is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs. In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.

  8. IFI6 Inhibits Apoptosis via Mitochondrial-Dependent Pathway in Dengue Virus 2 Infected Vascular Endothelial Cells.

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    Yiming Qi

    Full Text Available Dengue hemorrhagic fever (DHF/Dengue shock syndrome (DSS is a fatal infectious disease that demands an effective treatment. Interferon (IFN-stimulated genes (ISGs induced by dengue virus (DENV exert antiviral effects. Among ISGs, IFN-α inducible gene 6 (IFI6 was increased in DENV infected human umbilical vascular endothelial cells (HUVECs by microarray analysis in our previous study. However, its function is incompletely understood. In this study, we confirmed that IFI6 was markedly induced in DENV infection of both primary HUVECs and EA.hy926 cell lines. Recombinant EA.hy926 cell lines in which IFI6 was either over-expressed (IFI6+/+ or knocked-down (IFI6-/- were generated. The activation of caspase-3 and intrinsic apoptosis-related protein caspase-9 were down-regulated in IFI6+/+ but up-regulated in IFI6-/- cells at 24-48 hrs post-infection. After incubation with DENV for 48 hrs, the mitochondrial membrane potential (Δψ(m was more stable in IFI6+/+ cells but reduced in IFI6-/- cells, as assayed by fluorescence staining with JC-1. We observed that Bcl-2 expression was increased in IFI6+/+ and decreased in IFI6-/- cells. By contrast, Bax expression was decreased in IFI6+/+ and increased in IFI6-/- cells. It is presumed that the anti-apoptotic function of IFI6 is expressed by regulating the rheostatic balance between bcl-2/bax expression and inhibition of Δψ(m depolarization during DENV infection of vascular endothelial cells(VECs. In addition, the pro-apoptotic protein X-linked Inhibitor of Apoptosis (XIAP-Associated Factor 1(XAF1 expression had been reported to be up-regulated and led to the induction of apoptosis in DENV2-infected VECs,but the relationship between XAF1 and IFI6 dengue virus-induced apoptosis in VECs warrants further study.

  9. Could an experimental dengue virus infection fail to induce solid immunity against homologous viral challenge in non-human primates?

    Science.gov (United States)

    Valdés, Iris; Gil, Lázaro; Lazo, Laura; Marcos, Ernesto; Martín, Jorge; Suzarte, Edith; Castro, Jorge; Romero, Yaremis; Guillén, Gerardo; Hermida, Lisset

    2016-02-01

    There are several dengue vaccine candidates at advanced stages of development, but none of them are licensed. Despite the reactogenicity and immunogenicity profile in humans of the tetravalent ChimeriVax™ dengue vaccine candidate, in efficacy trials, it has failed to confer complete protection against dengue virus (DENV)-1 and DENV-2. However, full protection against the four serotypes had been observed previously in monkeys immunized with this vaccine candidate. Some authors have tried to explain this contradiction by hypothesizing that protection rates in non-human primates (NHPs) are associated with a lack of post-challenge anamnestic immune responses. Here, we studied the protection and anamnestic response patterns after homologous challenge in NHPs previously infected with DENV-2. Two immunization schemes were used, varying the viral doses and the intervals between them. Animals developed immunity against DENV-2 that provided full protection against reinfection with a homologous virus. However, all monkeys showed a significant increase in antiviral and neutralizing antibody titers after challenge. Our results suggest that sterilizing immunity could not be induced by infection with the virus despite the lack of detectable viremia in some animals in which an increase in antibody titer was observed. For this reason, we propose that the lack of an anamnestic neutralizing antibody response after challenge, as suggested by some authors, should be carefully reviewed as a criterion for evaluating the functionality of vaccine candidates.

  10. Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution

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    Saw, Wuan Geok; Tria, Giancarlo; Grüber, Ardina; Subramanian Manimekalai, Malathy Sony; Zhao, Yongqian; Chandramohan, Arun; Srinivasan Anand, Ganesh; Matsui, Tsutomu; Weiss, Thomas M.; Vasudevan, Subhash G.; Grüber, Gerhard

    2015-10-31

    Infection by the four serotypes ofDengue virus(DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all fourDengue virusserotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase–RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.

  11. Dengue vaccine: hypotheses to understand CYD-TDV-induced protection.

    Science.gov (United States)

    Guy, Bruno; Jackson, Nicholas

    2016-01-01

    Dengue virus (DENV) is a human pathogen with a large impact on public health. Although no vaccine against DENV is currently licensed, a recombinant vaccine - chimeric yellow fever virus-DENV tetravalent dengue vaccine (CYD-TDV) - has shown efficacy against symptomatic dengue disease in two recent Phase III clinical trials. Safety observations were also recently reported for these trials. In this Opinion article, we review the data from recent vaccine clinical trials and discuss the putative mechanisms behind the observed efficacy of the vaccine against different forms of the disease, focusing on the interactions between the infecting virus, pre-existing host immunity and vaccine-induced immune responses.

  12. Molecular Typing of Dengue Virus Circulating in Kolkata, India in 2010

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    Arindam Sarkar

    2012-01-01

    Full Text Available Dengue is one of the major public health threats in Kolkata. Every year, blood samples with dengue-like illness are referred to us from different medical colleges and hospitals in Kolkata for the detection of dengue infection in them. In 2010, a total of 378 samples were referred to us for that purpose. All the samples were tested for the detection of IgM antibodies by ELISA method, followed by RT-PCR test for the detection of serotypes. Only 173 samples were ELISA positive. Out of 378 samples, 108 were RT-PCR positive. Out of 108 samples, 74 samples had monotypic infection with different serotypes of DENV and 33 samples had dual infections with DENV-2 and DENV-3. Only one sample had the infection with DENV-1, DENV-2, and DENV-3. DHF was found mainly among the patients, infected with multiple dengue serotypes. Only 3 dengue monotypic infected patients had suffered from DHF.

  13. Synthetic strategy and antiviral evaluation of diamide containing heterocycles targeting dengue and yellow fever virus.

    Science.gov (United States)

    Saudi, Milind; Zmurko, Joanna; Kaptein, Suzanne; Rozenski, Jef; Gadakh, Bharat; Chaltin, Patrick; Marchand, Arnaud; Neyts, Johan; Van Aerschot, Arthur

    2016-10-04

    High-throughput screening of a subset of the CD3 chemical library (Centre for Drug Design and Discovery; KU Leuven) provided us with a lead compound 1, displaying low micromolar potency against dengue virus and yellow fever virus. Within a project aimed at discovering new inhibitors of flaviviruses, substitution of its central imidazole ring led to synthesis of variably substituted pyrazine dicarboxylamides and phthalic diamides, which were evaluated in cell-based assays for cytotoxicity and antiviral activity against the dengue virus (DENV) and yellow fever virus (YFV). Fourteen compounds inhibited DENV replication (EC50 ranging between 0.5 and 3.4 μM), with compounds 6b and 6d being the most potent inhibitors (EC50 0.5 μM) with selectivity indices (SI) > 235. Compound 7a likewise exhibited anti-DENV activity with an EC50 of 0.5 μM and an SI of >235. In addition, good antiviral activity of seven compounds in the series was also noted against the YFV with EC50 values ranging between 0.4 and 3.3 μM, with compound 6n being the most potent for this series with an EC50 0.4 μM and a selectivity index of >34. Finally, reversal of one of the central amide bonds as in series 13 proved deleterious to the inhibitory activity.

  14. Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints.

    Science.gov (United States)

    Sessions, October M; Wilm, Andreas; Kamaraj, Uma Sangumathi; Choy, Milly M; Chow, Angelia; Chong, Yuwen; Ong, Xin Mei; Nagarajan, Niranjan; Cook, Alex R; Ooi, Eng Eong

    2015-01-01

    Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients' sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.

  15. Epidemiology of Dengue Among Children Aged < 18 Months-Puerto Rico, 1999-2011.

    Science.gov (United States)

    Hause, Anne M; Perez-Padilla, Janice; Horiuchi, Kalanthe; Han, George S; Hunsperger, Elizabeth; Aiwazian, Jonathan; Margolis, Harold S; Tomashek, Kay M

    2016-02-01

    Dengue, a mosquito-borne viral illness caused by dengue virus types (DENV)-1 to DENV-4, is endemic in Puerto Rico. Severe dengue usually occurs in individuals previously infected with DENV or among infants born to previously infected mothers. To describe clinical features of dengue in infants, we retrospectively characterized dengue patients aged Dengue Surveillance System (PDSS) during 1999-2011. To determine frequency of signs, symptoms, and disease severity, case report forms and medical records were evaluated for patients who tested positive for dengue by reverse transcriptase polymerase chain reaction or anti-DENV immunoglobulin Menzyme-linked immunosorbent assay. Of 4,178 reported patients aged dengue, 23% had dengue with warning signs, and 33% had severe dengue. Mean age of patients with severe dengue was 8 months. Anti-DENV immunoglobulin G (IgG) titers were not statistically different in patients with (50%) and without (59%) severe dengue. In this study, one-third of DENV-infected infants met the severe dengue case definition. The role of maternal anti-DENV IgG in development of severe disease warrants further study in prospective cohorts of mother-infant pairs.

  16. The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

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    Adriana S Azevedo

    Full Text Available The dengue envelope glycoprotein (E is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2 and a chimeric yellow fever/dengue 2 virus (YF17D-D2. The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

  17. The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

    Science.gov (United States)

    Azevedo, Adriana S; Gonçalves, Antônio J S; Archer, Marcia; Freire, Marcos S; Galler, Ricardo; Alves, Ada M B

    2013-01-01

    The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

  18. Host cell responses to dengue virus infection

    NARCIS (Netherlands)

    Diosa Toro, Mayra

    2017-01-01

    Dengue (ook wel knokkelkoorts) is de meest voorkomende virale infectieziekte dat wordt overgedragen door muggen in de wereld met naar schatting 390 miljoen infecties per jaar. Ondanks de grote klinische impact en economische schade van het dengue virus is er nog steeds geen behandeling beschikbaar.

  19. Inhibition of dengue virus entry and multiplication into monocytes using RNA interference.

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    Mohammed Abdelfatah Alhoot

    2011-11-01

    Full Text Available BACKGROUND: Dengue infection ranks as one of the most significant viral diseases of the globe. Currently, there is no specific vaccine or antiviral therapy for prevention or treatment. Monocytes/macrophages are the principal target cells for dengue virus and are responsible for disseminating the virus after its transmission. Dengue virus enters target cells via receptor-mediated endocytosis after the viral envelope protein E attaches to the cell surface receptor. This study aimed to investigate the effect of silencing the CD-14 associated molecule and clathrin-mediated endocytosis using siRNA on dengue virus entry into monocytes. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression analysis showed a significant down-regulation of the target genes (82.7%, 84.9 and 76.3% for CD-14 associated molecule, CLTC and DNM2 respectively in transfected monocytes. The effect of silencing of target genes on dengue virus entry into monocytes was investigated by infecting silenced and non-silenced monocytes with DENV-2. Results showed a significant reduction of infected cells (85.2%, intracellular viral RNA load (73.0%, and extracellular viral RNA load (63.0% in silenced monocytes as compared to non-silenced monocytes. CONCLUSIONS/SIGNIFICANCE: Silencing the cell surface receptor and clathrin mediated endocytosis using RNA interference resulted in inhibition of the dengue virus entry and subsequently multiplication of the virus in the monocytes. This might serve as a novel promising therapeutic target to attenuate dengue infection and thus reduce transmission as well as progression to severe dengue hemorrhagic fever.

  20. Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo.

    Science.gov (United States)

    Williams, Katherine L; Wahala, Wahala M P B; Orozco, Susana; de Silva, Aravinda M; Harris, Eva

    2012-07-20

    The envelope (E) protein of dengue virus (DENV) is composed of three domains (EDI, EDII, EDIII) and is the main target of neutralizing antibodies. Many monoclonal antibodies that bind EDIII strongly neutralize DENV. However in vitro studies indicate that anti-EDIII antibodies contribute little to the neutralizing potency of human DENV-immune serum. In this study, we assess the role of anti-EDIII antibodies in mouse and human DENV-immune serum in neutralizing or enhancing DENV infection in mice. We demonstrate that EDIII-depleted human DENV-immune serum was protective against homologous DENV infection in vivo. Although EDIII-depleted DENV-immune mouse serum demonstrated decreased neutralization potency in vitro, reduced protection in some organs, and enhanced disease in vivo, administration of increased volumes of EDIII-depleted serum abrogated these effects. These data indicate that anti-EDIII antibodies contribute to protection and minimize enhancement when present, but can be replaced by neutralizing antibodies targeting other epitopes on the dengue virion.

  1. Substitution of the precursor peptide prevents anti-prM antibody-mediated antibody-dependent enhancement of dengue virus infection.

    Science.gov (United States)

    Wang, Ying; Si, Lu-Lu; Guo, Xiao-Lan; Cui, Guo-Hui; Fang, Dan-Yun; Zhou, Jun-Mei; Yan, Hui-Jun; Jiang, Li-Fang

    2017-02-02

    Antibody-dependent enhancement (ADE) is currently considered as the mechanism underlying the pathogenesis of severe dengue disease. Many studies have shown that precursor (pr) peptide-specific antibodies do not efficiently neutralize infection but potently promote ADE of dengue virus (DENV) infection. To explore the effect of pr peptide substitution on neutralization and ADE of DENV infection, the rabbit anti-prM polyclonal antibodies (pAbs) and anti-JEVpr/DENV-M pAbs were prepared, and the neutralization and ADE of these two pAbs were further compared. Here, we report that both anti-JEVpr/DENV-M and anti-prM pAbs exhibited broad cross-reactivity and only partial neutralization with four DENV serotypes and immature DENV. Rabbit anti-prM pAbs showed a significant enhancement in a broad range of serum dilutions. However, there was no statistically significant difference in the enhancing activity of rabbit anti-JEVpr/