Pan American Dhf

7/23/2019 Pan American Dhf

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Trial

Help to Hold the Hope1R13AI115934-01 to Mariano Garcia-Blanco and Carlos A. Sariol


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Dear Participants,

The main objective of this meeting is to promote theexchange of scientific information between academia,government and industry in the area of dengue research.This meeting has acquired an essential niche as one ofthe conferences available to the international Dengue

community. We expect it will serves a distinct functionby providing a mid-format meeting at which the latestunpublished research can be discussed in depth.

Particularly relevant are the unpublished results of allfive dengue vaccine candidates presently in clinicaltrials, including result from Phase III safety studyrecently completed in our Latin American Region. Weencourage all of you to promote the networking and to

establish new collaborations and to open newopportunities for the students and young investigators.


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Dear Participants,

The Pan American Dengue Research Network (Pan Dengue Net) welcomes all of you andappreciates your time and contributions, which have made the Fourth Meeting possible.

We feel it is essential to continue to have meetings that give the Pan American dengue researchcommunity the opportunity to integrate key aspects from different areas of dengue research(virology, immunology, epidemiology, vector biology, modeling among others), and attractparticipants from other disciplines as well, allowing valuable cross-fertilization.

These interactions are likely to result in recruitment opportunities for participants who are

seeking post-doctoral fellowships or new faculty positions, as well as in fostering North-Southproductive collaborations. We hope this meeting will contribute to meet these goals.

This year, our event has participants from at least 25 countries from the Americas, Europe, Asia,and Australia. By the end of September, we had 295 registered participants but more significant,67% are females, Congratulations!

This meeting would not be possible without the contribution of our Sponsors, particularly theBrazil Ministry of Health, the Instituto Evandro Chagas, and the Chief of Arbovirology andHemorrhagic Fevers Section and Chairman of this Meeting Dr. Pedro Vasconcelos and his team.

Also the contribution of Dr. Maurcio Lacerda Nogueira as Coordinator and member of theBrazilian crew has been key to the meeting organization.

The scientific success and quality of this meeting is largely attributable to Dr. Eva Harris (just Eva),Chairperson of the Scientific Committee, who as in previous meetings devoted a lot of energy toselect the Speakers, to select the members of the scientific committee and to put together theexciting Agenda of this meeting.

We are deeply in debt to all of you, members of our community, who took time away form yourdaily activities (academic, administrative, scientific, clinical, etc.) to be part of the Organizing andScientific committees (listed on this agenda) to guarantee the success of this Fourth meeting.

We are already working on the 2016 Pan Dengue Net Fifth meeting. Volunteers are more thanwelcome! Don't be shy, we will need a lot of help.

If you are a potential Sponsor, please add us to your contributions list for 2016!!

If you think you can help, please contact us with your comments, suggestions and ideas [email protected] or through our web site wwwpandenguenet.org


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Pedro F.C. Vasconcelos (Brazil) Chairman

Eva Harris (USA) Chairperson

Carlos Sariol (USA/PR) Acting Secretary

Jorge Osorio (USA/Colombia)

Aravinda de Silva (USA)

Jorge Muoz Jordan (CDC, USA)

Cristina Cassetti (USA, NIH)

Margarita Ossorio (USA, NIH)

Pedro F.C. Vasconcelos (Brazil) Chairman

Mauricio Lacerda Nogueira (Brazil) Coordinator

Carlos Sariol (USA/PR) Acting Secretary

Adriano Mondini (Brazil)

Daniele B. A. Medeiros (Brazil)

Eunice Lima (Brazil)

Joo Carlos Lopes (Brazil)

Sueli G. Rodrigues (Brazil)

Giovanini Evelin Coelho (Brazil)

Maria Guadalupe Guzman (Cuba)

Rosa M. del Angel (Mexico)

Cristina Cassetti (USA, NIH)

Margarita Ossorio (USA, NIH)


Mariano Garcia-Blanco (USA)

Janiffer Oliveira Chiang (Brazil)

Eva Harris (USA) Chairperson

Pedro F.C. Vasconcelos (Brazil) Mauricio L Nogueira (Brazil)

Laura White (USA)

Andrea Gamarnik (Argentina)

Irene Bosch (USA)

Juan Ernesto Ludert (Mexico)

Aravinda de Silva (USA)

Jorge Osorio (USA/Colombia)

Alan D. Barrett (USA)

Anna Fernadez Sesma (USA)

Cristovam P. Diniz (Brazil)

Duane Gubler (Singapore)

Elizabeth Hunsperger (USA)

Ernesto Marques Jr. (USA)

Mauro M. Teixeira (Brazil)

Nikkos Vasilakis (USA)

Ana B Perez (Cuba)

Luiz Tadeu Figueiredo (Brazil)

Mrcio Teixeira Nunes (Brazil)


Maria Guadalupe Guzman (Cuba)

Scott Halstead


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PROGRAMMonday 10/20/14 - Morning8:30 am - 12:45 pm

Virology / Antivirals- 85 minRichard Kuhn (USA)

Andrea Gamarnik (Argentina)

Guilherme Ferreira Silveira- Claudia Santos (Brazil)

Sebastian Aguirre (USA)

Andrea da Poain (Brazil)

Highlighted Posters (Poster Walk)

ImmunologyEmily Gallichotte (USA)Raphael Zellweger (USA)Cintia Marinho (Brazil)Mariana Gandini (Brazil)Daniel Limonta (Cuba)

Clinical ResearchCynthia Rodriguez (Honduras)Crystyan Siles (Peru)Carlos Narvaez (Colombia)Mayara Marques Carneiro da Silva (Brazil)

Virology/Antivirals

Pathogenesis

COFFEE BREAK 15 minutes

Virology / Antivirals- 95 minM. Garcia-Blanco (USA / Singapore)Aruna Sampath (Unither Virology, USA)Mariana Leguia (Peru)Elsa Damonte (Argentina)Maria Flechas (Colombia)Devika Sirohi (USA)

Monday 10/20/14 - Afternoon2:00 - 5:10 pmPathogenesis -80 min

Pedro Vasconcelos (Brazil)Beatriz Sierra (Cuba)

Irene Bosch (USA)

Yerly Useche (Colombia)

Henry Puerta Guardo (USA)

Immunology - 85 minErnesto Marques (Brazil)

Eva Harris (USA)

Daniela Weiskopf (USA) AmeIia Pinto (USA)

Davis Beltran (Panam)

LUNCH 12:45-2:00 PM


Tuesday 10/21/14 - Morning8:30 am - 12:40 pmImmunology/Diagnostics/ Prognostics- 85 minGuadalupe Guzman (Cuba)Elizabeth Hunsperguer (USA)Allan Brasier (USA)Monique da Rocha Queiroz Lima (Brazil)Parichat Duangkhae (USA)

Epidemiology/Phylogenetics - 85 minM. Nogueira Lacerda (Brazil)

Jorge Muoz (USA/Puerto Rico/CDC)

Leah Katzelnick (USA)

Magelda Montoya (USA)

Rosmari Rodriguez (Cuba)

Tuesday 10/21/14 - Afternoon2:00 - 5:10 pmVaccines - 75 min

Nicholas Jackson (Sanofi)Jorge Osorio (Takeda)

Stephen Whitehead/Ricardo Palacios (USA/Brazil)

Alexander Schmidt (GSK)

Beth-Ann Coller (Merck)

Vaccine/Modeling- 100 minAnna Durbin (USA)Aravinda de Silva (USA)Alex Perkin (USA)Diana Rojas (University of Florida/USA-Colombia)Leah Katzelnick (UK/USA)Laura White (USA)

Diagnostics/PrognosticsIrene Bosch (MIT/USA)Claire Huang (CDC/USA)Angela Argolo (Brazil)Diego Allonso (Brazil)Stalin Vilcarromero (Peru)

VectorNatapong Jupatanakul (USA)Jos Joaqun Carvajal Corts (Brazil)Luciana dos Santos Dias (Brazil)Kacey C. Ernst (USA)

Highlighted Posters (Poster Walk)

Epidemiology/PhylogeneticsRaquel Lana (Brazil)

Adrienne Guinard (GSK/Belgium)

Cynthia Jimenez (Colombia)

Mabel Carabali (Colombia)

Vaccine/ModelingHarry Partidos (Takeda)

Clemente Diaz (GSK/Puerto Rico)

Guido Camargo (Colombia)

Milene Silveira Ferreira (Brazil)

Melissa Mattocks (USA)

Alienys Izquierdo (Cuba)

LUNCH 12:45-2:00 PM

20 minutesCOFFEE BREAK


Wednesday 10/22/14 - Morning8:30 am - 12:40 pmDengue in Brazil Round Table- 80 minKleber Luz (Brazil)Marcio Nunes (Brazil)Giovanini Coelho (Brazil)

Tuesday 10/22/14 - Afternoon2:00 - 5:10 pm


Clinical Research/Cost Studies -90 minLuis Villar (Colombia)

Federico Narvaez (Nicaragua)

Eduardo Undurraga (USA/Chile)

Ivony Yireth Agudelo (Colombia)

Joao Bosco Siquiera (Brazil)

LUNCH 12:45-2:00 PM

Vector Biology/Ecology/Surveillance - 65 minSarah Anne Jablonski Guagliardo (USA)

Catalina Alfonso Parra (USA)

George Dimopholous (USA)

Amy Morrison (USA/Per)


Vector Control II- 105 min

Andrew R. McKemey (USA)Margaret de Lara Capurro (Brazil)Peter Ryan (Australia)Luciano Moreira (Brazil)

Round Table I

Roberto Barrera (USA/CDC/Puerto Rico)

Alvaro Eiras (Brasil)

Dawn Wesson (USA)

Round Table II

19:00-21:00Welcome Cocktail

Meeting Sessions

and Invited Speakers

SundayOctober 19th

18:00-18:15Opening Ceremony

18:15-19:00Keynote

POSTER SESSIONS5:15-6:45 pm

POSTER SESSIONS5:15-6:45 pm

BELM, BRAZIL | OCTOBER 19-22 | 2014

Silvio Urcuqui (Colombia)

Viviana Castilla (Argentina)

Antonio Dias (UK)

Cinthia L. Dionisio (Mexico)

Juan Ludert (Mexico)

Maria Paulina Cabarcas (Colombia)

Ruben Soto Acosta (Mexico)

Marciano Paes (Brazil)

Emiliana Silva (Brazil)

Victor Fiestas (Brazil)

Monique Trugilho (Brazil)

Jonas Conde (Brazil)


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VIROLOGY/ANTIVIRALS

INVITED SPEAKERSSTRUCTURE-FUNCTION ANALYSES OF THE DENGUE VIRUS NS1 PROTEIN

1,2 1 1 1 3 3 4 3Richard J. Kuhn , Joyce Jose , Jinsam Chang , Andrew Miller , David Akey , Clay Brown , Theodore C. Pierson , & Janet Smith1 2 3Markey Center for Structural Biology, Dept. of Biological Sciences, Bindley Bioscience Center, Purdue University, West Lafayette, IN; Life Sciences Institute,

4University of Michigan, Ann Arbor, MI, Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MDWe have recently solved the high-resolution x-ray crystal structures of the dengue virus and the West Nile virus NS1 proteins. The NS1 protein is found as a functionaldimer with a hydrophobic and hydrophilic face separated by an extended set of beta strands. The hydrophobic face is suggested to play an intimate role in RNAsynthesis by directly contacting membrane replication proteins. As NS1 is also secreted in the form of a lipoprotein complex, this face also serves to organize lipids inthe hexamer form of NS1. Several additional structural features of NS1 suggest a role in evading immune surveillance. Mutational and functional studies are be ingdone to interrogate the relationship between the structure and function of the NS1 protein. Surprisingly, many single point mutations render the virus restricted inreplication. Many of these detrimental substitutions were made in the beta roll and greasy nger domains. This region is suggested to play a role in RNA synthesis.Insights into the functional differences between the West Nile and dengue NS1 proteins are being pursued by exchanging regions or specic residues that differbetween the two proteins. Certain chimeras, while not expected to have structural differences, do present phenotype differences. Structural features will be presentedin the context of biological functions.

RNA STRUCTURE DUPLICATIONS IN THE DENGUE VIRUS GENOME FACILITATES HOST ADAPTATION

Sergio Villordo*, Claudia Filomatori* and Andrea GamarnikFundacin Instituto Leloir-CONICET, Buenos Aires, ArgentinaDengue virus cycles in nature between humans and mosquitoes, however, it is still unclear how the virus adapts to use different cellular machineries for replicationand overcome different types of antiviral responses. RNA viruses in general have high capacity to adapt to different environments due to the genetic diversity of viralpopulations. Using dengue virus, we recently found specic RNA sequences in the viral 3'UTR that are essential for viral replication in mosquito cells but dispensablefor replication in mammalian cells. These studies provided direct evidence for host-specic functions of viral RNA elements and raised the question whether viral RNAstructures are under specic selective pressures during host adaptation. To further these studies, we evaluated how RNA cis-acting elements evolve under single-host selective pressures in mosquito and mammalian cells and during host switch. Deep sequencing of dengue virus populations subjected to host adaptationshowed a strong selection of different mutations in the viral 3'UTR in each host, while cis-acting elements present at the 5' end of the genome remained unchanged.Secondary RNA structure analysis showed that viruses selected in mosquito cells contained mutations mapping to a single stem-loop structure, which was found tobe duplicated in mosquito-borne aviviruses. Using recombinant viruses and evaluation of tness parameters, we found that RNA duplication is required to toleratemosquito-adaptive mutations for replication in mammalian cells. Our ndings provide a novel model supporting the role of RNA duplications for efcient dengue virusreplication in multiple hosts.

THE LOVE-HATE RELATIONSHIP BETWEEN HOST PROTEINS AND DENGUE VIRUS RNAS1 2,3,4,5 1,2 1 3,4 3,4Mariano A. Garcia-Blanco , Katell Bidet , Alex Ward , Brandt Levitt , and Shelton Bradrick

1Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore, 2NUS Graduate School for Integrative Sciences and Engineering, National4University of Singapore, Singapore, 3Center for RNA Biology, Duke University Medical Center, Durham, North Carolina, USA, Department of Molecular Genetics and

Microbiology, Duke University Medical Center, Durham, North Carolina, USA, and 5Department of Medicine, Duke University Medical Center, Durham, North Carolina,USA.Viral RNA-host protein interactions are critical for replication of aviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogensincluding dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infectionand found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products ofseveral interferon stimulated genes (ISGs), and for efcient translation of PKR and IFITM2 mRNAs. This identies G3BP1, G3BP2 and CAPRIN1 as novel regulators ofthe antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic aviviral RNA (sfRNA), which bound to G3BP1, G3BP2and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation forinterferon stimulated gene expression and presents the rst mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells. We alsoexamined the function of ERI3, a host 3'-5' RNA exonuclease, which is required for dengue virus replication. We determined that this enzyme is required, paradoxically,to stabilize viral RNAs. In addition to presenting mechanistic data on the role of ERI3 in dengue virus infection we will present the results of our high throughput screenfor ERI3 inhibitors and their action as anti-dengue compounds.


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DEVELOPMENT OF AN IMINOSUGAR-BASED THERAPEUTIC FOR DENGUE VIRAL INFECTION1 3 1,2 2 3 1A Sampath , E. Plummer , K. Wareld , S. Enterlein , S. Shresta ; U. Ramstedt

1 2 3Unither Virology, Silver Spring, MD, IBT, Gaithersburg, MD, LIAI, La Jolla, CA.

Unither Virology has identied a lead iminosugar candidate, UV-4B for the treatment of Dengue viral (DENV) infection. UV-4B shows in vitro antiviral activity against a llfour DENV serotypes, including clinical isolates, when tested in a yield reduction assay. In vivo, UV-4B has shown efcacy in an antibody dependent enhancementmodel of DENV infection. In this model, the minimal effective dose for UV-4B was determined to be 20 mg/kg/dose, TID, via oral gavage with signicant protectionobserved when administered as late as 24-48 hours post infection.The proposed mechanism of action of UV-4B is that it inhibits the enzymatic activities of ER -glucosidases. Since the proposed mechanism of action is via a hostglycosylation pathway that affects infectivity and virus particle assembly, it is anticipated that development of viral resistance to UV-4B will be less likely to occur thanwith directly acting antiviral agents. In an in vitro study that evaluated the potential for DENV-2 to develop drug resistance to UV-4B, 38 cycles of treatment with UV-4Bdid not demonstrate any clear pattern for causing drug induced resistance when compared to the vehicle control. In vivo, the antiviral effect of UV-4B was sustainedthrough 5 passages of DENV-2 virus in a STAT1x2 knockout mouse model. In vitro and in vivo safety pharmacology studies have been conducted. In vitro assessmentsof protein binding, blood-to-plasma distribution, metabolic stability and inhibition potential of human cytochrome P450 enzymes have been performed. Thepharmacokinetic characteristics of UV-4B have been studied in mice, rats, and dogs. The toxicological prole of UV-4B has been evaluated in mice, rats, and dogs

have been found acceptable to proceed to rst in human (FIH) studies. An investigational new drug application (IND) for a FIH study has been approved with clinicaltesting initiated mid-2014. UV is also planning Phase 2 clinical trials to demonstrate efcacy of UV-4B in DENV infection. Acknowledgements: The project wassupported by Award from the National Institute of Allergy and Infectious Diseases, HHSN272201100030C

ABSTRACTS ORAL PRESENTATIONS

VIROLOGY/ANTIVIRALS

SINGLE POINT MUTATIONS IN THE HELICASE DOMAIN OF NS3 ENHANCE DENGUE VIRUS TYPE 1 REPLICATION AND BYPASS TYPE I IFN RESPONSE IN HUMANMONOCYTE-DERIVED DENDRITIC CELLS

Guilherme Ferreira Silveira, Daisy Maria Strottmann, Luana de Borba, Daniel Santos Mansur, Juliano Bordignon, Claudia Nunes Duarte dos Santos

Dengue is one of the most prevalent arboviral disease worldwide, with 2.5 billion people living in areas under risk of infection. Although dengue pathogenesis is not yetcompletely understood, several factors seem to be associated with dengue disease and the outcome of the infection is determined by the interplay of viral and hostfactors. In this study we evaluated the infection prole of two recombinant strains of dengue virus serotype 1 (DV-1) carrying single point mutations at NS3hel protein

L435S or L480S, in human monocyte-derived Dendritic Cells (mdDCs), targets for DV infections. Both mutant viruses could infect and replicate more efcientlyproducing higher viral progeny in infected mdDCs, compared to parental non-mutated virus (vBACDV1). Mutated DVs also induces a differential gene expressionresponse on mdDCs compared to vBACDV1 virus, with up-regulation of IFN signaling and pattern recognition receptors (PRR) canonical pathways. mdDCs infectedwith DV-1 NS3 mutated viruses expressed higher levels of TLR3 and 7 mRNAs and induce a pronounced production of type I IFN compared to parental virus. The type IIFN produced by the mdDCs can reduce infection rate, especially in DV-1 NS3 mutated strains. These results suggest that the type I IFN function was effective toreduce but not sufcient to prevent viral replication of mutated strains and demonstrate that single point mutations in NS3hel protein of DV-1 are sufcient to modifythe viral tness and bypass the type I IFN function in human primary cells.

DENGUE VIRUS COUNTERACTS THE CGAS/STING/IRF3 PATHWAY IN INFECTED HUMAN CELLS.1 1,2 1 1 1,2 1,2Sebastian Aguirre , Jenish Patel , Ana Maestre , Dabeiba Bernal-Rubio , Adolfo Garcia-Sastre , and Ana Fernandez-Sesma .

1Department of Microbiology and the Global Health and Emerging Pathogens Institute, Icahn Mount Sinai School of Medicine and Icahn Mount Sinai Graduate School of2Biomedical Sciences , New York, NY, USA

Dengue virus (DENV) has become a global threat infecting near to 390 million humans per year. To establish a productive infection in humans, DENV needs to

counteract the host innate immune system, particularly the type I interferon (IFN) system. It was demonstrated by our group and others that DENV interferes with both,the production and signaling of type I IFN pathways through the expression of viral proteins that specically target adaptor molecules involved in these essential hostresponses to pathogens. We showed that the DENV protease complex NS2B3 is able to interact with and cleave the adaptor molecule STING, inhibiting the activation ofIRF3 and subsequent type I IFN induction in infected cells. A recent report showed an anti avivirus activity of the newly described PRR cGAS, which after activationgenerate a second messenger (cGAMP) that in turn activates the adaptor STING and the subsequent induction of type I IFN. In order to characterize the role of cGASduring DENV infection we evaluated the ability of this protein to be activated and induce type I IFN during DENV infection in primary cells. We also investigated the roleof the NS2B3 protease complex in the inhibition of type I IFN induction induced by cGAS. We observed that DENV infection inhibits the sensing of cytosolic DNA indendritic cells. This new mechanism of inhibition by interfering with the cGAS/STING/IRF3 pathway is dependent of the DENV protease complex NS2B3. Furthermore,silencing of cGAS in human dendritic cells (DCs) resulted in a higher accumulation of DENV RNA after infection. These results suggest an active role of cGAS as asensor during DENV infection and conrm the role of DENV protease as a master regulator of the type I IFN response in DENV infected cells.


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DENGUE VIRUS CAPSID PROTEIN ABILITY TO TRANSLOCATE NUCLEIC ACIDS ACROSS MEMBRANES: IMPLICATIONS IN VIRUS ENTRY INTO HOST CELLS1 2 1 3 3 2 1Christine Cruz-Oliveira , Joo Miguel Freire , Thas M. Conceio , Daisy M. Strottmann , Claudia N. Duarte dos Santos , Miguel A. R. B. Castanho , Andrea T. Da Poian

Instituto de Bioqumica Mdica Leopoldo de Meis, UFRJ, Rio de Janeiro Brasil; Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal.Instituto Carlos Chagas Filho, Fiocruz-PR, Curitiba, BrasilFor all enveloped viruses, genome access to the cytoplasm depends on the fusion of viral envelope with a cellular membrane, a process triggered by a viral fusionprotein anchored at the virion envelope. In the case of dengue virus (DENV), it is currently established in the literature that, despite the different entry routes of viralparticle internalization, genome release into the cytoplasm occurs through envelope (E) protein-mediated membrane fusion. However, based on recent results, wehypothesize that DENV capsid (C) protein may cooperate with E protein during the fusion stages. We characterized the capsid proteins of all aviviruses assupercharged proteins, which are proteins with a high net charge/molecular weight (MW) that are able to efciently penetrate cells and deliver functional cargoes. Wefound that recombinant DENV C protein, as well as two synthetic peptides comprising C protein residues 45-72 and 67-100, were able to translocate shortoligonucleotide sequences (ssDNA and siRNA) and large nucleic acids (GFP-encoding plasmid) across cellular membranes. The GFP-encoding plasmid as well as thesiRNAs were not only transported into the cells, but also expressed efciently. In addition, DENV 2 C protein was able to internalize DENV 1 RNA (derived from DENV1infectious clone vBACDV1) in hepatic cells. After C protein-mediated transfection, DENV1 RNA expression was monitored using antibodies against DENV E proteinthrough microscopy and ow citometry studies. Besides efcient viral RNA expression and translation, transfection of DENV1 RNA with C protein promotes virus

assembly, since infectious DENV particles were detected in the medium at 96 hours post transfection. Taken together, these results reinforce the role of C protein inviral genome release into the cytoplasm.

ASSESSING DENGUE VIRUS-INDUCED CHANGES IN GENE EXPRESSION PROFILES VIA RIBOSOME PROFILING1 1 1 1 1 2Mariana Leguia , Diana Juarez , Michael Torres , Eric S. Halsey , Daniel G. Bausch , Anton Vila-Sanjurjo .

1 2U.S. Naval Medical Research Unit No. 6, Universidade de A Corua, PeruA majority of the research on dengue (DENV) virus-induced changes in gene expression has focused on the role of the adaptive immune response, which is undeniablyimportant. However, epidemiological data suggest that the host's genetic background may also contribute important susceptibility factors that could exert their effectin a manner independent from the adaptive immune response. Cellular responses with the potential to make a difference between life and death outcomes areultimately mediated by the actions of proteins encoded in the genome. Thus, an understanding of differential global gene expression at the proteome level is essentialto understand how DENV infection can result in dramatically different disease outcomes. Ribosome proling is a new technique that enables direct measurements ofprotein expression at the whole cell level. In so doing, it generates all the information needed for a comprehensive understanding of how global gene expression mayinuence particular disease phenotypes. We have recently completed the rst ribosome proling-based study of DENV-2 infected human cells. Our results indicate

that ribosome proling is a powerful tool to study changes in cellular dynamics upon DENV infection. Specically, we are able to pinpoint differentially regulated genesand corroborate previously identied putative predictors of disease progression. As a whole, these data sets elucidate differentially regulated genes in the context ofthe host's genetic background. Furthermore, this approach has the potential to enable discovery of genes not previously associated with particular disease states, andin so doing, lead to the development of improved vaccines, diagnostics and therapeutics.

DENGUE-3 VIRUS (DENV-3) ENTRY INTO THE HOST CELL: MECHANISM AND ANTIVIRAL TARGET FOR HEPARAN SULFATE-MIMICKING AGENTS

Elsa B. Damonte, Luana E . Piccini, Viviana Castilla.Laboratorio de Virologa, Departamento de Qumica Biolgica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN- CONICET, 1428Buenos Aires, ArgentinaDengue virus (DENV) entry into the host cell represents an attractive antiviral strategy for chemotherapy to suppress the beginning of infection. However, it appears tobe a very complex process regulated by several cell- and virus-dependent factors that may affect antiviral susceptibility. Here, we studied rst the mode of entry ofDENV-3 strain H87 into the host cell by analyzing the effect of pharmacological and molecular inhibitors of different endocytic pathways on viral infectivity and antigenexpression. Together, the results obtained demonstrated that DENV-3 entered mosquito C6/36 cells by clathrin-mediated endocytosis whereas the entry into monkey

Vero and human A549 cells occurred via a clathrin-independent, dynamin and acid pH-mediated route with a partial involvement of caveolae. The kinetics of viralentry, analyzed by using a resistance to ammonium chloride assay, indicated that virion penetration started at 5 min post-binding and the time for viral escape fromendosomes was about 15 min. The antiviral susceptibility of DENV-3 to entry inhibitors was next analyzed in both Vero and A549 cells. Diverse classes of sulfatedpolysaccharides (SP), compounds mimicking the cellular heparan sulfate residues, exhibited a potent and selective antiviral activity against DENV-3. Carrageenanswere the most active inhibitors with effective concentration 50% (EC50) values around 1 g/ml and selectivity indices (ratio cytotoxicity/antiviral activity) higherthan 1000. The target of these compounds was DENV-3 entry by dual blockade of virus adsorption and internalization of the nucleocapsid from endosomes.The comparison with our previous studies on Vero cell entry with DENV-1 (clathrin-mediated endocytosis and resistance to SP) and DENV-2 (non classical clathrin-and caveola-independent pathway and susceptibility to SP) conrms that diverse viral and cellular factors affect DENV entry and should be considered for evaluationof safe antiviral agents reactive against all DENV serotypes.


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IDENTIFICATION OF TERPENES INHIBITORS OF DENGUE VIRUS1 2 2 1Camila Flechas-Alarcn , Nerlis Pjaro-Castro , Jesus Olivero-Verbel , Sindi Velandia-Cruz1, Raquel Ocazionez-Jimenez .

Centro Nacional de Investigaciones para la Agroinsdustrializacin de Especies Vegetales Aromticas y Medicinales Tropicales (CENIVAM), 1Laboratorio de Arbovirus,Centro de Investigaciones en Enfermedades Tropicales (CINTROP), Universidad Industrial de Santander, Bucaramanga, Colombia. 2Grupo de Qumica Ambiental yComputacional, Facultad de Ciencias Farmacuticas, Universidad de Cartagena, Cartagena, Colombia.

Introduction - An antiviral drug is not currently available for treatment of dengue. Essential oils from aromatic plants can inhibit dengue virus infection and it has beenattributed to its chemical constituents. Hence, the identication of terpenes inhibitors of the virus could contribute to discovery of anti-dengue drugs. Methods - Theconcentration that inhibited 50% (IC50) DENV-2 replication was determined by using a virus-HepG2-based assay measuring the NS1-protein level. Potentialcytotoxic effects (CC50 values) were evaluated in ve human cell types by using MTT assays. Docking of the compounds in viral proteins was performed usingAutoDock Vina. Identication of protein residues interacting with the compound was carried out with LigandScout 3.0 software. Results - -Caryophillene, Citral andEstragole were identied as inhibitors of DENV-2 replication at IC50 of 22.5 5.6, 31.3 4.5 and 30.0 1.0 M, and with a SI value of 71.7, 8.0 and 6.7,respectively. Time-of-drug-addition studies revealed that the compounds act at the very early stages of the viral replication cycle. Preincubation of high-titer stocks ofvirus with compounds at the highest non-toxic concentration did not resulted in inactivation of the virus. (R)-(-)- and (S)-(-)-Carvone, Geranyl-acetate, Nerol,Carvacrol, p-Cymene, Thymol and-Mircene did not exhibit promising antiviral activity. All compounds were not cytostatic for human cells derived from kidney, liverand skin (CC50 between 200 and 902 M). The afnity scores for the compounds on C, prM-E, E, NS3-NS2B and NS5 viral proteins varied between -4.0 and -7.3kcal/mol. -Caryophyllene interacting with NS3-NS2B had the highest afnity (-7.2 kcal/mol). Conclusion - Promising terpenes inhibitors of dengue virus wereidentied which could be used as a starting point for consideration in designing effective drugs for dengue. This research was carried out thanks to nancial supportfrom the Colombian Institute of Science and Technology (Colciencias), Grant RC-245-2011 (Patrimonio Autnomo del Fondo Nacional de Financiamiento para laCiencia, Tecnologa e Innovacin, Francisco Jos de Caldas).

DENGUE VIRUS MATURITY DETERMINED BY MASS SPECTROMETRY AND INFLUENCE OF PRM CONTENT ON VIRUS BIOLOGY1, 2 2 2 1 3 1,2Devika Sirohi , Victoria Hedrick , Ernesto S. Nakayasu , Valorie D. Bowman , Catherine P. Riley & Richard J. Kuhn

1 2 3Markey Center for Structural Biology, Dept. of Biological Sciences, Bindley Bioscience Center, Purdue University, West Lafayette, IN, USA; Pathology AssociatesMedical Laboratory, Coeur d'Alene, ID, USA

Maturation of Dengue Virus (DENV), driven by structural rearrangements of Envelope (E) protein and furin protease-mediated cleavage of the precursor membrane(prM) protein, is required for particle infectivity. However, studies indicate that furin cleavage is inefcient in DENV compared to other aviviruses. As a consequence,DENV preparations are structurally diverse and comprise a continuum of particles with variable maturity from immature 'spiky' (180 prM/0 M) to fully mature 'smooth'

particles (0 prM/180 M). The presence of prM on particles alters the representation and accessibility of epitopes and inuences virus-antibody interactions, perhapsunfavorably for the host. Antibodies against prM exhibit weak neutralization activity and can potentially enhance infection. In fact, levels of prM antibody in patientshave been shown to have a positive correlation with disease severity. While the signicance of prM in disease pathogenesis is clear, the prM content of DENV virionsand how it modulates infectivity and disease outcome remains unknown. One of the bottlenecks is the lack of a robust method for quantitative determination of prMcontent on virions. We have developed mass spectrometry based sensitive assays for measuring the maturity of DENV virions. These are being used to probe the prMcontent of DENV produced in cell culture and we are developing protocols to study virus produced from natural infection of mosquitoes (vector) and humans (host). Inparallel, cryo-electron microscopy has been employed to provide a physical assessment of virus particles under analysis. Using these methods, we have comparedthe efciency of cleavage among different serotypes of DENV and are identifying factors that modulate the cleavage efciency. This presentation will focus on themethods developed for estimating DENV maturity, its applications in vaccine development, serotypic differences in maturity and our future efforts geared towardsestablishing a correlation between prM cleavage efciency and protective versus enhancing antibody response.

HIGHLIGHTED POSTERS

VIROLOGY/ANTIVIRALS

HP01Dengue virus inhibits miR-133a expression through its 3'UTR but miR-133a overexpression blocks dengue virus replication1 1 1,2 1 1,3 2 1Jorge Andrs Castillo , Juan Camilo Castrilln , Mayra Diosa-Toro , Juan Guillermo Betancur , St Laurent G 3rd , Jolanda M Smit , Silvio Urcuqui-Inchima

1 2Grupo Inmunovirologa, Facultad de Medicina, Universidad de Antioquia; Department of Medical Microbiology, University Medical Center Groningen, University of3Groningen, The Netherlands. St Laurent Institute, 9 Lewis St, Providence, RI 02906, USA

Dengue virus (DENV) is the most common vector-borne viral infection worldwide with approximately 390 million cases and 25,000 reported deaths each year. Despitethe high economic and social impact of DENV, neither vaccine nor antiviral drug therapy exists for any of the four dengue serotypes. MicroRNAs (miRNAs) are non-coding small RNA molecules responsible for the regulation of gene expression by repressing mRNA translation or inducing its degradation. miRNAs have beendescribed to poses antiviral activity towards several mammalian-infecting viruses. However, the role of miRNAs during DENV replication is not yet understood andwell-studied. Here, we analyzed miR-133a expression in Vero cells during DENV infection and we observed a rapid down-regulation of miR-133a early in infection. The3'UTR of DENV participates in this process by altering the expression of proteins involved in the miRNA biogenesis pathway. In addition, we observed that reduced


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levels of miR-133a augment expression of the polypyrimidine tract binding protein during the rst 12 hours after infection. Furthermore, transfection of Vero cells with

mature miR-133a was found to inhibit DENV replication, while inhibition of miR-133 expression with miR-133a antisense mimics, increased the viral replication.Taken together, our results indicate that miR-133a has anti-DENV activity. To the best of our knowledge, this is the rst study demonstrating that a cellular miRNA hasanti-DENV activity.

HP02

The heterogeneous nuclear ribonucleoprotein K is a cell factor required for dengue virus type 2 multiplication

Viviana Castilla, Jess E. Brunetti, Luis A. ScolaroLaboratorio de Virologa, Departamento de Qumica Biolgica, Facultad de Ciencias, Universidad de Buenos Aires, Argentina

Heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and K are cellular RNA-binding proteins that participate in mRNA splicing, trafcking and translation andhave been implicated in the multiplication of several cytoplasmic RNA viruses. To identify cellular factors involved in dengue virus (DENV) replication, here wecharacterized hnRNP A2 and K expression in Vero and A549 cells infected with DENV type 2 and we evaluated the effect of hnRNP K silencing on DENV-2 multiplication.We analyzed hnRNP intracellular localization by indirect immunouorescence assays, using anti-hnRNP and anti-viral glycoprotein E antibodies, at 2 or 5 days post-infection (dpi). Whereas hnRNP A2 showed a clear nuclear localization, similar to that observed in uninfected cells, the cytoplasmic translocation of hnRNP K was

evident at 5 dpi in both cell types. We also performed A549 cell transfection with a plasmid that allows the expression of T7-tagged hnRNP K and the re-localization oftransiently overexpressed hnRNP K to the cytoplasm was observed at 2dpi. The analysis by western blot showed no signicant differences in the total levels of hnRNPK expression between uninfected and DENV-2 infected Vero or A549 cells. Finally, A549 cells were transfected with small interfering RNAs (siRNAs) that target hnRNPK or small non-interfering RNA used as control, and at 24 h post-transfection cells were infected. The effectiveness of siRNA silencing was corroborated by westernblot and at 1 and 5 dpi, virus yield and the amount of glycoprotein E expressing cells were assessed by plaque assay and immunouorescence technique, respectively.A signicant reduction of 66.6% and 96.0% of virus yield was achieved at 1 and 5 dpi, respectively, and the amount of uorescent cells was also inhibited. The resultsobtained indicate that DENV-2 infection alters hnRNP K intracellular distribution and this hnRNP would play an important role in DENV-2 multiplication in cell cultures.

HP03

MicroRNA profiling associates miR-21 and miR-146a with dengue virus-induced apoptosis and replication in human hepatoma cells1 1 1,2 1Antonio Gregorio Dias Junior ; Daniele Barbosa de Almeida Medeiros ; Ana Ceclia Ribeiro Cruz ; Mrcio Roberto Teixeira Nunes ; Evonnildo Costa Gonalves3;

3 1 1,2Antnio Rosrio Carlos Vallinoto ; Edna Cristina Santos Franco ; Pedro Fernando da Costa Vasconcelos21Instituto Evandro Chagas/SVS/MS - Seo de Arbovirologia e Febres Hemorrgicas - Ananindeua, Par, Brazil. Universidade do Estado do Par - Belm, Par, Brazil

3Universidade Federal do Par - Belm, Par, Brazil

Dengue virus (DENV 1-4) is a mosquito-borne agent recognized by its impact on public health in tropical and sub-tropical countries. Infected individuals may presentsevere forms of the disease, namely dengue hemorrhagic fever and dengue shock syndrome. These are often associated with a subversion of the innate immuneresponses (e.g. due viral proteins inhibition of interferon - IFN - type I pathway; antibody dependent enhancement; host polymorphisms, etc.) along with aninammatory cytokine storm and organ impairment, where apoptotic cells might be found. To exploit possible mechanisms involved in these events, we infected IFN-decient Huh7.5 and IFN competent HepG2 human hepatoma cell lines with DENV2. In comparison, Huh7.5 produced the highest virus titers with caspase 3production and decreased cellular viability over time. In addition, an microRNA proling revealed miR-21 and miR-146a were upregulated at 48 hours post-infection(hpi) in Huh7.5 cells. Hence, we show DENV replication affects cellular microRNA proling in an IFN-decient environment, concomitant with peaks of detectableinfectious particles and caspase 3 detection.

HP04

Effect of dengue virus infection on calcium homeostasis in hepatic cultured cells1 2 3 1 4 2 1 1Cinthia L. Dionicio , Franshelle Pea ., Eda Benitez , Fernando Medina , Jos L. Zambrano , Marie C. Ruiz , Rosa M. Del Angel , Juan E. Ludert

1 3Department of Infectomics and Molecular Pathogenesis and Department for Biochemistry, Center for Research and Advanced Studies (CINVESTAV), Mexico City,Mexico; 2Center for Biochemistry and Biophysis and 4Center for Microbiology and Cell Biology, Venezuelan Institute for Scientic Research (IVIC), Caracas, Venezuela.email: [email protected]

Dengue is the mosquito-borne viral disease most important to humans. It produces a wide range of clinical conditions ranging from mild forms (dengue fever) to thehemorrhagic forms, known as severe dengue (DS). The dengue virus (DENV) belongs to the family Flaviviridae and 4 serotypes have been described. Ca2+ is asecond messenger controlling many intracellular processes in mammalian cells. Substantial evidence exists indicating that Ca2+ plays an important role in thereplication cycle of several viruses, including some Flavivirus. However, the importance of Ca2+ during the replication cycle of DENV is unknown. This study aims toexplore the possible involvement of Ca2+ and various mediators of this ion levels during DENV infection. Experiments carried out in Hep-G2 cells infected with DENV-4 (MOI=3) and loaded with Fura-2, showed that infection signicantly (p0.005) increased the permeability of the plasma membrane to Ca2+, and causes a


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signicant (p0.005) reduction in the levels of Ca2+ releasable from the endoplasmic reticulum, at 12 and 18 hpi. In addition, we observed an increase in the mRNA

levels of the proteins involved in capacitive entry, STIM1 and Orai1, at 18 and 24 hpi. However, WB analysis of Orai1 and STIM1 protein levels showed no changes in thelevels of these proteins along infection. The results suggest that the observed increase in plasma membrane permeability is due to an increase in the activation ofchannels, rather than an increase in protein production. Finally, it was observed that manipulation of the intra and extracellular levels of Ca2+ in infected Huh-7 cellsthrough treatments with Ca2+ chelators, such as BAPTA-AM and EGTA, reduced viral particle production in more than 1 log (p


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necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with

antioxidant and anti-inammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for24 and 48h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated withdifferent concentrations of NDGA. NDGA treatment signicantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment alsoled to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number duringDENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection bytargeting genome replication and viral assembly.

POSTERS SESSION VIROLOGY

V-P01

THE DENGUE VIRUS NON-STRUCTURAL PROTEIN 1 (NS1) INTERACTS WITH THE HUMAN RIBOSOMAL PROTEIN L18 IN HUH-7 INFECTED CELLS

Margot Cervantes-Salazar, Ariana Hurtado-Monzn, Antonio ngel-Ambrocio, Rubn Soto-Acosta, Patricia Bautista-Carbajal, Juan E. Ludert, 1Rosa Mara del ngel1Departamento de Infectmica y Patognesis Molecular. Centro de Investigacin y de Estudios avanzados del instituto politcnico Nacional (CINVESTAV-IPN), MxicoCity, MxicoThe persisting epidemics of dengue disease in the world as well as the increasing number of cases, especially of the severe forms of the infection, have augmented theinterest in the study of dengue virus (DENV) replicative cicle. The austerity of the viral genome makes it dependent on the participation of differents molecules andorganelles from the host for entry and replication. The NS1 protein plays important roles in viral replication and pathogenesis, however, the function of NS1 are not fullyunderstand. Thus the amein of this work was to identify proteins that interact with NS1 in infected cells. Using afnity chromatography and immunoprecipitationassay, approaches a total of 64 cell proteins were isolated and identied by mass spectrometry (MALDI-ToF MS / MS) from Huh-7 cells. Using the string 9.1 data basesit was found that many of them are functionally related. One of the proteins identied was the ribosomal protein L18. The L18 protein is part of the large ribosomalsubunit and it has been described that it is involved in translation initiation. Western-blot and confocal microscopy analysis indicated that the expression of L18protein is not altered during DENV infection, however L18 protein silencing by siRNA inhibited viral infection determined by viral yiel, NS1 secretion and qRT-PCRwithout any effect in total cellular protein synthesis. Additionally, silencing of L18 in Vero cells, stable trasfected of DENV 4 replicon, induce an inhibition in luciferaseactivity. All these results suggest that the ribosomal protein L18 is involved the replicative cycle of DENV.

V-P02

CALMODULIN INHIBITION REDUCES DENGUE VIRUS REPLICATION IN HEPATIC CELLSPatricia Bautista-Carbajal, Rubn Soto-Acosta, Margot Cervantes-Salazar, Antonio H. Angel-Ambrocio, Juan E. Ludert, Rosa Ma. del ngel11Departamento de Infectmica y Patognesis Molecular. Centro de Investigacin y de Estudios Avanzados del Instituto Politcnico Nacional (CINVESTAV-IPN), MxicoCity, MxicoDengue is the most important mosquito borne-viral disease in global public health.The virion contain an RNA genome of positive polarity that encodes 3 structural and7 nonstructural proteins. DENV replication occurs in close association with membranes, in particular derived from the endoplasmic reticulum (ER). The association ofthe replication complex to the ER has focused our attention on calcium sensor proteins such as calmodulin (CaM). This protein can turn on approximately 30 systemswith different targets, including calcium extrusion pumps and translate modest increases of intracellular Ca2+ in physiological responses. The aim of this work wasto analyze if CaM may have a direct role during dengue virus replicative cycle. Western blot analysis indicated that CaM levels increased up in Huh7 cells at 48 hourspost infection. Afterwards, infected cells were treated with the CaM antagonist W7 (25 M) for 48 hours after infection. Cell treatment resulted in a decrease in viralyield by 1 log. The reduction in viral yield was accompanied by a reduction in mRNA levels and NS1 secretion. In addition, confocal microscopy and ow cytometryanalysis of treated cells showed a reduction by about one third in the percentage of infected cells. Interestingly, confocal microscopy analysis of treated cells indicatedthat the expression levels of NS3 are reduced and the intracellular distribution of the E protein altered. Finally, W7 treatment of a VeroR cell line, stably transfected witha DENV4 replicon results in a reduction in luciferase activity used as an indicator of translation and replication. These results suggest the CaM is necessary for dengue

virus replication and presumably play a role in viral translation and replication.

V-P03

BIOLOGICAL ASPECTS OF THE INFECTION BY WMEL AND WMELPOP STRAINS OF WOLBACHIA IN BRAZILIAN AEDES AEGYPTI GENOTYPE1 2 1Heverton Dutra , Rafael de Freitas , Luciano Moreira

1 2Mosquitos Vetores: Endossimbiontes e Interao Patgeno Vetor, Centro de Pesquisas Ren Rachou - Fiocruz, Belo Horizonte, MG; Laboratrio de Transmissores deHematozorios, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ; BrazilWolbachia are obligate, intracellular, maternally-transmitted bacterial endosymbionts infecting around 70% of all known arthropods, causing a variety of biologicaleffects including "reproductive parasitism", which favors it spread in wild populations. Aedes aegypti do not harbor Wolbachia, but when wMel and wMelPop strainswere independently inserted into this species, they signicantly reduced the mosquito vectorial competence for DENV. Wolbachia has been trialed as a novel strategyfor reducing dengue incidence (ED = www.eliminatedengue.org). However, Wolbachia-based control will only be effective if the infection generated in the laboratory is


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able to xate into wild mosquitoes. Here we evaluated the effects of wMel and wMelPop strains on key biological parameters of recently backcrossed Brazilian A.

aegypti, in preparation for eld releases, reared under different stress conditions. For both strains, there was signicant difference in larval development time only inhighest stress condition, where infected individuals developed more rapidly than uninfected, regardless of sex. Regarding wMel infection, we observed severalphenotypic changes, ranging from no effect at all (fecundity, fertility), to partially detrimental (longevity and body size in highest stress condition), and partiallybenecial (increased levels of glycogen under the highest stress condition). About infection levels, there was generally, a higher bacterial concentration in the Brazilianmosquitoes over time for wMel strain compared to Australian mosquitoes, exhibiting maternal transmission and cytoplasmic incompatibility rate of 96% and 99,51%respectively. For wMelPop, there were benecial phenotypic effects: larval development time was lower in infected individuals, which showed an increased body sizeand glycogen content in highest stress condition compared to uninfected females. In conclusion, we highlight key phenotypic effects of Wolbachia on the biology ofBrazilian A. aegypti. We also think it contributes with relevant information regarding the benecial effects of wMel strain, being suitable for application in the eld,possibly controlling dengue outbreaks in Brazil.

V-P04

STRUCTURAL AND MOLECULAR CHARACTERIZATION OF DENV2 CAPSID PROTEIN AND ITS MUTANTS1 1 1 1Janaina Figueira Mansur , Renata Morgado Pereira , Rafael Mesquita Stoque , Ronaldo da Silva Mohana Borges

1

Laboratrio de Genmica Estrutural - Instituto de Biofsica Carlos Chagas Filho - Universidade Federal do Rio de Janeiro- Rio de Janeiro- Brazil.The process of DENV nucleocapsid assembly is not well understood. There are some studies that showed the presence of capsid protein at the cytoplasmic side of theendoplasmic reticulum (ER) membranes and that some substitutions and deletions at hydrophobic regions of capsid protein can disrupt its association withmembranes, suggesting that capsid-membrane interaction is mediated by conserved regions among residues 45 to 65. Furthermore, it was also shown that in theabsence of capsid protein there is production of subviral particles without RNA. These ndings suggest that the production of infectious viral particle is dependent onthe interaction between viral genome and capsid protein associated with ER membranes. The comprehension of the fundamental processes of the viral infection cyclecan provide potential targets for design of antiviral drugs. In this context, the present work aims to study the molecular and structural aspects of capsid protein andcharacterize its interaction with membranes. DENV2 capsid protein is a highly basic protein, composed exclusively of alpha-helix, that has about 12 KDa and isdimeric in solution. In this work, we have cloned, expressed and puried ve mutants of capsid protein (L81N, I88N, L50S, L54S, L50S/L54S). The oligomeric state ofthese proteins was analyzed by gel ltration chromatography and they showed to be dimeric as the wild type protein. L81N and I88N mutants were also analyzed byEGS cross-linking assay and the results corroborate with those obtained in the chromatography. The secondary structure of L81N and I88N were studied by circulardichroism and presented alpha-helix structure; however, the I88N mutant showed loss of secondary structure. Stability of these two mutants was analyzed by ureadenaturation assays monitoring the tryptophan uorescence. L81N and I88N mutants were much less stable than wild type, suggesting that hydrophobic residues atthe alpha-helix 4 are important to protein stability.

V-P05

DENGUE VIRUS INTERACTOME: INTERACTION OF NONSTRUCTURAL PROTEIN 5 (NS5) AND HUMAN PROTEINS1 1Estefania A. Aguilera1, Emiliana M. Silva , and Ronaldo Mohana-Borges

1Laboratrio de Genmica Estrutural- IBCCF - Universidade Federal do Rio de Janeiro, RJ, Brazil

Among DENV nonstructural proteins, NS5 is the most conserved protein in DENV serotypes. It is a bifunctional enzyme that contains the S-adenosyl methioninetransferase and RNA-dependent RNA polymerase (RdRp) catalytic domains. Although all suggested functions of NS5 are generally to occur in the cytosol NS5 hasbeen located in the cell nucleus. There is no role for nuclear localization of dengue virus NS5, however the interaction study of NS5 and cellular proteins it's reallyimportant for pathogenesis understanding of DENV infection. Thus, the objective of this work is to verify the role of NS5 and its RdRp and MTase domains on therecruitment and interaction with host proteins during the replicative cycle, since we believe that the understanding of this process is essential to development ofantiviral treatments and new therapies. In this study, we employed the Matchmaker Gold Yeast Two-Hybrid System to identify human proteins that interact with NS5protein. Y2HGold yeast cells were transformed with the pGBKT7-NS5 construct using lithium acetate method and mixed with cDNA human library provided in yeastY187, together, they mate to create diploids cells that contain four reporter genes: HIS3, ADE2, MEL1, and AUR1-C, that are activated in response to two-hybridinteractions. As result we screened 132 putative interactions between DENV NS5 protein and human proteins. We demonstrated that NS5 interacts with several

proteins that have diverse functions; including metabolic, regulatory, transcriptional factors and cytoskeletal proteins. Among the screened proteins our resultsshowed that NS5 interacts with tumor necrosis factor receptor superfamily member 6, cyclin-D-binding Myb-like transcription factor 1 and centromere protein F. Thenext step of this work will conrm some of these putative interactions by co-immunoprecipitation, pull-down, co-immunolocalization and mass spectrometry andthen investigate the role of these interactions in viral replication and pathogenesis.

V-P06

VERTICAL TRANSMISSION OF DENGUE VIRUS IN AEDES AEGYPTI CAPTURED IN PUERTO IGUAZ, MISIONES, ARGENTINA1 2 2Manuel Espinosa , Sergio Giamperetti , Marcelo Abril1, Alfredo Seijo

1 2Fundacin Mundo Sano, Servicio de Zoonosis, Hospital F.J. Muiz; Buenos Aires, Argentina

In recent years, there have been reports of transovarial or vertical transmission of the dengue virus in Aedes aegypti and A. albopictus. While it is still uncertain howthis can currently impact the incidence of outbreaks - where the typical form of transmission (human with viremia-vector-susceptible human) prevails --, this


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phenomenon could play a crucial role in maintaining the virus in the ecosystem during interepidemic periods, mainly in regions where circulation of the virus is

particularly seasonal in nature. As part of the Entomological Vigilance of Dengue Program implemented by the Mundo Sano Foundation in Puerto Iguaz, MisionesProvince, Argentina, genetic material for serotype DEN 3 was detected in male specimens of A . aegypti captured in the urban area, in the months following the dengueepidemic that affected the Tropic of Capricorn region during the summer-fall of 2009. This event represents the rst evidence of the vertical transmission phenomenondocumented in Argentina. The existence of this alternative way of transmission in a region that is close to the international border, with the presence of A. aegypti andA. albopictus, high human transit due to the great number of tourists visiting the Iguaz Falls National Park, and the history of circulation of the four serotypes of thedengue virus, as well as yellow fever, should be given epidemiological importance in order to stay vigilant regarding the incidence of dengue virus and other relatedavivirus.

V-P07

MODULATION OF DENV-2 REPLICATION VIA BIOCHEMICAL AND GENETIC INHIBITION OF CHOLESTEROL SYNTHESIS1 3 1 1 2 1Bryan L. Owen , Romn Vidaltamayo , Augusto Rojas , Hugo Barrera , Javier Ramos , Ana Rivas

1 2Biochemistry Department, Faculty of Medicine, Nuevo Leon Autonomous University; Infectology Department, Universitary Hospital Jos Eleuterio Martnez,3Monterrey, Nuevo Len; Research Department of Health Sciences at Universidad de Monterrey; Mexico

Pathogenesis of dengue virus (DENV) infection is still, not completely understood. Although Antibody dependent enhancement and immunology related factors havebeen appointed as causative for severe Dengue, several cases are not consistent with this hypothesis, suggesting that other mechanisms could be involved.Cholesterol levels have been reported to regulate DENV replication in vitro and proved to be necessary to successfully complete the viral life cycle. Also, Lovastatintreatment reduced viral RNA on a Dengue replicon system. The aim of this work is to evaluate the effect of cholesterol regulation on DENV replication cycle. Cholesterolsynthesis was biochemically (statins lovastatin, pravastatin and atorvastatin) and genetically (iRNA against HMG-CoA R) inhibited in non-infected and DENV infectedHuH-7 Hepatoma cells. Statin concentration was maintained by applying treatment every 24 h. Supernatant was collected every 24h up to 96h for cholesterol andDENV evaluation and replaced with fresh media containing treatment. DENV replication was evaluated using Anti Flavivirus Group antigen 4G2 and diaminobenzidine(DAB) ourophore. Viral titter was measured with the Plaque formation unit (PFU) assay in BHK-21 cells and cholesterol levels were evaluated using Caymanscholesterol uorometric assay. A reduction in 4G2 antigen was observed in all statin treated cells compared with the untreated control. DENV inhibition percentages forLOV, PRA and ATO were 30%, 40% and 20% respectively at 72h p.i. 12h pre-treatment with statins slightly reduced virus titter while post-infection continuoustreatment result on a marked reduction on DENV infective particles up to 40% for PRA, 30% for LOV and 20% for ATO at 96h p.i. Statin treatment also reducedIntracellular cholesterol levels in a time dependent manner simultaneously to DENV reduction. Genetic inhibition of cholesterol synthesis also reduces DENV titer. Ourresults indicate that cholesterol inhibition causes a reduction of DENV infectious particles and 4G2 aviviral antigen signal. Our results suggest that cholesterol isparticularly involved on DENV later stages of replication.

V-P08

TRANSPLACENTAL TRANSFER PROFILE OF DENGUE VIRUS-SPECIFIC IGG1, 2 1 2 1 1 2 1, 2 2Priscila Castanha , Eduardo J. M. Nascimento , Marli Cordeiro , Robyn Konicki , Guangchao Gu , Claudeir Silva Jr. , Ernesto Marques Jr. , Cynthia Braga

1Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA, USA; 2Oswaldo Cruz Foundation, Aggeu Magalhaes Research Center, Recife, BrazilPlacental transfer of maternal dengue virus (DENV)-specic IgG to the fetus likely plays an essential role in immunity and pathogenesis of DENV infection in infants.We investigated the placental transfer prole of DENV-specic IgG antibodies in maternal and umbilical cord blood sera pairs from parturients who took part of anongoing birth cohort study in Recife, Brazil. Serotype-specic antibody prole was determined by Plaque reduction neutralization test. In-house virus-capture ELISAwas used to estimate the levels of DENV-specic IgG (total as well as IgG1 and IgG4). Antibody titers were log-transformed and the transplacental antibody transfercalculated as ratio (value infant/value mother). From 200 parturient examined, 10.5% did not have detectable neutralizing DENV-specic antibodies to any of theDENV serotypes, 62.2% showed monotypic infection by DENV3, 22.8% the dual infection by DENV3/DENV4 and 4.5% other DENV serotype combinations. Dengue-specic IgG titers in the cord sera were higher than maternal levels (p=0.026), which is consistent with an active transport mechanism across the placenta. Anegative correlation between DENV-specic IgG maternal titers and placental transfer ratio of anti-DENV3 (r= -0.1846, p=0.022) and anti-DENV4 (r= -0.3249,p=0.015) antibodies was found, demonstrating that high levels of maternal anti-dengue IgG antibodies reduce the maternofetal transfer of DENV serotype-specic

IgG antibodies. Additionally, the titers of both isotypes IgG1 (r=0.6944, p


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detection of specic antibodies. Dengue IgA antibodies (Ab) could be a promising approach as an indicator of recent infection, and moreover, as possible marker for

disease severity. The purpose of this study was to determine the presence of dengue IgA-Ab and to correlate it with primary and secondary infection and diseaseseverity. The study population was 59 patients, clinically classied as DHF (n=55) and DSS (n=4) according to the WHO criteria and laboratory conrmed. Twentysamples were classied as primary infection and 39 as secondary infection. Dengue specic IgA-Ab was performed in plasma samples by IgA antibody-capture EIA(VIRCELL, S.L. Spain). The overall percentage of IgA-Ab and IgM-Ab positivity was 60% (n=35) and 86% (n=51) respectively. The presence of IgA-Ab wasstatistically signicant for secondary infections (31/39; 80%) when was compared with primary infection (4/20; 20%) OR=15.5, p=0.0001. Regarding diseaseseverity, 56% of IgA-Ab was found in DHF vs 100% in SSD and 51% of IgM-Ab in DHF vs 47% in DSS. Our ndings are in agreement with other studies, which havereported higher positivity of IgA-Ab detection in samples from secondary infections when compare with primary cases. There is not statistical signicant difference ofIgA-Ab according to disease severity, there is a tendency to nd higher presence of IgA-Ab positivity in DSS than in DHF (OR=7). Further analysis should be done todetermine the utility of IgA-Ab detection as a marker of diagnosis and/or prognosis in dengue disease.

V-P10

EXPRESSION LEVEL EVALUATION OF DGCR8 IN HUMAN CELLS DURING VIRAL INFECTION BY DENGUE VIRUS 41 1 1 1 1 1 1 1Samir Casseb , Karla de Melo , Gustavo Holanda , Bruno Tardelli , Maria Helena Mendona , Ana Celila Cruz , Pedro Fernando da Costa Vasconcelos

1

Instituto Evandro Chagas /SVS/MS, Ananindeua, Par, BrazilDengue virus and its four serotypes (DENV-1 to DENV-4) infect 390 million people and are implicated in at least 25,000 deaths annually, Dengue virus is the mostcommon disease in tropical and subtropical regions. In Brazil, DENV-4 reemerged in 2010 in Boa Vista and Cant municipalities Roraima State. Virus spread to othergeographic regions of Brazil with cases of infection start to be registered in the Northern (Roraima, Amazonas and Par State), Northeast (Bahia, Pernambuco andPiau State) and Southeast (Rio de Janeiro and So Paulo State). The Drosha-DGCR8 complex (Microprocessor) is required for microRNA (miRNA) biogenesis. DGCR8recognizes the RNA substrate, whereas Drosha works as an endonuclease. High-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) wereused to identify DGCR8 RNA targets in human cells. Unexpectedly, miRNAs were not the most abundant targets. DGCR8-bound RNAs also comprised several hundredmRNAs as well as small non-codius RNAs and long non-coding RNAs. In the present study, we investigated the correlate expression of DGCR8 during viral infection byDengue virus 4 in A549 cells. We used the method described by Johnson et al. 2005 for quantication of viral load, together with method for absolute quanticationcurve by RT-qPCR, quantication mRNA used for DGCR8 target and endogenous control RPL38 by RT-qPCR using SYBR Green method. Quantication of mRNA relatedto DGCR8, showed no changes from uninfected cells. Since its reduced expression during viral infection after three days post-infection, and its lower expression on thethird day, might be related with the low expression of DGCR8 with high viral replication rate. The low expression of DGCR8 may help the viral replication process; thepossible low expression of DGCR8 might be related not only with the biogenesis of miRNAs, but possibly to others small RNAs.

V-P11STRUCTURAL STABILITY OF DENGUE VIRUS RECOMBINANT NS1 PROTEIN

Diego R. Coelho, Diego Allonso, Ronaldo Mohana-Borges

Laboratrio de Genmica Estrutural, Instituto de Biofsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ, Brazil.The current literature about Dengue suggests that the recombinant NS1 protein expressed in bacteria (NS1bac) may have almost the same conformation of the wildtype form, except for the post-translational modications. Thus, the aim of this study was to characterize the structural stability of NS1bac in order to compare,hereafter, to the glycosylated protein. First, the NS1bac protein was expressed in E. coli, puried and refolded. Its monomeric population was then puried by sizeexclusion chromatography. The stability of tertiary and secondary structures was analyzed by uorescence spectroscopy and circular dichroism (CD), respectively.The results suggested that the structure of NS1bac is very stable against thermal denaturation, acidic pH, and increasing concentration of urea and guanidinehydrochloride. The secondary structure analysis by circular dichroism also revealed a typical spectrum of -helix and -sheet contents. In order to perform a futurecomparison, it was also developed a protocol for expression and purication of recombinant hexameric glycosylated NS1 protein (NS1mam). The NS1mam waspuried from BHK-21 cell culture transfected with recombinant vector containing the NS1 gene. The preliminary structural analysis also revealed a typical CDspectrum of -helix and-sheet, in agreement with recent crystallographic results published.

V-P12

LIVER INJURY IN EXPERIMENTAL DENGUE VIRUS INFECTION IN CALLITHRIX PENICILLATA1 2 3 3 1Daniele Henriques , Vera Barros , Paulo Castro , Gilmara Silva , Milene Ferreira1 and Pedro Vasconcelos

1 2 3Seo de Arbovirologia e Febres Hemorrgicas, Instituto Evandro Chagas, Ananindeua, Par; Seo de Patologia, Instituto Evandro Chagas, Ananindeua, PA;Centro Nacional de Primatas, Instituto Evandro Chagas, Ananindeua, PA; BrazilDengue viruses are globally the most important arboviruses causing human disease in more than 100 countries, and none vaccine is licensed for them. The lacks of anexperimental model that mimic human disease is of concern, missing information on the disease pathogenesis. This study aimed to evaluate pathological changes inthe liver of marmoset Callithrix penicillata sequentially infected with DENV3 and DENV2 Brazilian isolates obtained from fatal cases. C6/36 cells supernatant wereused to infect 26 marmosets subcutaneously with DENV3 (primary infection-PI) (3.23 x 103 PFU/ml); uninfected control animals (CA) were reserved up the end ofexperiment. Daily for seven days and at intervals of 10, 15, 20, 30, 45 and 60 days post-infection experimented animals were anesthetized and sacriced; secondary


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infection-SI with DENV2 (4.47 x 104 PFU/ml) was performed two months after PI of the remaining 13 non-sacriced animals. Liver of infected and CA animals were

processed for histopathological and immunohistochemical assays. Both PI and SI marmoset livers showed sinusoidal congestion; portal spaces presented discreteinammatory response in most PI liver samples, while for SI the inammatory inltrate was more evident in the parenchyma. In both experiments, the inammatoryinltrate was predominantly mononuclear; foci of hepatocellular necrosis were also observed. Other histological changes sporadically observed at different timeintervals include apoptotic Councilman corpuscles in midzone, hyperplasia and hypertrophy of Kupffer cells, hepatocellular swelling in the lobe areas I and III, andmegakaryocytes in both experiments; focal hemorrhage was only observed in a SI animal showing acute hepatic hemorrhagic failure. Samples of both experimentsshowed mild acute hepatitis. No pathologic changes were observed on CA livers. These ndings were consistent with those described for livers of human fatal dengue,indicating marmosets as suitable model to study dengue pathogenesis and thus may be a useful experimental model for dengue vaccine evaluation.

V-P13

CO-CIRCULATION OF DENGUE VIRUS 1, 2, 3 AND 4 IN SYMPTOMATIC PATIENTS DURING AN OUTBREAK, CONTAGEM, MINAS GERAIS, BRAZIL, 2013

Elisa Helena Paz Andrade, Ana Paula Pessoa Vilela, Julio Cesar Camara Rosa, Leandra Barcelos Figueiredo, Jaquelline Germano de Oliveira, Paulo Cesar PeregrinoFerreira, Claudio Antonio Bonjardim, Erna Geessien KroonUniversidade Federal de Minas Gerais, Belo Horizonte, Brazil

In dengue endemic countries the co-circulation of multiple Dengue virus (DENV) serotypes in the same area has been described. Contagem is a city located in the Stateof Minas Gerais, in the Southest region of Brazil. In 2013, Contagem underwent a dengue epidemic with 23.572 notied cases, of which three patients died. In thisstudy, we report the co-circulation of DENV1-4 in symptomatic patients during this outbreak. A total of 49 acute phase plasma samples from patients clinicallysuspected of dengue admitted to a hospital, located in Contagem, were collected during three weeks of May, 2013. All patients were residents of Contagem orneighboring cities. RNA of DENV serotypes 1, 2, 3 and 4 were detected by RT-semi-nested PCR targeting the C-prM region of its genome. Positive semi-nested PCRsamples were puried and used directly as template in sequencing reactions. Of 49 samples, 33 (67.3%) tested positive for DENV RNA by RT-semi-nested PCR. Ofthese, 20 DNA fragments were sequenced and 8 (40%) were identied as DENV-3, 5 (25%) as DENV-4, 4 (20%) as DENV-2 and 3 (15%) as DENV-1. The dataconrmed the co-circulation of the four DENV serotypes in one outbreak in a short time period. Financial support: CAPES, CNPq, MS/ SCTIE/ Decit/ FAPEMIG/ CNPq/PRONEX Dengue / INCT-Dengue

VP-14

MODULATION OF DENGUE VIRUS INFECTION BY STRESS GRANULES.

Araujo D.F.F., Assuno-Miranda I., Carneiro L.A.

Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.Stress granules (SGs) are cytoplasmic RNA granules that form in response to various types of cellular stress such as heat shock, hypoxia and radiation. Theirformation results in inhibition of translation initiation of new proteins with the phosphorylation of eukaryotic initiation factor elF2, as a major event. The role of SGssuppression in translation and in RNA silencing suggests that such structures may have an impact on viral replication. Several studies have demonstratedinteractions of these structures with several viruses during the course of infection. Recently, it was shown that dengue virus (DENV) infection negatively regulates theformation of SGs. Furthermore, co-localization between viral replication sites and proteins of SGs was also evidenced by immunouorescence, suggesting a possibleinteraction. Thus, we propose to investigate the role of SG formation in the viability of the host cell, the viral replication and assembly of new viral particles in the courseof infection by DENV2. Initially, we evaluated the replication cycle of DENV2 comparing infected wild type cells with infected cells in which no phosphorylation of elF2and consequently no formation of SGs, takes place. The study was performed by comparing cell viability, production of infectious particles, viral replication and theproduction of viral proteins in both cell types. It was found that in cells in which no phosphorylation of eIF2 takes place, there is a decrease in cell viability and in theproduction of infectious particles, despite a higher production of viral mRNA and proteins. Next stages of the project involve the study of the production of inammatorycytokines produced by DENV2 infection such as IL-6, KC and RANTES in both cell types and also to check the type of death induced by the virus and to identify whichstructures present in SGs are important for the completion of the viral life cycle.

V-P15

THE HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN K IS A CELL FACTOR REQUIRED FOR DENGUE VIRUS TYPE 2 MULTIPLICATION

Viviana Castilla, Jess E. Brunetti, Luis A. Scolaro.Laboratorio de Virologa, Departamento de Qumica Biolgica, Facultad de Ciencias, Universidad de Buenos Aires, Argentina.Heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and K are cellular RNA-binding proteins that participate in mRNA splicing, trafcking and translation andhave been implicated in the multiplication of several cytoplasmic RNA viruses. To identify cellular factors involved in dengue virus (DENV) replication, here wecharacterized hnRNP A2 and K expression in Vero and A549 cells infected with DENV type 2 and we evaluated the effect of hnRNP K silencing on DENV-2 multiplication.We analyzed hnRNP intracellular localization by indirect immunouorescence assays, using anti-hnRNP and anti-viral glycoprotein E antibodies, at 2 or 5 days post-infection (dpi). Whereas hnRNP A2 showed a clear nuclear localization, similar to that observed in uninfected cells, the cytoplasmic translocation of hnRNP K wasevident at 5 dpi in both cell types. We also performed A549 cell transfection with a plasmid that allows the expression of T7-tagged hnRNP K and the re-localization oftransiently overexpressed hnRNP K to the cytoplasm was observed at 2dpi. The analysis by western blot showed no signicant differences in the total levels of hnRNP


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K expression between uninfected and DENV-2 infected Vero or A549 cells. Finally, A549 cells were transfected with small interfering RNAs (siRNAs) that target hnRNP

K or small non-interfering RNA used as control, and at 24 h post-transfection cells were infected. The effectiveness of siRNA silencing was corroborated by westernblot and at 1 and 5 dpi, virus yield and the amount of glycoprotein E expressing cells were assessed by plaque assay and immunouorescence technique, respectively.A signicant reduction of 66.6% and 96.0% of virus yield was achieved at 1 and 5 dpi, respectively, and the amount of uorescent cells was a lso inhibited. The resultsobtained indicate that DENV-2 infection alters hnRNP K intracellular distribution and this hnRNP would play an important role in DENV-2 multiplication in cell cultures.

V-P16

VIRAL GROWTH CURVE FOR DETERMINING DAYS OF HARVEST OF DENGUE VIRAL STOCK IN VERO CELLS

Vivian Regina Silveira, Iray Maria Rocco, Rezolina Pereira dos Santos, Adriana Yurika Maeda, Fernanda Gisele da Silva, Terezinha Lisieux Moraes Coimbra, Ana LuciaRodrigues Oliveira, Maria do Carmo Sampaio Tavares Timenetsky.Insituto Adolfo Lutz, Coordenadoria de Controle de Doenas, Sao Paulo, Brazil.

Faced with the need for a dengue vaccine, Adolfo Lutz Institute directs its efforts to implement techniques to measure neutralizing antibodies. According to WHO, thecorrelation between the presence of neutralizing antibodies and virus protection against infection is not absolute and suggests that the Plaque ReductionNeutralization Test (PRNT) is the most widely accepted approach for measuring virus neutralization and protective antibodies. The deployment of PRNT implies the

production of viral antigen. Knowing the growth curve of these viruses is an important parameter to PRNT results as a large amount of inactive particles can result in afalsely low antibodies titers. Therefore, the best harvest day must occur during the late exponential growth phase. The aim of this study was to observe the growthcurve of these viruses and determine the best harvests days with titers around 105PFU/ml, verify the possibility of two different harvests in one bottle. Standardsstrains DENV1(Hawaii), DENV2(NGC), DENV3(H87) and DENV4(H241) adapted in Vero cells were used. Viral stocks were grown in Vero cells from the ATCC. Theconditions for amplication were standardized using a Multiplicity of Infection of about 10-2 to DENV3 and 10-3 to DENV1, 2 and 4. The contents of this viralpreparation was quantied by titration in 24 wells plates and revealed by immunostaining using monoclonal antibodies. The results demonstrate that it is possible tomake two harvests and suggest that the best days to harvest of DENV3 is the day 7 (104.90 PFU/ml) and a second harvest on day 12 (104.70PFU/ml). For DENV1, 2and 4 the best for the harvests days were the day 5 (105.69PFU/ml, 104.93PFU/ml and 105.87PFU/ml) and second harvest day 10 (105.38PFU/ml, 104.58PFU/ml e105.13PFU/ml) respectively.

V-P17

EXPRESSION OF THE DENGUE VIRUS NS1 PROTEIN IN HEPG2 CELLS AND ULTRASTRUCTURAL ASPECTS1 1 1 1 2 1Kssila Rabelo , Marciano Viana Paes , Adriana de Souza Azevedo , Edson Roberto A. de Oliveira , Ceclia Jacques G. de Almeida , Ada Maria de Barcelos Alves and

1Simone Morais da Costa1 2Laboratrio de Biotecnologia e Fisiologia de Infeces Virais, Instituto Oswaldo Cruz, Fiocruz,- RJ, Brasil. Laboratrio de Imunofarmacologia, Instituto Oswaldo Cruz,Fiocruz,- RJ, BrasilDengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in whichthe liver is normally affected. The dengue non-structural protein 1 (NS1) is essential for virus viability, although its function is not yet elucidated. It is found in infectedcells associated with plasma membrane and secreted into the circulation. Some reports indicate that NS1 can be used as a protective antigen, while others suggest itsinvolvement in viral pathogenesis. Therefore, studies concerning the effect of the NS1 in hepatic cells can elucidate some aspects of its role during the virus infection.In this work, we standardized an efcient protocol of HepG2 transfection, a human hepatocarcinome cell, with a plasmid encoding the dengue-2 virus NS1 protein andevaluated the effect of this protein in the cell ultrastructures. Such assays were necessary, since HepG2 cells are particularly difcult to be transfected. First, weevaluated three different transfection methods: Lipofectamine, FuGENE 6 and nucleofection, and the expression of NS1 were detected by immunouorescence andow cytometry. Subsequently, we analyzed ultrastructural changes in HepG2 cells expressing the recombinant protein by electron microscopy. Low levels of NS1expression were observed in cells transfected with Lipofectamine and FuGene, 24h post transfection. On the other hand, the efciency of nucleofection wasremarkable higher when compared to the others methods, in which approximately 59% of cells expressed the NS1. The analysis by electron microscopy suggested thatNS1 appears to induce some morphological alterations in HepG2 involving organelles as Endoplasmic Reticulum and Mitochondria, as well as in Lipid Bodiesproduction. Funding support: IOC-Fiocruz, PAPES-Fiocruz, FAPERJ, CNPq.

V-P18

THE DENGUE VIRUS NS3 PROTEIN IS LOCATED IN DETERGENT RESISTANT MICRODOMIANS OF HMEC-I INFECTED CELLS1 1 2 1Garca-Cordero J , Bueno-Cruz K , Gutirrez-Castaeda B , Gonzlez-Y-Merchand J3, Cedillo-Barrn L .

1 2 3Depto. Biomedicina Molecular, CINVESTAV, D.F.; Facultad de Estudios Superiores IZTACALA UNAM; Estado de Mxico; Dpto. de Microbiologa Molecular ENCB-IPND.F.; Mxico

Dengue is a viral disease transmitted by mosquitoes, the causative agent is an enveloped virus of positive-strand RNA and there are four serotypes (DEN-1 to 4). Theinteraction of virus with the host cell is still a matter of study. It is proposed that there are different kinds of cellular receptors including: heparan sulfate, lectins, DC-SIGN, heat shock proteins, among others. Several reports have shown that different life cycle events, including the process of virus replication, are held in


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membranous organelles. It has been shown that the complex formed by replicative NS3 and NS5 proteins is associated with membranes of the endoplasmic

reticulum. Cell membranes are characterized by the presence of dynamic structures of proteins and lipids known as lipid rafts or Resistant Microdomains Detergents(RMD) where events such cell signaling are take place. The goal of this work was to evaluate whether the dengue virus uses the lipid rafts during the events followingthe entry in a model of endothelial cells (HMEC-I). HMEC-I cells were infected with Dengue-2 virus and analyzed at 48h post-infection to evaluate the level ofcolocalization of a nonstructural protein NS3 (as part of the replication complex) and lipid rafts markers residents such as caveolin and otillin. Confocal microscopyresults show a high level of colocalization between lipid rafts resident proteins and viral NS3 protease, but not for the transferrin receptor as no resident marker of lipidrafts. However when the extraction of detergent resistant microdomains (MRD) was performed through sucrose gradients and by evaluating NS3 distribution in each ofthe MRD fractions, it was observed that NS3 localises to the lipid rafts region. Furthermore, when NS3 was immunoprecipitated with caveolin, it was observed that it isindeed associated only with caveolar rafts. Moreover, it was found that lipid rafts could be playing an important role during viral assembly due to the fact that whencells were treated with an inhibitor of cholesterol synthesis (Lovastatin) it was observed that there was a reduction in viral infectious progeny. Finally, it was observedthat NS5 protein did not colocalize with caveolar lipid rafts, which suggests that lipid rafts may only participate during the processing of the polyprotein. In conclusion,it was found that NS3 viral protein is localized in a protein complex associated to caveolin and that this complex affects the assembly of dengue virus in HMEC-I cells.

POSTERS SESSION ANTIVIRALS

A-P01VITAMIN D INHIBITS DENGUE VIRUS INFECTION IN THE HUMAN MONOCYTIC CELL LINE U937-DCSIGN BY INDUCING AUTOPHAGY.1 1 1 1 1Antonio H. Angel-Ambrocio , Margot Cervantes-Salazar , Rubn Soto-Acosta , Patricia Bautista-Carbajal , Rosa M Del Angel

1Centro de Investigacin y de Estudios Avanzados Del Instituto Politcnico Nacional. Mxico D. F.

The 1,25-dihidroxy-calcitriol, vitamin D3 (VD3) has widely spread effects on cellular differentiation and proliferation, and immune responsiveness inmonocytes/macrophages, dendritic cells and lymphocytes. The use of VD3 has been proposed for the treatment of autoimmune, bacterial and viral infectiousdiseases. Previous work from our group showed that treatment with VD3 resulted in a signicant reduction in the number of DENV U937 and HUH7 infected cells. Themechanism involved in the antiviral action of VD3 is not fully understood but it is known that VD3 induces autophagy in many cells lines and this has been themechanism proposed to explain VD3 reduction in virus replication. Autophagy has been shown to facilitate DENV replication in some cells lines but to impede it inothers. In this study, we sought to characterize VD3-induced antiviral mechanisms in DENV infected monocityc cells, using the highly permissive cell line U937-DCSIGN. Cells in culture were infected with DENV and treated either with VD3, rapamycin (an inductor of autophagy), 3-methyladenine (3MA, an inhibitor ofautophagy), VD3 plus 3-methyl adenine or left untreated as controls. Treatment with either VD3 or rapamycin at non-toxic concentrations induced autophagy andresulted in an inhibition of DENV replication, while 3MA treatment has not effect. Interestingly, the combination of VD3 with 3MA treatment only partially revert the

antiviral effect observed with VD3. We also found that VD3 induces the secretion of human cathelicidin, a protein that has direct antimicrobial activity. Our resultssupport the idea that autophagy is one of the mechanisms induced by VD3 to inhibit DENV infection; however, the secretion of cathelicidin may also be an anti-DENVmechanism induced by VD3 in monocytic cells. These ndings provide insights into novel approaches to treat the severe forms of dengue disease.

A-P02

SYNTHETIC COMPOUNDS OBTAINED IN HOUSE INHIBIT DENGUE VIRUS INFECTION IN VITRO1 2 1 2 1 1Andrea C. Koishi , Tas A. Corra , Paula R. Zanello , Mauro V. Almeida , Juliano Bordignon , Claudia N.D. dos Santos

1 2Instituto Carlos Chagas, Brazil; Universidade Federal de Juiz de Fora, BrazilDengue is a signicant public health problem worldwide caused by a mosquito-borne Flavivirus. In Brazil, until the 18 epidemiological week of 2014 there were394.614 cases registered, being 176 cases of severe dengue, 2.302 cases of dengue with warning signs and 104 conrmed deaths. Currently the four virus serotypesare circulating in the country in the proportion of DENV1 (85.5%), DENV4 (11.7%), DENV2 (2.4%) and DENV3 (0.4%). Despite the important social and clinical impactof the disease, there are no vaccines or specic antiviral therapies for its prevention and treatment. Considering the above, drug discovery research for dengue is ofutmost importance. Here we propose the evaluation of the antiviral activity of synthetic compounds obtained in house. The antiviral activity screening was performed

using an in situ ELISA assay that uses the human-derived hepatoma Huh7.5 lineage infected with DENV-1/FGA/89 as substrate. This assay was developed andvalidated to this purpose. Compound cytotoxicity was assessed by the MTT and Neutral Red assays. Selected hits were tested in human peripheral blood mononuclearcells (PBMCs) infected with DENV-4/TVP360, and infection and cell viability (7-AAD and Anexin V staining) were evaluated by ow cytometry. Out of 49 compoundsscreened with the maximum non-toxic dose (MNTD) in Huh7.5, fteen were able to reduced DENV-1 infection in at least 70%. The ones that presented SI valuesgreater than 5 were chosen for further analysis. In order to evaluate the antiviral activity in a more relevant physiological system, PBMCs were infected with DENV-4and treated for 120h. Treatment with three compounds were able to lower infection to levels compared to IFN-2A (reference compound) with little cytotoxic e ffect.This study shows that compounds obtained in house reduced DENV infection in human cell lineage and primary cell culture, being promising for the development of adengue antiviral drug.


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A-P03

MARCGRAVIACEAE-ORIGINATED COMPOUNDS DEMONSTRATED ANTIVIRAL ACTIVITY AGAINST DENV-2 IN PRIMARY HUMAN MONOCYTES INFECTED IN VITRO1 1 1 1 1Luciana Gomes Fialho ; Cntia da Silva Mello ; Vagner Pereira da Silva ; Maria Raquel Figueiredo ; Claire Fernandes Kubelka

1Fiocruz, Rio de Janeiro - RJ - BrasilIntroduction: The discovery of medicinal plants with antiviral activity against Dengue virus (DENV)-2 is important since the viral load and replication generate immuneresponses leading to severe disease outcomes. The use of the virus main target cells in this study, like primary monocytes, reproduces events occurring with denguepatients. Marcgraviaceae, has been evaluated for its biodynamic activities, due to its almost complete chemical and pharmacological uniqueness. The aim of thiswork was to determine the antiviral potential of a Marcgraviaceae sp. Methods and results: Primary human monocytes were infected with DENV-2 (strain 16681). Weanalyzed for three days the iNOS expression by ow cytometry, while cell culture supernatants were evaluated for NS1, by ELISA and for NO2-, by Griess reaction. Thevirus induced expression/production of all factors evaluated at 48 hours post-infection and this time-point was adopted for antiviral assays with plant fractions.Monocytes treated after infection either with buthanolic fraction or with 89-98 subfraction, both derived from a leave crude ethanolic extract were able to reducesignicantly the NS1 production (50% and 79%, respectively). The 89-98 subfraction increased NO production in 55%. Afterwards, we evaluated if these compoundscould be inhibiting the infection to new cells. Monocytes were infected and

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