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3rd ICGEB WORKSHOP ON HUMAN RNA VIRUSES APRIL 3–5, 2012

SCIENTIFIC PROGRAM AND ABSTRACTS

Fundación Instituto Leloir Patricias Argentinas 435 C1405BWE, Buenos Aires, Argentina Tel: +54–11–5238–7500

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ORGANIZERS: Alessandro Marcello International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

Oscar Burrone International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

Andrea V. Gamarnik, Fundación Instituto Leloir, Buenos Aires, Argentina

LOCAL ORGANIZING COMMITTE: Fundación Instituto Leloir, Buenos Aires, Argentina Liliana H. Alonso Vanina M. Luciano Luana de Borba Sergio M. Villordo Ezequiel J. Aranovich

Néstor G. Iglesias Federido A. de Maio Laura A. Byk Leopoldo G. Gebhard

ACKNOWLEDGMENTS:

The organizers acknowledge with gratitude to TWAS, Academy of Sciences for Developing World.

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INDEX SCIENTIFIC PROGRAM .......................................................................................................... 5! Tuesday – April 3rd ............................................................................................................................. 5! Wednesday – April 4th ........................................................................................................................ 6! Thursday – April 5th ............................................................................................................................ 7! ORAL PRESENTATIONS ........................................................................................................ 8! #1 – Modeling the macromolecular assembly of the HIV-1 provirus.................................................. 8! #2 – Activity of the Human Immunodeficiency Virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by ires trans-acting factors................................................................... 9! #3 – Classification of dengue fever patients based on gene expression data using support vector machines................................................................................................................................. 10! #4 – Dynamics of the RNAi-mediated antiviral immunity ................................................................. 11! #5 – Activation of the innate immune response against DENV in normal non-transformed human fibroblasts................................................................................................................................ 12! #6 – Rotavirus infection of cells in culture induces activation of RhoA and changes in the actin and tubulin citoskeleton ................................................................................................................. 13! #7 – Development of an artificial microrna-mediated antiviral strategy against foot-and-mouth disease virus ........................................................................................................................... 14! #8 – Pathogenesis of low pathogenic avian influenza virus (LPAIV) in chickens in a model of coinfection with chicken anemia virus (CAV) .......................................................................... 15! #9 – Longitudinal study of HIV-1 ENV diversity in viral populations with different cell tropism ........ 16! #10 – Phylogenetic analysis of bovine leukemia viruses isolated in south america reveals diversification in seven distinct genotypes .............................................................................. 17!

POSTERS – ARENAVIRIDAE................................................................................................ 18! #1 – A N-terminal coiled coil motif is essential for Tacaribe virus nucleoprotein functionality ......... 18! #2 – Translational strategy of Junin virus: participation of the viral nucleoprotein in the translation initiation complex .................................................................................................................... 19! #3 – Molecular determinants of Arenavirus Z protein Homo-oligomerization and l Polymerase binding .................................................................................................................................... 20! #4 – Acridone derivatives as inhibitors of Junín virus RNA synthesis.............................................. 21!

POSTERS – BUNYAVIRIDAE................................................................................................ 22! #5 – The Andes Hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism .............................................................................................................. 22!

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POSTERS – FLAVIVIRIDAE .................................................................................................. 23! #6 – TIA-1 is a novel host factor involved in TBEV replication......................................................... 23! #7 – Distribution and molecular characterization of Hepatitis C Virus (HCV) genotypes in patients with chronic infection from Pernambuco State, Brazil............................................................. 24! #8 – Genetic variation in host il28b predicts extrahepatic HCV infection......................................... 25! #9 – Differential replication and plaque morphology of infectious clones of Dengue Virus 2 .......... 26! #10 – Functional elements at the C-terminus of DENV capsid protein ............................................ 27! #11 – Study of the antiviral activity of 1-Indanone Thiosemicarbazones against BVDV.................. 28! #12 – The involvement of P2X7 receptor in human monocytes infected with Dengue-2-virus: preliminary studies .................................................................................................................. 29! #13 – Characterization of Dengue Virus RNA replication elements................................................. 30! #14 – Modeling gene sequence changes over time in type 3 Dengue Viruses from South America: diversification, rates and population dynamics. ...................................................................... 31! #15 – Putative role of the capsid protein in Dengue cellular infection: fusion mechanism reinterpreted............................................................................................................................... 32! #16 – Lipid rafts association with the protein NS3 and dsRNA of dengue virus in cells HMEC-I .... 33! #17 – Exploring different strategies to express Dengue Virus envelope protein in plant systems... 34! #18 – Dynamic trafficking of proteins and Flavivirus RNA between replication compartments and the cytosol............................................................................................................................... 35! #19 – A detailed comparative analysis on the overall codon usage patterns in West Nile Virus: bias and host influence in virus codon usage pattern..................................................................... 36! #20 – Construction and characterization of a stable subgenomic replicon system of a Dengue Virus type 3 strain (BR DEN3 290-02) ............................................................................................. 37! #21 – Characterization of Dengue Virus type 3 entry in Vero and A549 cells ................................. 38! #22 – Vitamin D3 reduces Dengue Virus infection and cytokine production from cultured cells, a potential therapeutic strategy for dengue treatment ............................................................... 39! #23 - Early molecular markers predictive of Dengue Hemorrhagic Fever ....................................... 40! #24 – The role of heterotypic DENV-specific CD8+T lymphocytes in the pathogenesis of Dengue Hemorrhagic Fever ................................................................................................................. 41! #25 – Different conformations of the Dengue Virus RNA genome are crucial for infectivity ............ 42!

POSTERS – HEPADNAVIRIDAE........................................................................................... 43! #26 – HDAG-l variants in covert Hepatitis D and HBV occult infection among amerindians of argentina: new insights ........................................................................................................... 43!

POSTERS – HEPEVIRIDAE................................................................................................... 44! #27 – Genetic variability of Hepatitis E virus in Uruguay ................................................................. 44! POSTERS – OPHIOVIRIDAE................................................................................................. 45! #28 – Study of genome organization and expression of Citrus psorosis virus RNA 1 ..................... 45!

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POSTERS – ORTHOMYXOVIRIDAE..................................................................................... 46! #29 – Mice vaccination with hydrostatic pressure-inactivated H3N8 protects against experimental Flu ........................................................................................................................................... 46! #30 – Nanobodies with in vitro neutralizing activity protect mice against H5N1 Influenza Virus infection................................................................................................................................... 47! #31 – A combinatorial antiviral approach against Influenza A Virus using ribozyme and siRNA..... 48!

POSTERS – PAPILLOMAVIRIDAE ....................................................................................... 49! #32 – Modulation of the intrinsically disordered HPV E7 oncoprotein by calcium ........................... 49! POSTERS – PARAMYXOVIRIDAE........................................................................................ 50! #33 – Impaired RIG-i-dependent antiviral cytokine responses at birth: implications for susceptibility to acute RSV disease early in life? ......................................................................................... 50! #34 – Purification and characterization of recombinant Human Respiratory Syncytial Virus NS1 protein ..................................................................................................................................... 51!

POSTERS – PICORNAVIRIDAE............................................................................................ 52! #35 – Epidemiological surveillance of Poliovirus in the region of Southern cone of America and Bolivia, period 2007-2010 ....................................................................................................... 52! #36 – Characterization of protein-protein interactions during transcription and replication of Footand-Mouth Disease Virus........................................................................................................ 53!

POSTERS – REOVIRIDAE..................................................................................................... 54! #37 – Analysis of the host-pathogen interaction established in plants infected by Mal de Río Cuarto Virus (MRCV, Fijivirus, Reoviridae) ........................................................................................ 54! #38 – HSV-1-based replication-defective particles and amplicons as transgenic vectors expressing antigens from human and animal pathogens.......................................................................... 55! #39 – Molecular characterization of Rotavirus and Norovirus in patients with acute gastroenteritis in Salto City, Uruguay ................................................................................................................. 56!

POSTERS – RETROVIRIDAE................................................................................................ 57! #40 – Inhibition of Human Immunodeficiency Virus type 1 replication by a non-immunomodulatory mechanism GBV-C NS5A protein-mediated........................................................................... 57! #41 – Interaction between HIV bound to erythrocytes with monocytes, macrophages or dendritic cells......................................................................................................................................... 58! #42 – Spermatozoa efficiently transmit HIV-1 to dendritic cells and elicit their phenotypic maturation ................................................................................................................................................ 59! #43 – Analysis of molecular mechanisms that contribute to the development of HTLV-1 associated pathologies.............................................................................................................................. 60!

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POSTERS – TOGAVIRIDAE .................................................................................................. 61! #44 – Activity of Venezuelan Equine Encephalitis (VEE) complex viruses in North-Central provinces of Argentina............................................................................................................................. 61!

POSTERS – VIRUS AND CANCER....................................................................................... 62! #45 – LXCXE-motif mediated interaction of viral proteins with the Retinoblastoma Tumor suppressor: a common theme in dna and rna virus-host interactions .................................... 62! #46 – Genetic transference medicines designed to target gynecological cancers .......................... 63! #47 – Evaluation of the potential use of Rotavirus as oncolytic virus in the murine myeloma cell line Sp2/0 Ag14. ............................................................................................................................ 64!

INDEX OF ABSTRACTS ....................................................................................................... 65 SPEARKERS CONTACT INFORMATION............................................................................. 66! PARTICIPANTS CONTACT INFORMATION......................................................................... 68 LOCALIZATION MAPS .......................................................................................................... 70

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3rd ICGEB WORKSHOP ON HUMAN RNA VIRUSES Fundación Instituto Leloir, Buenos Aires, Argentina April 3-5, 2012

SCIENTIFIC PROGRAM TUESDAY – APRIL 3rd 9:00 to 9:45 – Lecture 1 “Antiviral innate immunity against viruses: Interferons” Dr. Takashi Fujita 10:00 to 10:45 – Lecture 2 “Role of trim factors in innate immunity” Dr. Adolfo Garcia-Sastre 11:00 to 11:30 – Coffee break 11:30 to 12:15 – Lecture 3 “Viral modulation of host responses” Dr. Shahid Jameel 12:30 to 13:15 – Short talks of selected abstracts “Modeling the Macromolecular Assembly of the HIV-1 Provirus” – Matías Machado, Uruguay “Activity of the human immunodeficiency virus type 1cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors” – Maricarmen Vallejos García, Chile “Classification of dengue fever patients based on gene expression data using support vector machines” – Ana Lisa Gomes, Brazil 13:15 to 14:30 – Lunch (Posters area) 14:30 to 15:15 – Lecture 4 “Molecular basis of arenavirus replication” Dr. Nora Lopez 15:30 to 16:15 – Lecture 5 “Viroplasms, the engine of Rotavirus replication“ Dr. Francesca Arnoldi 16:30 to 17:00 – Coffee break 17:00 to 17:45 – Lecture 6 “Vaccines based on self-amplifying alphavirus RNA replicons” Dr. Christian Mandl

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WEDNESDAY – APRIL 4th 9:00 to 9:45 – Lecture 7 “The HCV Pandemic - New Options for Treatment” Dr. John McLauchlan 10:00 to 10:45 – Lecture 8 “Sensing viral RNA in cytoplasm and activation of the interferon system” Dr. Takashi Fujita 11:00 to 11:30 – Coffee break 11:30 to 12:45 – Short talks of selected abstracts “Dynamics of the RNAi-mediated Antiviral Immunity” – Juan Mondotte, France “Activation of the innate immune response against DENV in normal non-transformed human fibroblasts” – José Bustos-Arriaga, Mexico “Rotavirus infection of cells in culture induces activation of RhoA and changes in the actin and tubulin citoskeleton” – Jose Luis Zambrano Rouvier, Venezuela “Development of an artificial microRNA-mediated antiviral strategy against foot-and-mouth disease virus” – María Inés Gismondi, Argentina 13:00 to 14:30 – Lunch (Posters area) 14:30 to 15:15 – Lecture 9 “Influenza viruses: from genes to disease” Dr. Adolfo Garcia-Sastre 15:30 to 16:15 – Lecture 10 “Unravelling host factors involved in Rotavirus replication cycle” Dr. Francesca Arnoldi 16:30 to 17:00 – Coffee break 17:00 to 17:45 – Lecture 11 “Functional microRNA generated from a cytoplasmic RNA virus” Dr. Christian Mandl 18:00 to 18:45 – Lecture 12 “Control gene expression in coronaviruses” Dr. Luis Enjuanes 18:45 to 20:00 – Poster session – Wine and “Picada”

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THURSDAY – APRIL 5th 9:00 to 9:45 – Lecture 13 “Hepatitis viruses: diverse viruses that target the same organ” Dr. Shahid Jameel 10:00 to 10:45 – Lecture 14 “The Role of Host Processes in the HCV Life Cycle” Dr. John McLauchlan 11:00 to 11:30 – Coffee break 11:30 to 12:15 – Lecture 15 “RNA structures and the regulation of dengue virus replication” Dr. Andrea Gamarnik 12:30 to 13:15 – Short talks of selected abstracts “Pathogenesis of low pathogenic avian influenza virus (LPAIV) in chickens in a model of coinfection with Chicken Anemia Virus (CAV)” – Agustina Rimondi, Argentina “Longitudinal study of HIV-1 ENV diversity in viral populations with different cell tropism” – Paula Aulicino, Argentina “Phylogenetic analysis of bovine leukemia viruses isolated in South America reveals diversification in seven distinct genotypes” – Gonzalo Moratorio, Uruguay 13:15 to 14:30 – Lunch (Posters area) 14:30 to 15:15 – Lecture 16 “Molecular basis of coronavirus virulence: development of a recombinant vaccine to prevent the severe and acute respiratory syndrome” Dr. Luis Enjuanes 15:30 to 16:15 – Lecture 17 “Selective degradation of proteins within the secretory pathway and in vivo biotinylation of viral proteins” Dr. Oscar Burrone 16:30 to 17:00 – Coffee break 17:00 to 17:45 – Lecture 18 “Viral replication in 4D: tracking viral RNA genomes in living cells” Dr. Alessandro Marcello 18:00 – Closing Discussion

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ORAL PRESENTATIONS #1 – MODELING THE MACROMOLECULAR ASSEMBLY OF THE HIV-1 PROVIRUS MACHADO Matías R.1, Sergio Pantano1 1 Biomolecular Simulation Group, Institut Pasteur de Montevideo, Mataojo 2020, 11400, Montevideo, Uruguay. Contact: [email protected] The proviral stage of the HIV-1 is of high relevance to the infection and prevalence of this virus. While integrated, the viral genome uses the host´s machinery to self replicate. In particular conditions, the switch to a latent state of no transcription allows the virus to escape from the immune system and antiretroviral therapies.1,2 Many efforts have been done to characterize the proteins interactions, epigenetic signals and chromatin organization events in the latent and active states of transcription.2-4 In that sense, many pieces of this huge puzzle have been collected during the time. As a consequence, several interaction schemes were drawn regarding the many factors participating in these processes.2,5,6 The idea of translating a classic scheme of molecular biology into a structural model is not new and there are several examples in the literature.7 However, so far there is no such attempt for HIV-1. In the present work we show for the first time a structural picture of the HIV-1 provirus on its latent state (Figure 1). To build the model we review data from varied techniques of molecular8-10 and structural biology11-14, which were integrated though computational modeling tools. The pseudo-atomic resolution of our model allows to characterize possible protein-protein contacts, exposed DNA sequence and to determine volume excluded regions. We expect this structural view will help in understanding the biology and mechanism behind the HIV-1 infection.

Figure 1. Structural model of HIV-1 provirus on its latent state. The chromatin (blue) and some host proteins are highlighted References [1] Marcello A. Retrovirology. 2009, 6:11. [2] Le Douce V, Herbein G, Rohr O, Schwartz C. Retrovirology. 2010, 7:32. [3] Pereira LA, Bentley K, Peeters A, Churchill MJ, Deacon NJ. Nucleic Acids Res. 2000, 28:663. [4] Imai K, Ochiai K. J Oral Sci. 2011, 53:1. [5] Trono D, Van Lint C, Rouzioux C, Verdin E, BarréSinoussi F, Chun TW, Chomont N. Science. 2010, 329:174. [6] Hakre S, Chavez L, Shirakawa K, Verdin E. Curr Opin HIV AIDS. 2011, 6:19. [7] Panne D, Maniatis T, Harrison SC. Cell. 2007, 129: 1111. [8] Verdin E, Paras P Jr, Van Lint C. EMBO J. 1993, 12:3249. [9] Demarchi F, D'Agaro P, Falaschi A, Giacca M. J Virol. 1993, 67:7450. [10] Perkins KJ, Lusic M, Mitar I, Giacca M, Proudfoot NJ. Mol Cell. 2008, 29:56. [11] Davey CA, Sargent DF, Luger K, Maeder AW, Richmond TJ. J Mol Biol. 2002, 319:1097. [12] Bushnell DA, Westover KD, Davis RE, Kornberg RD. Science. 2004, 303:983. [13] Stroud JC, Oltman A, Han A, Bates DL, Chen L. J Mol Biol. 2009, 393:98. [14] Ferré-D'Amaré AR, Pognonec P, Roeder RG, Burley SK. EMBO J. 1994, 13:180.

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#2 – ACTIVITY OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 CELL CYCLEDEPENDENT INTERNAL RIBOSOMAL ENTRY SITE IS MODULATED BY IRES TRANS-ACTING FACTORS VALLEJOS Maricarmen1, Jules Deforges2, Terra-Dawn M. Plank3, Alejandro Letelier1, Pablo Ramdohr1, Christopher G. Abraham4, Fernando Valiente-Echeverría1, Jeffrey S. Kieft3,5, Bruno Sargueil2 and Marcelo López-Lastra1. 1

Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de

Investigaciones Médicas, Facultad de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile,

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CNRS UMR 8015, Laboratoire de cristallographie et RMN Biologique,

Universite´ Paris Descartes, 4 avenue de l’Observatoire, 75270 Paris Cedex 06, France, 3Department of Biochemistry and Molecular Genetics, 4Department of Microbiology and 5Howard Hughes Medical Institute, University of Colorado Denver School of Medicine, Aurora, CO, 80045, USA. The full-length mRNA of the human immunodeficiency virus type 1 (HIV-1), member of Retroviridae family and AIDS etiological agent, harbors an internal ribosomal entry site (IRES) element within its 5´ untranslated region (5’UTR) that allows the recruitment of the translational machinery. This IRES drives the synthesis of Gag structural protein and is active during the G2M cell cycle phase. The molecular mechanisms governing IRES function are not understood. Here we show that translation initiation mediated by the HIV-1 IRES requires the participation of trans-acting cellular factors other than the canonical translational machinery. To gain insight into structure-function relationship of the HIV-1 IRES, we modeled the RNA structure of the HIV-1 5’leader. We used standard´ chemical and enzymatic probes and an “RNA SHAPE” analysis to model the structure of the HIV-1 5´ leader and we show, by means of a footprinting assay, that G2/M extracts provide protections to regions previously identified as crucial for HIV-1 IRES activity. The introduction of mutations designed to alter the structure of the HIV-1 5´ leader did not significantly affect IRES activity suggesting that the structurefunction relationship within the HIV-1 IRES is not as rigid as has been described for other viral IRESes. Finally, we used a proteomic approach to identify cellular proteins within the G2/M extracts that interact with the HIV-1 5´ leader. Together, data show that HIV-1 IRES-mediated translation initiation is modulated by cellular proteins. This work was supported by Fondecyt 1090318.

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#3 – CLASSIFICATION OF DENGUE FEVER PATIENTS BASED ON GENE EXPRESSION DATA USING SUPPORT VECTOR MACHINES. GOMES AL1, Wee LJ2, Khan AM3, Gil LH4, Marques ET Jr4,5, Calzavara-Silva CE6, Tan T2. 1

Federal Rural University of Pernambuco, Recife, Brazil

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National University of Singapore, Singapore.

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Perdanga Univeristy, Kuala Lumpu, Malaysia

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Aggeu Magalhães Research Center- CPqAM/FIOCRUZ, Recife, Brazil.

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University of Pittsburgh, United States of America

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Rene Rachou Research Center – CpqRR/ FIOCRUZ, Belo Horizonte, Brazil.

Symptomatic infection by dengue virus (DENV) can range from dengue fever (DF) to dengue haemorrhagic fever (DHF), however, the determinants of DF or DHF progression are not completely understood. It is hypothesised that host innate immune response factors are involved in modulating the disease outcome and the expression levels of genes involved in this response could be used as early prognostic markers for disease severity. mRNA expression levels of genes involved in DENV innate immune responses were measured using quantitative real time PCR (qPCR). Here, we present a novel application of the support vector machines (SVM) algorithm to analyze the expression pattern of 12 genes in peripheral blood mononuclear cells (PBMCs) of 28 dengue patients (13 DHF and 15 DF) during acute viral infection. The SVM model was trained using gene expression data of these genes and achieved the highest accuracy of approximately 85% with leave-one-out cross-validation. Through selective removal of gene expression data from the SVM model, we have identified seven genes (MYD88, TLR7, TLR3, MDA5, IRF3, IFN-alpha and CLEC5A) that may be central in differentiating DF patients from DHF, with MYD88 and TLR7 observed to be the most important. Though the individual removal of expression data of five other genes had no impact on the overall accuracy, a significant combined role was observed when the SVM model of the two main genes (MYD88 and TLR7) was re-trained to include the five genes, increasing the overall accuracy to approximately 96%. Here, we present a novel use of the SVM algorithm to classify DF and DHF patients, as well as to elucidate the significance of the various genes involved. It was observed that seven genes are critical in classifying DF and DHF patients: TLR3, MDA5, IRF3, IFN- alpha, CLEC5A, and the two most important MYD88 and TLR7. While these preliminary results are promising, further experimental investigation is necessary to validate their specific roles in dengue disease.

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#4 – DYNAMICS OF THE RNAI-MEDIATED ANTIVIRAL IMMUNITY MONDOTTE Juan A. and Maria Carla Saleh Institut Pasteur, Viruses and RNAi Group, CNRS URA 3015, France It has been shown that in insects RNAi mediates antiviral immunity. The specificity determinants of the RNA silencing response are small interfering RNA (siRNA), which are derived from the invading viral genome. After being uploaded into the RNA induced silencing complex (RISC), these virus-specific siRNAs selectively target viral RNA by base-pairing for RISC-mediated cleavage and degradation. Recently it has been shown that infected Drosophila flies spread a systemic silencing signal that elicits a protective RNAi-dependent immunity throughout the organism. This suggests that the cellautonomous RNAi response is insufficient to control a viral infection and that insects also rely on a systemic immune response to fight against such infections. It is accepted that insects lack adaptive immunity and rely on the innate immune system to mount defense responses against pathogens. For decades, it was assumed that the innate immune system is hard-wired and unable to establish immunological memory. Interestingly, memory-like responses have been described in invertebrates, a phenomenon that was termed “immune priming” that challenged the dogma that invertebrates are incapable of adaptive immune responses. However, the existence of a mechanism of long-lasting antiviral RNAi immune responses in insects has never been explored. In this context, insects would be able to mount a dynamic antiviral RNAi response that may translate in a long-term protection that is transmitted to the progeny and confers cross-protection against related viruses. In this work we presented evidence for self-immune and cross immune protection in Drosophila. Our results indicate that Drosophila cells are protected from re-infection with the same virus, and also the existence of cross-protection by different viruses. Whether this immune protection is RNAi-mediated and the mechanism allowing its establishment is currently under study. By employing arboviruses and the insect model Drosophila melanogaster, we expect to provide extensive information concerning the basic mechanism of the RNAi-based antiviral immunity in insects. Studying the antiviral RNAi response in arthropods could unravel the mechanism by which insects harbour viruses and may identify new approaches to control virus replication in insect vectors that transmit disease-causing virus to humans.

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#5 – ACTIVATION OF THE INNATE IMMUNE RESPONSE AGAINST DENV IN NORMAL NONTRANSFORMED HUMAN FIBROBLASTS BUSTOS-ARRIAGA, José 1, Jazmín García-Machorro 1, Moises León-Juárez 1, Julio García-Cordero 1, Leopoldo Santos-Argumedo 1, Leopoldo Flores-Romo 2, Rene A. Méndez-Cruz 3, Francisco J. Juárez-Delgado 4, Leticia Cedillo-Barrón 1*. 1 Departamento de Biomedicina Molecular Centro de Investigación y de Estudios Avanzados, México Distrito Federal 2 Departamento de Biología Celular Centro de Investigación y de Estudios Avanzados, México Distrito Federal 3 Laboratorio de Inmunología UMF de la FES Iztacala Universidad Autónoma de México, Tlalnepantla Estado de México 4 Departamento de Cirugia, Hospital Santa Maria Ticomán, México Distrito Federal. INTRODUCTION. When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus. An important question is whether more abundant non-hematopoietic cells such as fibroblasts become infected, their role in antiviral innate immunity. OBJETIVE. Evaluate the response of human dermal fibroblast to the infection with DEN viruses and the differences between donors. METHODS. The infection was measured by FC, IFA and plaque assay, cytokine levels were measured by ELISA and intracellular FC, levels of expression of PRRs were measured by PCR and FC and finally the localization of the transcription factors were evidentiated by IFA. RESULTS. Fibroblasts freshly released from healthy skin and infected with DENV-2 shown viral antigen at 48h pi. Primary skin fibroblast cultures were established and subsequently infected. The percentage of envelope protein positive cells and the PFUs in the supernatant of 10 cultures established from different donors shown similar infection levels however the intensity of the response, evaluated as production of IFN!, TNF", defensin 5 (HD5) and ! defensin 2 (H!D2) was different between cultures. The infection up regulate the expression of the viral RNA PRRs; TLR3 and RIG- 1, but not Mda5 and induce increased nuclear translocation of, NFkB p50 subunit, IRF3, but not IRF7. Twenty-four hours after infection, 34% of the 60% of skin primary fibroblasts that expressed viral NS3 protein, showed a TUNEL positive signal as detected by immunofluorescence. The maximum infected cell count (68% of the antigen-positive cells) was reached at 48 h after infection. CONCLUSIONS. We demonstrated the susceptibility to DENV infection by primary dermal fibroblasts from normal human skin. Our results suggest that the fibroblasts, even that are equally permissive to the infection between donors, might be one of the host factors involved in the susceptibility/resistance to dengue infection in the early stages of virion inoculation.

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#6 – ROTAVIRUS INFECTION OF CELLS IN CULTURE INDUCES ACTIVATION OF RhoA AND CHANGES IN THE ACTIN AND TUBULIN CITOSKELETON ZAMBRANO ROUVIER, Jose Luis1, Orlando Sorondo1,2, Ana Alcala1, Esmeralda Vizzi1, Yuleima Diaz3, Marie Christine Ruiz4, Fabian Michelangeli4, Ferdinando Liprandi1, Juan E. Ludert5. 1

Lab. Biología de Virus. Centro de Microbiología y Biología Celular, Instituto Venezolano de

Investigaciones Científicas (IVIC), Caracas, Venezuela. 2

Escuela de Biología, Universidad Central de Venezuela (UCV), Caracas, Venezuela.

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Molecular Biology Institute. University of Bergen Thormøhlensgate 55, N-5008 Bergen, Norway

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Lab. Fisiología Gastrointestinal. Centro de Bioquímica y Biofísica, Instituto Venezolano de

Investigaciones Científicas (IVIC), Caracas, Venezuela. 5

Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y Estudios

Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Ciudad de México, México. Rotavirus infection induces an increase in [Ca2+]cyto, which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* nonneutralizing Mab, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca2+ chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca2+ homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.

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#7 – DEVELOPMENT OF AN ARTIFICIAL MICRORNA-MEDIATED ANTIVIRAL STRATEGY AGAINST FOOT-AND-MOUTH DISEASE VIRUS GISMONDI María Inés, Sebastián Asurmendi, Oscar Alberto Taboga Biotechnology Institute, National Institute of Agriculture Research (INTA), Castelar, Argentina Foot-and-mouth disease is a highly contagious viral disease of cloven-hoofed animals caused by foot-andmouth disease virus (FMDV. The disease is a primary animal health concern worldwide due not only to the productivity losses but also to the international trade restrictions associated with it. The virus belongs to the Aphtovirus genus within the Picornaviridae family and consists of seven serotypes and multiple subtypes. The viral particle contains a single-stranded positive-sense RNA genome of approximately 8500 nt in length, surrounded by an icosahedral capsid composed by four viral proteins. Upon entry in a susceptible cell, the FMDV RNA is translated into a single polyprotein that is cleaved to originate mature structural and non-structural viral proteins that mediate replication and encapsidation of the viral genome. Given that FMDV replicates and spreads extremely rapidly and that current vaccines are effective but require seven days to induce protection, there is a need to develop new antiviral tools that provide early protection in susceptible animals. Our current work is aimed at developing an antiviral strategy against FMDV based on RNA interference, in particular mediated by artificial microRNAs (amiR). Using the siRNA target finder (Ambion) software, putative amiR targets within the FMDV (serotype A) genome were predicted and three 21-nt regions were chosen (regions 3D1A and 3D2A within the 3D-coding sequence and region 3UTRA located at the 3’ non-coding portion of FMDV genome). Several transgenic cell lines constitutively expressing an amiR 100% complementary to any of the FMDV target sequences were established and cloned. Expression of the amiRs in transgenic cell lines was demonstrated by qPCR and sequencing. Transgenic cell lines were co-transfected with plasmids pRLUC/FMDV (encoding FMDV target sites 3D1A, 3D2A or 3UTRA downstream of Renilla luciferase) and pFLUC (encoding firefly luciferase), and luciferase activity was determined in cell lysates 24 h after transfection. Normalized luciferase activity decreased significantly in two amiR3D1A+ and in one amiR3D2A+ cell lines as compared with the same cells transfected with a control plasmid, whereas it remained unaffected in one amiR3D2A+ and in two amiR3UTRA+ cell lines. In both amiR3D1A+ cell lines analyzed, silencing was sequence specific, since the co-transfection of a reporter plasmid containing the homologous target sequence of FMDV serotype O (which contains 3 nucleotide changes as compared to 3D1A) did not result in luciferase activity reduction. To evaluate the antiviral activity of amiR3D1A+ cell lines, a one-step growth curve of the highly virulent FMDV/A/Arg2001 strain was performed. At the experimental conditions used, both transgenic lines were unable to control virus replication, as demonstrated by quantitation of intracellular FMDV genomes by qPCR and intracellular virus titration. Moreover, viral growth was similar in amiR+ and BHK21 cells. Analysis of the 3D1A sequence in supernatants of infected transgenic cell lines showed that the target sequence was not mutated after viral replication, ruling out viral escape to silencing. These results indicate that, although functional, the sole expression of amiR3D1A does not suffice to control replication of FMDV/A/Arg2001 in transgenic cells. Experiments including transfection of naked FMDV RNA in transgenic lines and in vitro determination of target site accesibility are in progress.

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#8 – PATHOGENESIS OF LOW PATHOGENIC AVIAN INFLUENZA VIRUS (LPAIV) IN CHICKENS IN A MODEL OF COINFECTION WITH CHICKEN ANEMIA VIRUS (CAV)

RIMONDI Agustina1, Kemin Xu2, María Isabel Craig1, Hongxia Shao2, Valeria Olivera1, Marcela Uhart3, Daniel R. Perez2, Ariel Pereda1 1-Laboratorio Aves y Porcinos, Instituto de Virología CICVyA - Instituto Nacional de Tecnología Agropecuaria (INTA) Castelar, Buenos Aires, Argentina. 2-Virginia-Maryland Regional College of Veterinary Medicine and Department of Veterinary Medicine, University of Maryland, CollegePark, USA. 3-Global Health Program, Wildlife Conservation Society, CABA, Argentina. Influenza is an infectious disease caused by a RNA virus from the Orthomyxoviridae family, whose genome is composed of 8 segments of single stranded and negative RNA. Extensive epidemiological studies have revealed that influenza viruses can infect a wide range of hosts, including many types of birds, humans, pigs, horses, dogs, cats, and other mammals. Of these species, aquatic birds are considered the natural reservoir of avian influenza A viruses, as all 16 known hemagglutinin (HA) subtypes (H1 to H16) and all 9 known neuraminidase (NA) subtypes (N1 to N9) have been recognized in these hosts. In general, wild bird infection with Avian Influenza Virus (AIV) is asymptomatic, while poultry infection with AIV can produce a variety of symptoms ranging from asymptomatic or mild presentations (Low Pathogenic Avian Influenza Virus or LPAIV), to an acute and fatal disease (High Pathogenic Avian Influenza Virus or HPAIV). Therefore, extensive animal influenza virus surveillance activities have been carried out in Southern China, East Asia, Northern Europe, Australia, and North America, with the aim of exploring the ecology of AIV in wild aquatic birds and to generate epidemiological data for an early warning of the introduction of AIV into commercial poultry. Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. In these years, several AIVs of different subtype (H4N2, H4N6, H5N3, H6N2, H6N8, H7N9, H13N9) were isolated from various species of wild aquatic birds. Recently, we characterized 5 AIV of the H6 subtype isolated from rosy-billed pochards because they were the subtype most consistently isolated. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. We also investigated the replication and transmission of these H6 Argentine AIVs in chickens and compared these strains to prototypic H6 strains from North America and Eurasia. Animal studies suggest a limited adaptation of these viruses to poultry and that additional molecular changes would be required for these viruses to replicate and transmit efficiently in chickens. According to the OIE, the current health situation in Argentina with respect to the AIV is free status, because the agent has not been yet described in poultry. However, the risk of possible introduction and spread of this agent requires the existence of an active surveillance system and an important control on the health status of birds in commercial flocks. In Argentina this is carried out by SENASA. Is important to mention that most of the poultry production is intensive, altering the immune system of birds and consequently the health status of poultry farms. Infectious agents are one of many factors that may affect their immune system, and within these, Chicken Anemia Virus (CAV) cause an important immunosuppressive disease that predispose affected flocks to acquire different secondary opportunistic infections. Although it is known there is a high incidence of CAV in commercial flocks in Argentina, knowledge is lacking about the importance of the immunosuppression caused by this agent in the evolution and pathogenesis of AIV. Therefore, in order to be able to answer this question and collaborate with the design of control and prevention strategies to protect commercial poultry farms in our country, we study the pathogenesis and transmissibility of local isolates of LPAIV in immunocompetent or experimentally immunosuppressed animals with CAV. In our opinion, the immunosuppressive model with CAV will highlight the close relationship between AIV and the host´s immune system to develop a wide range of syptoms. Our study also highlights the importance of the continuous surveillance of AIVs in South America as an integral part of worldwide efforts to better understand the ecology of these viruses and to prevent the emergence of novel, potentially pandemic strains.

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#9 – LONGITUDINAL STUDY OF HIV-1 ENV DIVERSITY IN VIRAL POPULATIONS WITH DIFFERENT CELL TROPISM AULICINO Paula, Andres Rossi, Cintia Crudeli, Carlos Rocco, Andrea Mangano, Luisa Sen Hospital de Pediatría “Juan P. Garrahan”. Laboratorio de Biología Celular y Retrovirus, Buenos Aires, Argentina HIV-1 can use CCR5, CXCR4, or both as cellular co-receptors for viral entry. Viral variants capable of using CCR5 are classified as R5 or NSI (Non-syncytium-inducing) and predominate at early stages of infection, while those capable of using CXCR4 produce a cytopathic effect on the cell line MT-2 and are classified as X4 or SI (syncytium-inducing). The latter appear late in infection and are associated with AIDS. Our aim was to study the frequency and evolution of viral subpopulations with different tropism within an individual. We looked for genotypic changes in the HIV-1 env gene that could be associated with viral tropism. Peripheral blood mononuclear cells (PBMCs) were isolated from an HIV-1 vertically infected patient at 12, 16, and 19 years of age. HIV-1 was isolated by co-cultivation of equal amounts of PBMCs from the patients and previously phytohemagglutinin (PHA) stimulated PBMCs from HIV seronegative blood donors, at a final concentration of 2#106 cells/ml. To determine SI or NSI viral phenotype, culture supernatants with p24 Ag$200 pg/ml were used to infect MT-2 cells and syncytium formation was registered microscopically at 3–4 day intervals for 14–21 days. The viral phenotype was classified as SI at 12 and 19 years (SI12y.o. & SI19y.o.), and NSI at 16 years (NSI16y.o.). DNA was extracted from PBMCs to amplify a 2877 bp segment including the complete HIV-1 env gene. The PCR products were cloned in PCR-2.1 TOPO vector, and transfected into in DH5" competent bacteria. Between 11 and 14 plasmids were purified and sequenced from each sample. Sequences were aligned with GeneCutter Tool (http://www.hiv.lanl.gov/content/sequence/GENE_CUTTER/cutter.html)

and

edited

with

BioEdit.

Phylogenetic relationships between clones were evaluated by Neighbor-Joining with the program MEGA 5.05. PSSM and Geno2Pheno algorithms were used to predict the viral tropism from the HIV-1 env (loop V3) sequence. Results showed that the frequency of X4 clones according to genotype-based prediction was: 100% for SI12y.o., 28% for NSI16y.o., and 50% for SI19y.o. Phylogenetic analysis revealed that X4 clones from samples obtained at 12 and 19 y.o. clustered independently, suggesting a different evolutionary origin. A subcluster of X4 (NSI16y.o.) was observed within the X4 (SI19y.o.) cluster, indicating the pre-existance of minority X4 strains at 16 years that could account for the expansión of X4 clones observed at 19 years. Regions V1, V2, V3 and C2 of the HIV-1 env were the main contributors to the tree topology The results evidence a great genotypic diversity within a same phenotypically-defined viral population and suggest that HIV-1 strains with equal tropism observed at different stages of disease can have an independent evolutionary origin within the same individual.

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3 ICGEB Workshop on Human RNA Viruses Fundación Instituto Leloir, Buenos Aires, Argentina ____________________________________________________________________________________________________________

#10 – PHYLOGENETIC ANALYSIS OF BOVINE LEUKEMIA VIRUSES ISOLATED IN SOUTH AMERICA REVEALS DIVERSIFICATION IN SEVEN DISTINCT GENOTYPES MORATORIO Gonzaloa,b #, Gonzalo Obala, Ana Dubraa,b, Agustín Correac, Sergio Bianchia,d, Alejandro Buschiazzoe, Juan Cristinab, Otto Pritscha,f* a

Unidad de Biofísica de Proteínas. Institut Pasteur de Montevideo. Mataojo 2020, 11400 Montevideo,

Uruguay. b

Laboratorio de Virología Molecular. Centro de Investigaciones Nucleares. Facultad de Ciencias, Iguá

4225, 11400 Montevideo, Uruguay. c

Unidad de Proteínas Recombinantes. Institut Pasteur de Montevideo. Mataojo 2020, 11400

Montevideo, Uruguay. d

Departamento de Fisiopatología, Hospital de Clínicas, Universidad de la República, Av. Italia s/n,

11600 Montevideo, Uruguay e

Unidad de Cristalografía de Proteínas. Institut Pasteur de Montevideo. Mataojo 2020, 11400

Montevideo, Uruguay. f

Departamento de Inmunobiología. Facultad de Medicina. Universidad de la República, Av. General

Flores 2125, 11800 Montevideo, Uruguay Bovine leukaemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus of the family Retroviridae. Recent studies revealed that BLV strains can be classified into six different genotypes and raised the possibility that another genotype may exists. In order to gain insight into the degree of genetic variability of BLV strains circulating in the South American region, a phylogenetic analysis was performed using gp51 env gene sequences. The results of these studies revealed the presence of seven BLV genotypes in this geographic region and the suitability of partial gp51 env gene sequences for phylogenetic inference. A significant number of amino acid substitutions found in BLV strains isolated in South America map in the second neutralization domain of gp51. A 3D molecular model of BLV gp51 revealed that these substitutions are located on the surface of the molecule. This may provide a selective advantage to overcome immune host neutralization.

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3 ICGEB Workshop on Human RNA Viruses Fundación Instituto Leloir, Buenos Aires, Argentina ____________________________________________________________________________________________________________

POSTERS – ARENAVIRIDAE #1 – A N-TERMINAL COILED COIL MOTIF IS ESSENTIAL FOR TACARIBE VIRUS NUCLEOPROTEIN FUNCTIONALITY D’ANTUONO Alejandra, Sabrina Foscaldi, Eugenia Loureiro and Nora López CEVAN-ICT Milstein CONICET. Buenos Aires, Argentina. Introduction. Arenaviruses, such as Tacaribe virus (TCRV) are enveloped viruses with a negativesense RNA genome, which encodes four proteins: the viral RNA polymerase (L protein), a matrix protein (Z), the precursor (GPC) of the envelope glycoproteins and the nucleocapsid protein (N). The N protein tightly binds to genomic and antigenomic RNAs forming nucleocapsids, which act as templates for both transcription and replication of viral genomes mediated by the virus polymerase. Besides, the ability of N to take part in multiple protein-protein interactions may be important in several steps of the viral life cycle. We have shown that the N-terminal region of N contains a coiled coil motif domain which is essential for N self-interactions. Also we have defined key hydrophobic amino acids within this motif as being essential for N-N interaction and its functional relevance. Results. Recently published crystallographic data for full-lenght nucleoprotein of Lassa virus was used as template to establish the spatial disposition of TCRV N protein residues. In this model two tight dimerization interfaces were predicted.

One of them comprising amino acids 195 to 259 and the other one

residues 281 to 287. Key amino acids within the putative interfaces were replaced by the non-polar amino

acid

Alanine.

The

ability

of

point

mutants

to

self-interact

was

evaluated

by

coimmunoprecipitation and their capacity to support viral RNA synthesis was assessed using a TCRV minireplicon system. Conclusions. We predict N-N dimerization interfaces based on TCRV N protein model and Lass N crystal structure superimposition. Functional analysis of residues within these regions revealed their relevance in N-N interaction as well as for its role in viral genome replication.

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3 ICGEB Workshop on Human RNA Viruses Fundación Instituto Leloir, Buenos Aires, Argentina ____________________________________________________________________________________________________________

#2 – TRANSLATIONAL STRATEGY OF JUNIN VIRUS: PARTICIPATION OF THE VIRAL NUCLEOPROTEIN IN THE TRANSLATION INITIATION COMPLEX 1

LINERO, Florencia; 2Welnowska, Ewelina; 2Carrasco, Luis; 1Scolaro Luis A.

1. Laboratorio de Virología, Dpto. Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. 2. Laboratorio de Virología, Instituto de Biología Molecular “Severo Ochoa”, Universidad Autónoma de Madrid, España. Translation efficiency of viral proteins is a key factor defining both cytopathogenicity and virulence of viruses, which are obligate intracellular parasites and entirely dependent on the cellular translation machinery to synthesize their proteins. This dependence has led them to develop different strategies of translational reprogramming to ensure an effective competition of viral mRNAs with cellular mRNAs. In order to study the translation strategy used by the arenavirus Junin (JUNV), aetiological agent of argentine haemorrhagic fever, we analyzed the initial steps of protein translation during JUNV infection focusing on the participation of the nucleoprotein (N) as a translational viral factor. Previous results obtained in our laboratory showed that JUNV is able to translate in a cap-independent mode despite the presence of Cap-structures in the 5´end of its mRNAs. In this respect the inhibition of the mTOR/raptor complex or the expression of a dominant negative of 4E-BP did not alter the synthesis and expression of JUNV proteins, indicating that the participation of the eIF4E factor, would not to be required for JUNV protein translation. eIF4E is a canonical translation factor that, along with eIF4A and eIF4GI, constitutes the eukaryotic initiation translation complex. In order to analyze the involvement of eIF4GI, we evaluated the effect of silencing this factor with specific small interfering RNAs, as well as, the cleavage of this factor by the expression of the poliovirus 2A protease. Both strategies showed a significant reduction in JUNV protein translation evaluated by immunofluorescence and radioactive labelling assays, respectively. Similar results were obtained for eIF4A, in which a significant reduction in JUNV replication was observed by treatment with the inhibitor hippuristanol, as well as by the expression of a dominant negative for eIF4A. These results suggest an alternative translational strategy used by JUNV in which the participation of eIF4GI and eIF4A, but not eIF4E, would be required to translate JUNV proteins. In view of these results we hypothesized the involvement of a viral protein in the translation initiation complex of JUNV mRNAs. The colocalization of N with eIF4GI strongly suggests that this viral protein would be part of the translation complex of viral mRNAs. According to these results we observed the presence of N in eIF4GI and eIF4A immunoprecipitated complexes suggesting that this viral protein would be part of the viral translation complex. Based on the above results we proposed a translational reprogramming strategy for JUNV in which the capbinding factor, eIF4E, would be replaced by N protein through its interaction with eIF4A and eIF4GI factors constituting the translation complex for viral proteins.

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#3 – MOLECULAR DETERMINANTS OF ARENAVIRUS Z PROTEIN HOMO-OLIGOMERIZATION AND L POLYMERASE BINDING LOUREIRO Eugenia, Wilda M, Levingston J, D`Antuono A, Marino C and Nora Lopez. Centro de Virología Animal (CEVAN), Instituto de Ciencia y Tecnología "Dr. Cesar Milstein", Consejo Nacional de Ciencia y Tecnología (CONICET), Saladillo 2468, Ciudad Autónoma de Buenos Aires, C1440FFX, Argentina. The arenavirus Z is a zinc-binding RING protein that has been implicated in multiple functions during the viral life cycle. These roles of Z involve interactions with viral and cellular proteins that remain incompletely understood. In this regard, Z inhibits viral RNA transcription and replication through direct interaction with the viral L polymerase. Here, we defined the L-binding domain of Tacaribe virus (TCRV) Z protein and the structural requirements mediating Z homo-oligomerization. By using site directed mutagenesis, coimmunoprecipitation, and functional assays, we showed that residues R37, N39, W44, L50 and Y57, located around the zinc-coordination site I, play a critical role in Z-L interaction. We also found that Z protein from either TCRV or the pathogenic Junin virus (JUNV), selfassociates into oligomeric forms in mammalian cells. Importantly, mutation of the myristoylation site, the strictly conserved residue G at position 2, severely impaired the ability of both TCRV Z and JUNV Z to self-interact as well as their capacity to accumulate at the plasma membrane, strongly suggesting that Z homo-oligomerization is associated to its myristoylation and cell membrane targeting. In contrast, disruption of the RING structure or substitution of W44 or N39, which are critical for L protein recognition, did not affect Z self-binding. Overall, the data presented here indicate that homooligomerization is not a requirement for Z-L interaction and Z-mediated polymerase activity inhibition.

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#4 – ACRIDONE DERIVATIVES AS INHIBITORS OF JUNÍN VIRUS RNA SYNTHESIS SEPÚLVEDA CS1, García CC1, Fascio ML2, D´Accorso NB2, Docampo Palacios ML3, Pellón RF3, Damonte EB1 1

Laboratorio de Virología, Dpto. de Química Biológica, FCEyN, UBA, Argentina.

2

CIHIDECAR (CONICET), Dpto. de Química Orgánica, FCEyN, UBA, Argentina.

3

Centro de Química Farmacéutica, La Habana, Cuba.

Arenaviruses are enveloped viruses containing a bipartite, single-stranded RNA genome, with ambisense coding strategy. Five arenaviruses are known to cause severe hemorrhagic fevers in humans, including Junín virus (JUNV) agent of Argentine hemorrhagic fever (AHF), but at present no reliable drug therapy is available. In the search of novel antiviral compounds against these pathogenic agents, a screening of antiviral activity of diverse N-substituted acridone derivatives identified a group of 10-allyl-9-(10H)-acridones as effective inhibitors of arenaviruses. The compound 10-allyl-6-chloro-4methoxy-9(10H)-acridone, designated 3f, was the most potent and selective antiviral agent against the infection of Vero cells with New and Old World arenaviruses. The effectiveness of 3f to inhibit JUNV multiplication was not importantly affected by the initial virus inoculum, with similar dose response curves in virus yield inhibition assays performed in Vero cells in the range of 0.2-40 plaque forming units (PFU)/cell. Mechanistic studies demonstrated that 3f did not affect virus entry, but it was able to interfere with JUNV intracellular multiplication through a strong inhibition of viral RNA synthesis, with about 3 log reduction in the amount of viral RNA quantified by real time RT-PCR in compound-treated cells relative to non-treated cells. The addition of exogenous guanosine rescued the infectivity of JUNV in 3f-treated cells in a dose-dependent manner, but the reversal was partial, suggesting that the inhibition of the cellular enzyme inosine monophospahe dehydrogenase (IMPDH) and thereby the reduction of GTP pool contributed to the antiviral activity of 3f, but it was not the only operative mechanism. The comparison of 3f with two other viral RNA inhibitors, ribavirin and mycophenolic acid, showed that ribavirin did not act against JUNV through IMPDH inhibition whereas the anti-JUNV activity of mycophenolic acid was mainly targeted to this enzyme. In conclusion, these results are consistent with a more than single mode of action for 3f, as reported for other acridones. A possible cellular target is represented by the enzyme IMPDH whereas another still unidentified and presumably viral target may also contribute to the RNA inhibition of arenaviruses.

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3 ICGEB Workshop on Human RNA Viruses Fundación Instituto Leloir, Buenos Aires, Argentina ____________________________________________________________________________________________________________

POSTERS – BUNYAVIRIDAE #5 – THE ANDES HANTAVIRUS NSs PROTEIN IS EXPRESSED FROM THE VIRAL SMALL MRNA BY A LEAKY SCANNING MECHANISM SOLÍS Loretto1, Jorge Vera-Otarola1, Karla Pino1 y Marcelo López-Lastra1. 1

Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de

Investigaciones Médicas, Facultad de Medicina, Pontificia Universidad Católica de Chile. Andes Virus (ANDV) is a member of the Hantavirus genus the Bunyaviridae family. The viral negativesense RNA genome is comprised of three segments Large (L), Medium (M) and Small (S). The small messenger RNA (SmRNA) that encodes the nucleocapsid (N) protein, exhibits an overlapping (+1) open reading frame (ORF), predicted to encode a putative nonstructural protein (NSs). In this study we evaluated if the ANDV NSs protein was expressed in the context of a viral infection. Our results show that the NSs protein is found in ANDV infected cells. Experiments conducted to understand the mechanism of translation initiation driving NSs protein expression from the SmRNA suggest that recognition of the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream the N protein initiation codon through a leaky scanning mechanism. This study was funded by the Proyecto Instituto Milenio P09/016-F IMII, ICM, and PHS grant 2U01AI045452-11. LS Holds a CONICYT doctoral fellowship.

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POSTERS – FLAVIVIRIDAE #6 – TIA-1 IS A NOVEL HOST FACTOR INVOLVED IN TBEV REPLICATION ALBORNOZ, A, Miorin, L. & Marcello, A. Laboratory of Molecular Virology, the International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy. Tick-borne encephalitis virus (TBEV) is a positive-single stranded RNA virus belonging to the Flaviviridae family. The replication of TBEV occurs in the cytoplasm of infected cells. The contribution of host RNA-binding proteins to flaviviral replication is an important issue under investigation. Tintracellular antigen-1 (TIA-1) is a stress-induced RNA-binding protein shown to be involved in the repression of initiation of translation of cellular mRNAs. When cells are subjected to UV radiation, heat shock, oxidative stress, as well as to certain viral infections, TIA-1 migrates from the nucleus and accumulates to form cytoplasmic foci named stress granules (SG). TIA-1 knockdown by RNAi led to a moderate increase of TBEV replication. During TBEV infection, cytoplasmic TIA-1 was recruited at perinuclear sites of viral replication decreasing TIA-1-SG formation. This effect was TIA-1 specific since analysis of other SG proteins like G3BP1 showed SG formation without redistribution of this protein to sites of viral replication. Moreover, heat-shock treatment of infected cells showed inhibition of TIA-1-SG formation over time compared to mock cells. Again this effect was TIA-1 specific, since G3BP1-SG formation did not show the same behavior. These data indicate that TIA-1 is a cellular restriction factor of TBEV that is recruited to viral replication sites.

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#7 – DISTRIBUTION AND MOLECULAR CHARACTERIZATION OF HEPATITIS C VIRUS (HCV) GENOTYPES IN PATIENTS WITH CHRONIC INFECTION FROM PERNAMBUCO STATE, BRAZIL ALVARADO-MORA, Mónica Viviana1*, Izolda Maria Moura2, Livia de Souza Botelho1, Edmundo Lopes3, Flair José Carrilho1, João Renato Rebello Pinho1 1

Laboratory of Tropical Gastroenterology and Hepatology, Department of Gastroenterology, School of

Medicine, University of São Paulo, São Paulo SP, Brazil. 2

Central laboratory of Clinical Hospital, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.

3

Department of Clinical Medicine, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.

Email: [email protected] Introduction: Hepatitis C virus (HCV), first identified in 1990, is a single strand RNA virus in the family Flaviviridae. HCV is a public health problem throughout the world and, 3% of the world population is infected with this virus. It is estimated that 3-4% millions individuals are being infected every year. The sequencing of HCV isolates has identified 6 genotypes with more than 70 subtypes. Sequencing of NS5B region has been standardized and used for identification of HCV subtypes for epidemiological applications. In Brazil, it has been estimated that around 1.5% of Brazilian populations is anti-HCV positive and the Northeast region showed a higher prevalence among the Brazilian regions. Objective: The aim of this study was to characterize the HCV genotypes circulating in Pernambuco state (PE), Brazil located in the Northeast region of the country. Methods: This study was carried out in Pernambuco state and including 85 anti- HCV positive patients (63 from Recife and 22 from other regions of Pernambuco) collected between 2004 and 2011. Forty-three were males and 42 were females, with age ranging from 26 to 65 years old. Fifty-eight patients were HCV monoinfected, 25 present antibodies to Schistosomiasis (HCV/EHE) and 2 were HCV/HIV coinfected. Fifty-three patients were treatment naïve and 32 have been submitted to treatment with pegylated interferon (Peg-INF) and Ribavirin. Furthermore, fibrosis stage indexes were obtained from liver biopsies performed in 51/85: F0= 3 (5.88%), F1=14 (27.4%), F2=18 (35.3%), F3=12 (23.5%) and four patients (7.84%) had inconclusive result. To perform the phylogenetic analysis, HCV RNA extraction was carried out from 140?l of serum using QIAamp® viral RNA kit (QIAGEN). The reverse transcriptase reaction was performed using the enzyme Reverse Transcriptase Moloney Murine Leukemia Virus (MMLV) and random primers. A fragment of 380bp of HCV NS5B region was amplified by Nested PCR for genotyping analysis. Viral sequences were characterized by phylogenetic analysis using reference sequences obtained from the Genbank (n=224). Sequences were aligned using Muscle software and edit in the SE-AL program. Phylogenetic analysis was conducted using Bayesian Markov chain Monte Carlo simulation (MCMC) using BEAST v.1.5.3. The maximum clade credibility (MCC) tree was obtained from summarizing the substitution trees and then it was removed 10% of burn-in using Tree Annotator v.1.5.3. Results: From 85 samples, 63 (74.1%) were positive to NS5B fragment and successfully sequenced. Subtype 1b was the most prevalent in this population (42- 66.7%), followed by 3a (16-25.4%), 1a (4-6.3%) and 2b (11.6%). Twelve HCV/EHE patients were infected with subtype 1b and seven with subtype 3a, respectively. Conclusions: Brazil is a large country with many different population backgrounds; a large variation in the frequencies of HCV genotypes is predictable throughout its territory. This study reports HCV genotypes from Pernambuco state where subtype 1b was found to be the most prevalent. Also, phylogenetic analysis suggesting the presence of the different HCV strains circulating within this population to the present.

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#8 – GENETIC VARIATION IN HOST IL28B PREDICTS EXTRAHEPATIC HCV INFECTION ANGULO Jenniffer1,3, Karla Pino1, Francisco Biel2, Alejandro Soza2 and Marcelo López-Lastra1 1

Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia (IMII), Centro

de Investigaciones Médicas, y

2

Departamento de Gastroenterología, Facultad de Medicina, Pontificia

Universidad Católica de Chile, Santiago, Chile.

3

Programa de Doctorado en Microbiología (USACH).

Hepatitis C virus (HCV) is mainly hepatotropic, however several reports document the presence of genomic viral RNA in extrahepatic sites including peripheral blood mononuclear cells (PBMCs). Sequence analysis revealed that HCV variants isolated from PBMCs differ from those circulating in plasma. This observation supports the notion of HCV replication in PBMCs. In consequence infection of PBMCs by HCV might represent a viral strategy to evade the host immune response and might also allow HCV to escape antiviral therapy. In this study we evaluated the relationship between the presence of HCV RNA in PBMCs and the response to PegIFN/RBV treatment in chronically infected patients. Results showed a negative correlation suggesting that detection of HCV RNA in PBMCs prior to the initiation of PegIFN/RBV treatment is a negative predictor of achieving a sustained virological response (SVR) (p=0.07). However, not all chronically HCV infected patients exhibited viral RNA in PBMCs, suggesting that host factors might be involved in viral extrahepatic infection. Interested by this possibility we next studied the association between a single nucleotide polymorphism (SNP) (rs8099917), known predictor of antiviral therapy outcome, and the occurrence of HCV-RNA in PBMC in a cohort of 85 chronically infected patients. Results revealed an association between the G allele (GT/GG genotype) and the presence of HCV-RNA in PBMC (OR: 4.073, 95% CI: 1.312-12.64, p= 0.0222). Results indicate that the presence of a G allele in rs8099917 predicts the susceptibility of PBMC to be infected by HCV. Thus, host factors are clearly associated with the ability of HCV to infect and most probably replicate in PBMCs, a phenomenon that may explain the association of SNP rs8099917 with the antiviral therapy outcome. CONICYT Doctoral Fellowship 21090131 P09/016-F IMII

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#9 – DIFFERENTIAL REPLICATION AND PLAQUE MORPHOLOGY OF INFECTIOUS CLONES OF DENGUE VIRUS 2 ARANOVICH, Ezequiel J.; Mondotte, Juan A.; Gamarnik, Andrea V. Laboratorio de Virología Molecular, Fundación Instituto Leloir, Buenos Aires, Argentina. Dengue is a mosquito-borne virus that belongs to the Flaviviridae family together with other important human pathogens such as yellow fever virus, West Nile virus, and Japanese encephalitis virus. Dengue virus infections could result in mild or severe clinical symptoms associated with fever, hemorrhagic fever or death. While in recent years there have been numerous studies on the pathogenesis of DENV, the factors that lead to different outcomes of the disease remain unclear. The aim of this work is to perform a comparative study of the replication properties of an Asian DENV2 infectious clone 16681 (from an isolate associated with hemorrhagic fever) with an Asian / American infectious clone 67655 of DENV2 from an Argentinean isolate (associated with a mild case disease). The two infectious clones were used to generate viral RNA by in vitro transcription, and transfected into mammalian cells (BHK21), human (A549 and Raji DC-SIGN) and Aedes albopictus (C6/36). Infectious viral particles were recovered in each case, which were used to obtain viral stocks. The replication of both viruses was characterized by one-step growth curves, plaque morphology, immunofluorescence as a function of infection, and accumulation of viral RNA by real time RT-PCR. Interestingly, while the infectious clone 16681 replicated to high titers in both mammalian and mosquito cells, the 67655 clone replicated much less efficiently in mammalian cells but retained high titers in mosquito cells. In addition, there was a great difference in the plaque morphology between these two viruses in mammalian cells. Unexpectedly, the virus that replicated to low titers in mammalian cells showed considerable larger plaques, suggesting that (although with lower replication) the 67655 virus was more cytopathic, leading to increased cell death and resulting in larger plaques. In order to define the genetic determinants of the differential phenotype, in particular the plaque morphology, we are constructing chimeric viruses between the 16681 and 67655 infectious clones. These chimeras are currently being tested for their ability to replicate in different host cells and their plaque phenotypes. We conclude that there are significant differences in the replication of the two DENV clones analyzed and that there are viral factors in these two clones that differently affect the host cell.

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#10 – FUNCTIONAL ELEMENTS AT THE C-TERMINUS OF DENV CAPSID PROTEIN BYK, Laura A.; Marcelo M. Samsa; Andrea V. Gamarnik Laboratorio de Virología Molecular, Fundación Instituto Leloir, Buenos Aires, Argentina. Dengue virus (DENV) is a serious human pathogen; each year an estimated 50 million DENV infections occur, with over 500.000 cases hospitalized for dengue hemorrhagic fever. DENV is an enveloped virus with a single stranded positive sense RNA genome of approximately 11kb. Only one ORF has been reported in the viral genome, in which a polyprotein is codified. Its N-terminus contains the information for the synthesis of the structural proteins: capsid (C), membrane (prM) and envelope (E). Following these three, the 7 non-structural (NS) proteins are codified. Once the NS proteins accumulate, the viral genome is used as a template for its amplification. During the new particle formation process, C protein recruits the viral RNA. This mechanism known as nucleocapsid formation is still undescribed. The mature C is a highly basic protein of 12 kDa that forms homodimers in solution. The monomer contains four alpha helices: a1, a2, a3 y a4 and the first 20 amino acids are unstructured in solution. It has been proposed that the highly basic a4-a4’ region interacts with the viral RNA. However, the determinants for this interaction have not been described yet. With the aim of analyzing the role in encapsidation of the positively charged residues located at DENV C C-terminus, four mutants were designed (CT1-4) containing 2 or 3 basic residues substitutions in each case. The mutants were introduced in a DENV infectious clone, which includes a reporter system (RDV) recently developed in our lab. Following luciferase activity as a function of time it is possible to distinguish between the different steps in the viral cycle (translation, RNA synthesis and viral propagation). Furthermore, by infecting fresh cells with the transfection supernatants, RDV allows to study the release of infectious viral particles to the culture media. Transfecting BHK cells with the viral RNA synthesized in vitro, containing or not the mutations in C protein, we were able to determine that even though the RNA translation and genome amplification were not affected, the viral particle formation process was seriously compromised for the CT2, CT3 and CT4 mutants. This effect is less dramatic for CT1. However, in the mosquito cell line C6/36 not only the mutants’ genome translation and RNA amplification were not affected, but also the viral particle production levels were similar to the wild type. These results provide a strong evidence of the role of the basic residues located at DENV C Cterminus in infectious viral particle formation, and indicate a differential requirement of these structural elements in different host cells.

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#11 – STUDY OF THE ANTIVIRAL ACTIVITY OF 1-INDANONE THIOSEMICARBAZONES AGAINST BVDV CASTRO, Eliana F.; Cavallaro Lucía V. Cátedra de Virología. Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires. Bovine viral diarrhea virus (BVDV) is an important pathogen of cattle and a surrogate model for identifying antiviral agents potentially active against hepatitis C virus (HCV). In this work, the antiviral activity against BVDV of a series of 1-indanone thiosemicarbazones and the mechanism of action of the most potent compound: 5,6-dimethoxi-1-indanone thiosemicarbazone (TSC), were investigated. TSC was highly selective against BVDV and inhibited the synthesis of the viral genome both in cell culture and within isolated viral replication complexes. We selected resistant viruses (BVDV-TSCr) in which we identified two mutations in the RNA-dependent RNA polymerase (RdRp). These results together with the results of the docking of TSC in the crystal structure of the RdRp of BVDV, allowed us to define this enzyme as the target of the antiviral activity of TSC. The resistance to TSC was stable, and when BVDV-TSCr were propagated in absence of compound, different replication patterns and a possible change in the cytopathic phenotype were observed. Finally, we evaluated the antiviral activity against BVDV of TSC combined with Ribavirin (drug used to treat HCV infections) and a synergistic effect was demonstrated. In this work, TSC is identified as a new nonnucleoside polymerase inhibitor of the polymerase of BVDV and a potential antiviral activity against HCV is proposed.

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#12 – THE INVOLVEMENT OF P2X7 RECEPTOR IN HUMAN MONOCYTES INFECTED WITH DENGUE-2-VIRUS: PRELIMINARY STUDIES CORRÊA, Gladys1*, Mariana Gandini1, Carina Zogovich1, Robson Coutinho-Silva2 and Claire F. Kubelka1 1

Laboratório de Imunologia Viral – Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Brazil

2

Laboratório de Imunofisiologia – Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do

Rio de Janeiro, Brazil. *[email protected] Extracellular ATP (ATPe) via P2X7 receptors is an important component of the inflammatory response to infection. Studies have shown that ATPe can also have a direct effect on microbial infection by modulating membrane trafficking in cells that contain vacuoles that harbor intracellular pathogens, such as Mycobacteria, Chlamydiae and Toxoplasma. Dengue virus (DENV) infection is a mosquitoborne disease that has spread very fast in the last 50 years and it is a major public health problem.Dengue viruses are single stranded RNA viruses that belong to the family Flaviviridae. Following the bite of a mosquito, usually A. aegypti or A. albopictus, DENV can cause a wide range of mild-to-severe illnesses. The aim of this study was to determinate the P2X7 receptor involvement during infection by DENV-2. Material and Methods: Human monocyte-enriched PBMC were cultured for a 24h period at 37°C. Cells were treated with ATP before or after DENV infection for 30 min. The supernatants were collected and stored at -20ºC for further analysis such as ELISA (viral NS1) or CBA assay (IP-10 and MCP-1). Monocytes were fixed with 2% formaldehyde and processed for flow cytometry, with Dengue-complex and Alexa 647 antibodies. Infected monocytes were also submitted to permeabilization assay with incorporation of ethidium bromide observed by fluorescence microscopy. Results: We observed that the treatment with ATP before infection can decrease the virus antigen detection. We also observed that chemokines related with dengue severity, like IP-10 and MCP-1 presented reduced levels after ATP treatment. Conclusion: We concluded that the P2X7 receptor maybe involved with inhibition of Dengue virus replication. The study of purinergic signaling during dengue fever is a very promising field. Financial support: FAPERJ, CAPES, CNPq and FIOCRUZ.

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#13 – CHARACTERIZATION OF DENGUE VIRUS RNA REPLICATION ELEMENTS DE BORBA, Luana1, Sergio M. Villordo1 and Andrea V. Gamarnik1 1

Laboratorio de Virología Molecular, Fundación Instituto Leloir, Buenos Aires, Argentina.

Dengue virus (DENV) is an arthropod-borne flavivirus that belongs to the family Flaviviridae, and it is transmitted to humans by bite of the mosquito vector Aedes aegypti. The DENV genome is a positive sense single-stranded RNA molecule that encodes a single open read frame (ORF), which is flanked by two untranslated regions (5’ and 3’UTR). The ORF translation generates a single polyprotein that is cleaved by host and virus-derived proteases to produce three structural (C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). It is known that several elements located in the viral 5’ and 3’UTRs and within the N-terminal capsid coding region play important role for the regulation of RNA translation and replication; (a) the 5’ complementary sequence (CS) element found within the N-terminal part of protein C that interact with 3’CS, identified within the 3’UTR, upstream of the conserved stem-loop structure (3’SL); (b) the 5’UAR sequence, located just upstream of the translation initiator AUG at the 5’UTR that is complementary to the 3’UAR, a region present within the stem of the 3’SL; (c) a small motif located downstream of the AUG, designated the 5’ downstream AUG region (5’DAR) that is predicted to be complementary in the 3’ end (3’DAR); (d) the 5’ stem-loop A (5’SLA) that interacts with the viral RNA-dependent RNA polymerase; and (e) the stem-loop cHP, found in the capsid coding region. Despite the many identified elements, the replication, translation or even encapsidation mechanisms of the dengue virus are not fully understood. Our goal is to perform a biochemical and biological characterization of an RNA element located in the coding sequence of the capsid protein, just downstream of the cHP element (nucleotides 45 to 198). Preliminary results, using a recombinant DENV RNA encoding a deletion of nucleotides 45 to 198, showed a delay in viral replication in mammalian cells to a level that was about 40 fold less than that observed for a WT virus at 24h post transfection (hpt), with a slight recovery up to 72 hours. However, it was not possible to detect infectious particles in these supernatants. Similarly, no viral replication or propagation was detected in mosquito cells up to 120hpt. Our findings suggest that this RNA element, present in the capsid coding sequence, plays a role in modulating DENV replication in different host cells. Further experiments are being conducted to better elucidate the mechanisms of action of these new cis-acting RNA elements in DENV life cycle.

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#14 – MODELING GENE SEQUENCE CHANGES OVER TIME IN TYPE 3 DENGUE VIRUSES FROM SOUTH AMERICA: DIVERSIFICATION, RATES AND POPULATION DYNAMICS. FAJARDO, Alvaro†, Alvaro Ramírez†, Zoila Moros, Marlene Gerder, Gerson Caraballo, Daria Camacho, Guillermo Comach, Victor Alarcón, Julio Zambrano, Rosa Hernández, Gonzalo Moratorio, Ferdinando Liprandi and Juan Cristina Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, UdelaR We have obtained nucleotide sequences corresponding to NS5 regions (RNA dependent RNA polymerase), prM-C (pre-membrane and capsid) and E (Envelope) of DENV. This allowed us to study the genetic variability of DENV, as well as to characterize the strains circulating in the region and establish its molecular epidemiology. Also, using Bayesian coalescence analyses we studied the evolutionary rates and population dynamics of DENV in the South American region. In particular we focused on the study of variants of DENV serotype 3 (DENV-3), since it is one of the most prevalent serotypes in South America. The main results obtained were: 1) Study of the phylogenetic relationships between DENV-3 strains isolated in Ecuador and Venezuela. All strains of DENV-3 circulating in the Latin American region that were included in these studies belong to genotype III. This particular genotype has been associated with large outbreaks of dengue hemorrhagic fever (DHF) and therefore with more severe clinical conditions that can cause death. In addition, we could appreciate a great diversity of this genotype in the Americas. This fact is related with epidemiological data that indicate a higher incidence of this genotype with most severe forms of the disease. 2) Estimation of evolutionary rates and population dynamics of DENV-3 in South America. The genotype III of DENV-3 has been expanding in the region, and its evolutionary rates are comparatively higher than those reported for other DENV serotypes. This is consistent with recent studies and is also associated with the epidemiological data of this genotype.

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#15 – PUTATIVE ROLE OF THE CAPSID PROTEIN IN DENGUE CELLULAR INFECTION: FUSION MECHANISM RE-INTERPRETED FREIRE, J.M.1, Veiga, A.S.1, Conceição T.M.2, Santos, N.C.1, Kowalczyk, W.2, Andreu D.2, Da Poian, A.T.3 and Castanho, M.1 1

Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas

Moniz, 1649-028 Lisbon, Portugal, 2Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil, 3Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, E-08003 Barcelona, Spain e-mail: [email protected] Dengue Virus (DV) causes about 20.000 deaths due to viral haemorrhagic fever pathology and infects 50-100 million people every year. No effective treatment is available and several aspects of the viral multiplication and infectivity remain unclear. The functionalities of the capsid protein (DVCP), for instance, remain elusive. Two peptides derived from two conserved domains of DVCP as well as the full protein were studied in the presence of oligonucleotides and biological membrane models (Large Unilamellar Vesicles) using Zeta-Potential, Fluorescence Spectroscopy and Confocal Microscopy in order to elucidade DVCP role in DV infection. Peptides R and M, these being respectively the putative DVCP RNA- and membrane-binding domains, show affinity for model lipid bilayers and for biological membranes (BHK, PBMC and HepG2 cells). The results obtained reveal that DVCP, so far assigned to structural functions, may play a key role during the entry steps of DV in Cell infection. The biological implications of these results will be discussed, including pinpointing novel antiviral selective targets. Keys Words: Dengue virus, Capsid Protein, Peptide, Spectroscopy, Lipid Membranes Acknowledgements: PTDC/QUI/69937/2006 and PTDC/QUI-BIQ/112929/2009 from Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal), FP7-PEOPLE IRSES (International Research Staff Exchange Scheme) project MEMPEPACROSS (EU), FP7-HEALTH-F32008-223414 (LEISHDRUG) project from the EU, and by the Brazilian Funding Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and National Institute of Science and Technology in Dengue (INCT-Dengue). FCT-MEC for JMF PhD grant SFRH/BD/70423/2010. WK is a fellow in the Juan de la Cierva Program of the Spanish Ministry of Science and Innovation.

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#16 – LIPID RAFTS ASSOCIATION WITH THE PROTEIN NS3 AND DSRNA OF DENGUE VIRUS IN CELLS HMEC-I JUÁREZ Moises León1, Garcia-Cordero Julio1, Gutiérrez-Castañeda Benito2, González-Y-Merchand Jorge3, Cedillo-Barrón Leticia1 1

Depto. Biomedicina Molecular, CINVESTAV, D.F, México.

2

Facultad de Estudios Superiores IZTACALA UNAM; Estado de México, México.

3

Dpto. de Microbiología Molecular ENCB-IPN D.F. México.

Dengue is a mosquito-borne viral disease and the causative agent is an enveloped virus of positivestrand RNA of the same name. The interaction of virus with the host cell is still a matter of study. Several reports have shown that different events of the virus replicative are carried out in membranous organelles. It has been shown that the replicative complex consisting proteins NS3 and NS5 is associated with membranes of the endoplasmic reticulum. Cell membranes are characterized by the presence of dynamic structures componed by proteins and lipids called lipid rafts. Where different process occur such cell signaling. The goal of my work is to assess whether the dengue virus uses a cellular lipid rafts during the enter process by using a model of Endotelial cells. According with ours results HMEC-I cells were infected with Dengue-2 virus and analyzed 48 h post-infection by microscopy to assess the degree of colocalization of nonstructural protein 3 (NS3) and intermediate replicative (dsRNA), which is part of the replication complex residents marker lipid rafts such as caveolin, cholera toxin receptor. Confocal microscopy results show 40% colocalization between markers of lipid rafts with viral protease NS3, but not for the transferrin receptor is a molecule absent in lipid rafts. In addition it was also observed that the protein caveolin colocalizes with replicative intermediate (dsRNA). It is suggested that the complex of dengue virus replication takes place in these lipid microdomains.

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#17 – EXPLORING DIFFERENT STRATEGIES TO EXPRESS DENGUE VIRUS ENVELOPE PROTEIN IN PLANT SYSTEMS MARTINEZ Carolina Andrea1, Ana María Giulietti1, Hugh Mason2, Julián Rodríguez Talou1 1

Industrial Microbiology and Biotechnology Area, School of Pharmacy and Biochemistry, University of

Buenos Aires, Buenos Aires, Argentina. 2

The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA.

Considerable research effort has been made towards developing subunit dengue virus (DV) vaccines using different expression systems. In addition, there is currently a requirement for a cost-effective and reliable diagnostic reagent to be used in the detection of DV in human population. Dengue virus envelope protein (E) is the main protein associated with immunity induction and has been shown to be an effective vaccine candidate and a promising antigen for diagnostic kits. Numerous reports are available describing the production of recombinant E-protein in different expression systems, such as yeast, baculovirus, Escherichia coli and mammalian cells. In this way, our laboratory has long been interested in developing plant cell platforms to express DV proteins as a future subunit vaccine and diagnostic kit component. With the aim of exploring different expression strategies, DV recombinant proteins were produced using tobacco plants and plant cell suspension cultures. Both, plant cell suspension cultures and tobacco plants studied were suitable systems for the production of the recombinant DV-2 E protein. Besides, a truncated version of the E-protein (Et) was designed to be expressed alone and co-expressed with dengue virus structural proteins C and prM (CprMEt). As well, the critical domain III of E-protein was fused to the carrier hepatitis B core antigen (HBcoreDV2d3). The recombinant proteins were successfully produced in the different plant systems and were reactive with the anti-E antibody. The HBcore-DV2d3 fusion was reactive with both anti-E and anti-HBcore antibodies. While electron microscopy studies are currently in progress, preliminary sucrose gradient sedimentation analysis showed that the plant-produced HBcore-DV2de fusion protein had a particulate nature.

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#18 – DYNAMIC TRAFFICKING OF PROTEINS AND FLAVIVIRUS RNA BETWEEN REPLICATION COMPARTMENTS AND THE CYTOSOL 1

MIORIN Lisa, 1Amelina Albornoz, 1,2Paolo Maiuri, 1Alessandro Marcello

1

Laboratory of Molecular Virology, The International Center for Genetic Engineering and

Biotechnology (ICGEB), Padriciano, Trieste, 34149 - Italy. 2

Current address: Systems Cell Biology of Cell polarity and Cell division, Institut Curie, CNRS,

UMR144, 26 rue d'Ulm, 75005 Paris - France. RNA viruses induce host cell membranes’ rearrangements to replicate efficiently and possibly to escape from innate immunity. In this work we exploited the tick-borne encephalitis flavivirus as a model to address the dynamics of viral RNA in virus-induced cytoplasmic compartments. We initially observed a consistent delay of interferon induction following virus replication. Delay in interferon induction correlated with a defect in pattern recognition receptor’s signaling. However, viral proteins couldn’t directly inhibit the pathway suggesting an indirect mechanism. In order to dissect the role of replication compartments in this delay we explored protein and viral RNA trafficking in the living cell. We found that cytoplasmic sites of replicated viral RNA accumulation are able to exchange proteins with the cytosol. However, viral RNA trafficking to the cytosol was significantly impaired. We conclude that these dynamic replication compartments established early during infection allow exchange of proteins with the cytosol, but this is insufficient to trigger IRF-3 signaling and interferon induction.

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#19 – A DETAILED COMPARATIVE ANALYSIS ON THE OVERALL CODON USAGE PATTERNS IN WEST NILE VIRUS: BIAS AND HOST INFLUENCE IN VIRUS CODON USAGE PATTERN. MORATORIO Gonzaloa, Andrés Iriarteb,c, Pilar Morenoa, Héctor Mustob and Juan Cristinaa* a

Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias,

Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. b

Laboratorio de Organización y Evolución del Genoma, Instituto de Biología, Facultad de Ciencias,

Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. c

Laboratorio de Evolución, Instituto de Biología, Facultad de Ciencias, Universidad de la República,

Iguá 4225, 11400 Montevideo, Uruguay. West Nile virus (WNV) is a member of the family Flaviviridae and its genome consist of an 11-kb single-stranded, positive-sense RNA. WNV is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in horses and humans, which serve as incidental dead-end hosts. Understanding the extent and causes of biases in codon usage is essential to the understanding of viral evolution. In this study, we performed a comprehensive codon usage analysis of 449 WNV strains, for which complete genome sequences are available. Effective number of codons (ENC) indicates that the overall codon usage among WNV strains is only slightly biased. The frequencies of codon usage in WNV ORFs are significantly different than the ones used by human or Culex pipiens quinquefasciatus vector cells. Furthermore, triplet choices are also strongly influenced by the underlying biases in base composition. The relative dinucleotide abundances suggest that codon usage in WNV can also be strongly influenced by biases in dinucleotide frequencies. No correlation was found between the number of isoacceptor tRNAs on human cells and codon usage of the WNV included in these studies, revealing a poor adaptation of the virus to the respective host tRNA pool. Taking together, the results of this work suggest that WNV genomic biases are the result of the evolution of genome composition, the need to escape the antiviral cell responses and a dynamic process of mutation and selection to re-adapt its codon usage to different environments.

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#20 – CONSTRUCTION AND CHARACTERIZATION OF A STABLE SUBGENOMIC REPLICON SYSTEM OF A DENGUE VIRUS TYPE 3 STRAIN (BR DEN3 290-02) MOSIMANN Ana Luiza Pamplona1, Luana de Borba1,3, Juliano Bordignon1, Peter W. Mason2, Claudia N. Duarte dos Santos1 1

Instituto Carlos Chagas, Rua Prof. Algacyr Munhoz Mader, 3775, Curitiba, Paraná, Brazil.

2

Department of Pathology, University of Texas Medical Branch, 3.206B Mary Moody Northen Pavilion,

301 University Boulevard, Galveston, TX 77555-0436, USA. 3

Present address: Fundación Instituto Leloir, Av. Patricias Argentinas 435, Buenos Aires, Argentina

Dengue viruses cause the most common arboviral disease afflicting men. They belong to the genus Flavivirus, family Flaviviridae and are single stranded RNA viruses with a genome of ~11kb. Its genome is flanked by non-translating regions (NTRs) and codes for tree structural (C, prM and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) which are translated into a polyprotein and processed by viral and host proteases. Until now there is no specific treatment for the disease and no efficient vaccine is available. The clinical manifestations can range from asymptomatic to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The mechanisms involved in the disease pathogenesis are not well understood and are still a challenge. The outcome seems to be the result of a combination of viral and host factors. In this context the study of molecular markers of virulence can contribute to a better understanding of dengue pathogenesis and eventually direct interventions such as antiviral therapy. In this sense the use of a subgenomic replicon system represents a powerful research instrument as it can be used: to define the role of mutations in the non-structural proteins, to study viral replication and even to test antiviral drugs. Our objective was to generate and characterize biologically a replicon generated from a recent clinical isolate of dengue virus serotype 3. To achieve our objective we first cloned different RT-PCR fragments from the non-structural proteins in the pGEM-T-easy vector. All clones were sequenced and used to assemble the replicon in a pBAC vector containing a T7 RNA polymerase promoter, a ribozyme sequence and a chloranphenicol resistance gene. All cloning procedure was done using Top10 E. coli strain. After the generation of a stable clone, it was used for in vitro transcription and transfection in C6/36 mosquito cells. Cells were analyzed by indirect immunofluorescence using dengue 3 polyclonal antibodies in different time points (48, 72, 96 and 120h) post-transfection. The results showed that the replicon was efficiently transfected and replicated in the cells. We also observed that the non-structural proteins could be recognized by the polyclonal antibody, but no virus particle was produced. As a perspective, we intend to assay mutations involved in a neurovirulent viral phenotype in the non-structural proteins that could modulate virus replication and be involved in dengue pathogenesis.

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#21 – CHARACTERIZATION OF DENGUE VIRUS TYPE 3 ENTRY IN VERO AND A549 CELLS PICCINI LE, Castilla V, Damonte EB Laboratory of Virology, Department of Biological Chemistry, School of Sciences, University of Buenos Aires. Dengue Virus (DENV) is an arthropod-borne virus of the family Flaviviridae that can cause in the man a feverish acute benign disease and self-limited, the dengue fever, or the more severe and potentially lethal forms of dengue hemorrhagic fever/dengue shock syndrome. The knowledge of the viral multiplication cycle is indispensable to the finding of potential target for antiviral agents. Virus entry is an attractive target for antiviral intervention that gained attention in recent years to block the initiation of infection. The entry of DENV into the host cell appears to be a very complex process that started to be studied in detail in recent years. We have previously demonstrated a differential mode of infective entry for DENV-1 and DENV-2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV-2, into different types of cells. The aim of the present study is to characterize DENV- 3 internalization process in mammalian Vero and A549 cells, using pharmacological inhibitors of different endocytic pathways. We evaluated the effect of non-cytotoxic concentrations of ammonium chloride (lysosomotropic compound), concanamycin A (specific inhibitor of vacuolar-proton ATPase), chlorpromazine and dansylcadaverine (inhibitors of clathrin-dependent endocytosis) and dynasore (dynamin inhibitor) on DENV-3 multiplication by performing a virus yield inhibition assay. Ammonium chloride, concanamycin A and dynasore inhibited virus replication in a dose-dependent manner in both cell systems. On the contrary, treatment with chlorpromazine and dansylcadaverine caused an increase in virus titres. Similar results were obtained when we analyzed the effect of these treatments on the expression of DENV-3 E glycoprotein using an indirect immunofluorescence assay. We next corroborated that the effect of ammonium chloride, concanamycin A and dynasore was exerted at the internalization of DENV-3 by performing an infectious centre assay. We also investigated the effect of nystatin and methyl-beta-cyclodextrin, two sterol-binding drugs that function as inhibitors of caveolae-mediated endocytosis, on DENV-3 internalization and we found a partial inhibition of infectious centre formation when either Vero or A549 cells were treated with the compounds previous to virus infection. We also determined that among the assayed inhibitors, only nystatin and methyl-beta-cyclodextrin exhibit direct viral inactivation effect since incubation of viral particles with these compounds in the absence of cells caused a strong reduction in virus infectivity. Thus, our results indicate that the entry of DENV-3 into Vero and A549 cells occurs through an acid pH-dependent endocytic pathway, dependent on dynamin and independent of clatrhin. These results differ from those previously reported for DENV-1 and DENV-2 confirming the use of alternatives internalization routes by different DENV serotypes.

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#22 – VITAMIN D3 REDUCES DENGUE VIRUS INFECTION AND CYTOKINE PRODUCTION FROM CULTURED CELLS, A POTENTIAL THERAPEUTIC STRATEGY FOR DENGUE TREATMENT PUERTA-GUARDO, Henry1, Sergio Isaac De la Cruz Hernández1,3, Victor H. Rosales2, Juan E. Ludert1, Rosa María del Angel1 1

Departamento de Infectómica y Patogénesis Molecular,

Investigación y Estudios Avanzados,

3

2

Laboratorios centrales Centro de

Departamento de Virología, Instituto de Diagnostico y

Referencia Epimiológicos (InDRE), México, D.F., Mexico ([email protected]) Dengue is the most important mosquito-borne viral infection to humans and constitutes a global public health problem. Yet, neither vaccine nor effective therapeutics is currently available for dengue virus (DENV). Vitamin D is known as a modulator of calcium homeostasis, and newer evidence proposes that it also plays a role in cellular growth, differentiation, and immunomodulation. In addition, recent evidence suggests that the active form of vitamin D3 [1α,-25-dihydroxyvitamin D3 (VD3)] may influence virus replication3. Thus, the aim of this work was to evaluate the effect of VD3 treatment on dengue virus infection in the human hepatic cell line Huh-7 and on virus infection and cytokine production in the human monocytic U937 cells. Treatment of either of these cell lines with VD3 resulted in a significant reduction in the number of infected cells and virus yield, in effective conditions where cell viability was not affected. Viral replication in monocytic cells was more susceptible to the treatments than replication in the hepatic cells. In addition, VD3 treatment significantly reduced the levels of proinflammatory cytokines (TNF-α, IL-6, IL-12p70 and IL-1β) produced by U937 cell upon infection. These results suggest that VD3 may represent a potentially useful antiviral compound to avoid or reduce the severe clinical forms of the dengue.

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#23 - EARLY MOLECULAR MARKERS PREDICTIVE OF DENGUE HEMORRHAGIC FEVER 1

CALZAVARA-SILVA, Carlos E., 2Mayara M. C. Silva, 3Eduardo J. M. Nascimento, 4Ana L. V.

Gomes, 4Rita C. C. Maia, 2Bartolomeu Acioli-Santos, 2Laura H. V. G. Gil, 2,3Ernesto T. A. Marques Jr. 1

Laboratory of Cellular and Molecular Immunology, Rene Rachou Research Center, Oswaldo Cruz

Foundation, Brazil. 2Laboratory of Virology and Experimental Therapy, Aggeu Magalhães Research Center, Oswaldo Cruz Foundation, Brazil. 3Center for Vaccine Research, Department of Infectious Diseases and Microbiology, Biomedical Science Tower, Pittsburgh, USA. 4Department of Veterinary Medicine, Federal Rural University of Pernambuco, Brazil. The triage and management of acute dengue patients during outbreaks is a challenging problem. Most of the dengue fever cases are benign, however some cases develop into a severe and possibly lethal vasculopathy, known as dengue hemorrhagic fever (DHF) or severe dengue. Early symptoms of mild dengue and hemorrhagic fever are very similar, therefore, a differential diagnosis able to foreseeing the propensity of a patient to develop the severe symptoms of dengue could be very useful to proper patient care. On the other hand, the molecular events triggered in human cells after infection by dengue virus (DV) have not been completely elucidated yet. To define the molecular factors involved in the evolution of dengue fever to DHF could permit the development of therapeutic strategies and effective diagnostic methods for high-risk patients and consequently reduce the mortality caused by DV. By using microarray, our group has already found several genes differentially expressed among mild and severe dengue patients and also febrile non-dengue patients. Using cDNA obtained from peripheral blood mononuclear cells (PBMC) of DENV-infected individuals as templates for quantitative real-time PCR reactions, we have been quantified the mRNA expression levels of about 30 genes involved in activation of innate immune response, complement cascade, apoptosis process and interferon pathways. The results showed, during the initial days of DENV infection, an overexpression of IFN-alpha, especially in patients whose developed DHF suggesting that exacerbated levels of IFN-alpha could be related to the development of severe symptoms of dengue. In a later phase of infection the IFN-alpha response is reduced while the IFN-beta expression levels remained high. During all stages of infection analyzed in this study we observed low levels of IFN-gamma response. The qPCR assays also showed that five of the tested genes were consistently differentially expressed between dengue and DHF samples, (PRDX4 and MAGED1 overexpressed in DHF samples and CFD, PSMB9 and FcGR3b overexpressed in mild dengue-patient samples) being useful to putatively compose a panel of “dengue hemorrhagic fever signature genes”. Despite the fact that these results grant further validation studies, this panel of candidate prognostic markers demonstrated its potential clinical use in a rapid assay based in blood samples.

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#24 – THE ROLE OF HETEROTYPIC DENV-SPECIFIC CD8+T LYMPHOCYTES IN THE PATHOGENESIS OF DENGUE HEMORRHAGIC FEVER TALARICO, Laura B.1; Hijano, Diego R.1,2; Batallé, Juan P.1; Díaz, Leandro M.1; Mauri, Aldana1; Acosta, Patricio L.1; Lawrence, Andrea2; Zea-Hernández, Johanna A.2; Melendi, Guillermina A.1; Polack, Fernando P.1,2 1

Fundación Infant, Buenos Aires, Argentina. 2Department of Pediatrics, Vanderbilt University, Nashville,

TN, USA. Dengue Virus (DENV) is the most common arboviral cause of illness worldwide. Around 100 million individuals are infected every year. Despite its importance, there is currently no licensed vaccine for prophylaxis or drugs to treat the infection. Annually, 500,000 DENV infections lead to dengue hemorrhagic fever (DHF), with a fatality rate ranging from 1% to 10%. The main hypothesis on the mechanism of illness for DHF involve enhancement of infection mediated by heterotypic antibody and memory T lymphocytes cross reactivity. We recently established a novel immunocompetent mouse model of DHF which shows key signs of disease in humans: thrombocytopenia, capillary leakage, internal hemorrhages, and transaminitis. The aim of the preset work was to study the role of DENV-specific CD8+ T lymphocytes in DHF pathogenesis. C57BL/6 and %MT (B cell deficient) mice were inoculated intraperitoneally (i.p.) with 106 PFU of DENV-1, DENV-2 or placebo. After 60 days mice were challenged with DENV-2. Thrombocytopenia, transaminitis and elevation of creatine kinase (CK) and lactate dehydrogenase (LDH) levels in serum were detected in C57BL/6 mice with DHF. Cellular infiltration in liver, megakaryocytes in spleen, higher viral titer in liver and longer bleeding time were found in mice with DHF. Marked increase in megakaryocytes in spleen, deposits of a homogeneous proteinaceous material in kidney, and extra-medullary hematopoietic centers in liver were found in %MT mice infected sequentially with DENV-1/DENV-2. DENV-2 was detected in liver and kidney. In CD8+T and CD4+/CD8+T lymphocytes depleted C57BL/6 mice sequentially infected with DENV-1/DENV-2 we found significantly low levels of transaminases, CK, LDH, and higher platelet count, while CD4+T lymphocytes depleted mice showed moderately low levels of the same enzymes. Moreover, we found increased levels of CK and LDH and low platelet count in mice that received CD8+T cells from DENV-1 infected mice and later infected with DENV-2. The dominant H-2b restricted CD8+T lymphocyte responses as a result of sequential DENV-1/DENV-2 infection were dominated by CTLs directed against epitopes of NS4b protein. These results show that CD8+T lymphocytes play a critical role in DHF pathogenesis, while CD4+T and B lymphocytes are less critical for the development of severe disease.

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#25 – DIFFERENT CONFORMATIONS OF THE DENGUE VIRUS RNA GENOME ARE CRUCIAL FOR INFECTIVITY VILLORDO Sergio M., Diego E. Alvarez, Andrea V. Gamarnik Fundación Instituto Leloir, Buenos Aires, Argentina The structure and plasticity of viral plus strand RNA genomes are fundamental for the multiple functions of these molecules. Local and long-range RNA-RNA interactions provide the scaffold for interacting proteins of the translation, replication, and encapsidation machinery. Using dengue virus as a model, we investigated the relevance of the interplay between two alternative conformations of the viral genome during replication. Flaviviruses require long-range RNA-RNA interactions and genome cyclization for RNA synthesis. In our studies, we defined a sequence present in the dengue virus 3’UTR that overlaps two mutually exclusive structures. This sequence can form an extended duplex by long range 5’-3’ interactions in the circular conformation of the RNA or fold locally into a small hairpin (sHP) in the linear form of the genome. A mutational analysis of the sHP structure revealed an absolute requirement of this element for viral viability, suggesting the need of a linear conformation of the genome. Viral RNA replication showed high vulnerability to changes that alter the balance between circular and linear forms of the RNA. Mutations that shift the equilibrium towards the circular or the linear conformation of the genome spontaneously revert to sequences with different mutations that tend to restore the relative stability of the two competing structures. We propose a model in which the viral genome exists in at least two alternative conformations and the balance between these two states is critical for infectivity.

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POSTERS – HEPADNAVIRIDAE #26 – HDAg-L VARIANTS IN COVERT HEPATITIS D AND HBV OCCULT INFECTION AMONG AMERINDIANS OF ARGENTINA: NEW INSIGHTS DELFINO Cecilia M1,2, María E. Eirin1, Carolina Berini1, Richard Malan3, Emiliano Gentile2, Amalia Castillo2, Williams Pedrozo3, Ramón Krupp3, Jorgelina Blejer4, José R.Oubiña2, Verónica L. Mathet2 and Mirna M. Biglione1. 1

Biomedical Research on Retrovirus and AIDS Institute (INBIRS), University of Buenos Aires (UBA).

Ciudad Autónoma de Buenos Aires, Argentina. 2

Institute of Medical Microbiology and Parasitology (IMPAM) UBA – National Research Council

(CONICET), Ciudad Autónoma de Buenos Aires, Argentina. 3

Central Blood Bank of Misiones province, Posadas, Misiones, Argentina.

4

Serology Area, Transfusion Medicine Section, Favaloro Foundation, University Hospital. Ciudad

Autónoma de Buenos Aires, Argentina. Background: Hepatitis D virus (HDV) is a defective virus that requires the helper function of hepatitis B virus (HBV) for its assembly and transmission. The HDV particle consists of an outer envelope of HBV surface proteins, while the genomic RNA is associated with two isoforms of the delta antigen, HDAg-S and HDAg-L. The HDAg-S plays an essential role in transactivating the replication of the HDV RNA, while HDAg-L interacts with HBsAg in the assembly of HDV and is a dominant repressor of HDV RNA replication. Guidelines suggest that all HBsAg-positive patients should be tested for anti-HDV IgG antibodies and to confirm active hepatitis D virus (HDV) infection by detection of HDV RNA by reverse transcriptase (RT) Polymerase chain reaction (PCR). Objectives: The aim of this study was to determine the serological prevalence and molecular features of HDV within an Amerindian community from Argentina exhibiting positivity for HBsAg and/or anti-HBc total Ig. Study design: Forty six plasma samples were tested for the detection of total anti-HDV antibodies by ELISA. Concomitantly, a partial RNA region coding for the delta antigen (HDAg) was amplified by RT-nested PCR (RT-nPCR). In silica translation of DNA sequences into the amino acid (aa) sequence of HDAg-S (aa110 to 198) and HDAg-L (aa 110 to 214) was performed. Results: Out of 46 HDV non-reactive samples by ELISA, 3 were HDV RNA positive by RT-nPCR. These samples were anti-HBc only positive, 2 of them identified as cases of occult B infection (OBI). The 3 cases were HBeAg negative and showed normal ALT/AST levels. All sequences were ascribed to HDV genotype 1, but exhibited nucleotide differences in HDAg-L coding region, among which, mutations at codons 197 and 201 -reportedly known to promote in vitro an unsuitable interaction with HBsAg- were observed. Conclusions: These results provide evidence of covert HDV infection even among OBI, highlighting the need to reevaluate the currently applied guidelines for HDV diagnostic algorithms, as well as to explore if the observed mutations promote any effect on HDV pathogenesis.

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POSTERS – HEPEVIRIDAE #27 – GENETIC VARIABILITY OF HEPATITIS E VIRUS IN URUGUAY RAMOS Natalia1, Santiago Mirazo1, José Russi2, Juan Arbiza1. 1. Sección Virología. Facultad de Ciencias, Uruguay. 2. British Hospital, Uruguay Hepatitis E infection is caused by Hepatitis E virus (HEV), the sole member of Hepeviridae family. Viral genome structure consists of three discontinuous open reading frames (ORFs). The virus infection is moderately severe and usually self-limiting, with death rate of