Research Group Information
(RG-LVT-Lisboa-750018-3140)
Designation:
Virology
Principal Investigator:
Celso Vladimiro Ferreira de Abreu Cunha
Location of Group:
Instituto de Higiene e Medicina Tropical - Universidade Nova de Lisboa
Keywords:
Hepatitis D virus
,Nucleocytoplasmic transport
,HIV
,Molecular epidemiology
Funding, sources, dates
Gene expression changes induced by the hepatitis delta virus. I- Analysis of the cellular proteome. POCI/SAU-IMI/55112/2004. FCT/FEDER, 99.056,00 euro
EC (Multicenter projects SPREAD, EuroHIVResistance, CASCADE and CHAIN). The SPREAD project finished in 2005. The others are currently ongoing.
FCT (Fundação para a Ciência e Tecnologia) (two grants for PhD abroad)
ANRS (Association Nacionale pour la Recherche du SIDA)
GlaxoSmithKline Foundation
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Group Team
List of Researchers in the Group:
001. Celso Vladimiro Ferreira de Abreu Cunha
( Cat.: Professor Auxiliar
Gr. Acad.: Doutoramento )
List of Collaborators (w/PhD):
001. Perpetua da Conceicao Rodrigues Gomes Cavaco Silva
( Cat.: Investigador Principal
Gr. Acad.: Doutoramento )
List of Collaborators (w/o PhD):
001. Ricardo Jorge Gonçalves Ornelas Camacho
( Cat.: Investigador Principal
Gr. Acad.: Mestrado )
002. Ana Leonor Vidal Gomes Casaca
( Cat.: Não aplicável (bolseiro)
Gr. Acad.: Licenciatura )
003. Carolina Alpalhão Mantero de Mendonça Alves
( Cat.: Não aplicável (bolseiro)
Gr. Acad.: Mestrado )
004. Marta Maria Lavouras Mendes
( Cat.: Não aplicável (bolseiro)
Gr. Acad.: Mestrado )
005. Natalia Maria Bezerra de Freitas
( Cat.: Não aplicável (bolseiro)
Gr. Acad.: Licenciatura )
006. Sérgio Miguel Regufe da Mota
( Cat.: Não aplicável (bolseiro)
Gr. Acad.: Licenciatura )
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Objectives & Achievements
Objectives:
HDV: To contribute to a deeper understanding of fundamental processes, at the molecular level, that mediates host-pathogen interactions.
HIV: Study of HIV-1 resistance to antiretrovirals, focusing on non-B genetic variants; study of HIV-2 resistance to antiretrovirals; molecular epidemiology of HIV-1 infection in Portugal; surveillance of resistance transmission.
Main Achievements:
This report concerns the activities of two subgroups studying HDV and HIV, respectively. The HIV subgroup, coordinated by R Camacho, integrated CMDT-LA since the beginning of the Center, and the HDV subgroup, coordinated by C Cunha, was integrated in CMDT-LA in 2006.
HDV: We have identified and characterized the nuclear localization signal (NLS) of the delta antigen showing that it consists of aa 66-75 (Alves et al.). We additionally identified a constitutive transport element (CTE) in the antigenomic RNA of the HDV also showing that it is localized in positions... (Freitas et al., manuscript in preparation). These two findings allowed us to identify and characterize the amino acid and nucleotide sequences, respectively, that mediate the nucleocytoplasmic traffic of HDV RNP particles during infection.
Moreover, we performed a comprehensive proteomic analysis of human liver cells transiently transfected with all the HDV components, separately, as well as of a HDV cDNA stably transfected cell line (Mota et al., J Prot. and Mota et al., submitted).This allowed us to get a global picture of gene expression changes, at the protein level, induced during HDV replication. Consequently, potential mechanisms of pathogenesis uncovered during this work can now more deeply be investigated.
HIV: The research performed by the HIV team led to the identification of novel HIV-1 resistance mutations and resistance pathways in subtypes A, C, F, G and CRF02_AG. Additionally, novel resistance mutations in HIV-2 were identified. The quantification of resistance transmission in Portugal as well as the characterization of HIV-1 genetic variants circulating in the country are also significant contributions of this team.
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Group Productivity
Publications in peer review Journals (3000 ca.) (Up to a max of 10. Always indicate at the end of the citation, impact factor of the journal (IF=) and number of citations (nº C=). Give title and full citation in original language. DO NOT translate)
Alves, C., Freitas, N., Cunha, C.. Characterization of the nuclear localization signal of the hepatitis delta virus antigen. Virology 370 (1): 12-21. (IF: 3.7; NC: 0)
Mota, S., Mendes, M., Penque, D., Coelho, A. V., Cunha, C.. Changes in the proteome of Huh7 cells induced by transient expression of hepatitis D virus RNA and antigens. Journal of Proteomics 71; 71-79. (IF: na)
Gomes P, Abecasis A, Almeida M, Camacho RJ, Mansinho K. “Transmission of HIV-2”, The Lancet Infect Dis. 2003 Nov; 3: 534-536. (IF: 10.5; NC: 7)
Abecasis A, Paraskevis D, Epalanga M, Fonseca M, Burity F, Bartolomeu J, Carvalho AP, Gomes P, Vandamme AM, Camacho RJ. HIV-1 genetic variants circulation in the North of Angola. Infection, Genetics and Evolution 5 (3), 231–237, 2005 (IF: 2.4)
Abecasis A, Deforche K, Snoeck J, Ghidey W, Carvalho AP, Gomes P, Camacho RJ, Vandamme A-M. Protease Mutation M89I/V is Linked to Therapy Failure in Patients Infected with the HIV-1 Subtypes C, F and G. AIDS 2005, 19: 1799-1806 (IF: 5.5; NC: 1)
Gomes P, Palma AC, Cabanas J, Abecasis A, Carvalho AP, Ziermann R, Diogo I, Gonçalves F, Lobo CS, Camacho RJ: Comparison of the COBAS TAQMAN HIV-1 HPS with VERSANT HIV-1 3.0 Assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes. J Virol Methods, 2006 (IF: 1.8; NC: 5)
Abecasis AB, Deforche K, Bacheler LT, McKenna P, Carvalho AP, Gomes P, Vandamme A-M, Camacho RJ: Investigation of Baseline Susceptibility to protease inhibitors in HIV-1 subtypes C, F G and CRF02_AG. Antiviral Therapy, 2006: 11(5): 581-9 (IF: 5.9; NC: 7)
Camacho RJ, Vandamme A-M: Antiretroviral Resistance in Different HIV-1 Subtypes: Impact on Therapy Outcomes and Resistance Testing Interpretation: Current Opinion on HIV & AIDS, 2007, 2:123-129 (IF: na)
Palma AC, Araujo F, Duque V, Borges F, Paixao MT, Camacho R; on behalf of the Portuguese SPREAD Network. Molecular epidemiology and prevalence of drug resistance-associated mutations in newly diagnosed HIV-1 patients in Portugal. Infect Genet Evol. 7 (2007) 391 – 398 (IF: 2.4; NC: 3)
J. Vercauteren, K. Deforche, K. Theys, M. Debruyne, J.L. Duque, S. Peres, A.P. Carvalho , K.Mansinho, A.-M. Vandamme, RJ. Camacho: The incidence of multidrug and full class resistance in HIV-1 infected patients is decreasing overtime (2001-2006) in Portugal. Retrovirology, 2008 5:12 (IF: 4.0; NC: 0)
Master and Ph.D. thesis completed (3000 ca.)
Marta Mendes – Influence of the HDV genomic and antigenomic and antigenomic RNA expression in human liver cells proteome. Master degree in Biotechnology, Instituto Superior Técnico.
Carolina Alves – Study of the nuclear import mechanism of the hepatitis delta virus antigen. Master degree in Biotechnology, Instituto Superior Técnico.
Sérgio Mota – Influence of siRNAs in the replication of hepatitis delta virus and analysis of gene expression in infected cells. PhD thesis, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa.
Patents/propotypes (2000 ca.)
Carvalhal, Ana Verónica; Lima, Carlos; Basto, Vera; Cunha, Celso; Escada, Pedro; Cruz, Helder; Cruz, Pedro. Adult human neural stem/progenitor cells from the olfactory epithelium and olfactory lamina propria, process for its obtention, proliferation in serum free culture medium and differentiation and utilization for transplantation. International Publication Number WO 2007/020611 A3
Organization of conferences (2000 ca.)
Co- Organization: V European HIV Drug Resistance Workshop, March 2007, Cascais, Portugal
Co-Organization: 13th International Workshop on virus evolution and molecular epidemiology; September 2007, Lisbon, Portugal,
Internationalization (2000 ca.) (Collaborative publication, Research, Graduate Training Networks or other forms of participation of the Research Group at the international level)
The hepatitis D subgroup established a permanent collaboration with the Department of Virology of the Fox Chase Cancer Centre, USA (Dr. John Taylor). In this context, a PhD student, Carolina Alves, started the work at Fox Chase aiming to characterize the HDV RNA transcription pre-initiation complexes. This work is planned for 24 months in the US laboratory and will then be completed in Lisbon. Additionally, both laboratories are working towards a comprehensive characterization of the gene expression changes induced during HDV replication using a recently developed Tet-inducible cell culture system developed in Taylor’s lab. Our group is performing the proteome analysis by LC-MS and the US group is performing microarray analysis. To perform the proteome analysis, collaboration was also established with the group of Jesus Vazquez in Madrid who developed specific software for data analysis of O18 labelled samples. Recently, this group established collaboration with the University of Heidelberg aimed to study intracellular virus traffic.
The HIV subgroup is a partner of the EC funded European projects SPREAD, EuroHIVResistance, CASCADE. Moreover, this team has two well established permanent collaborations with the Rega Institute of Virology, University of Leuven, Belgium, and the University Hospital Américo Boavida, Luanda, Angola. Occasional collaborations with the University of Stanford, CA, USA are also currently ongoing.
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Future Research
Objectives:
HDV - In the near future, we plan to continue the proteome analysis of HDV expressing cells using the Tet-inducible cell line above referred. This collaborative project will hopefully allow us to better characterize the mechanisms of replication and pathogenesis of the virus which may be indicative of a better and specific therapy not currently available. We additionally plan to start the characterization, including genotyping, of the circulating strains in Portugal using a collection of HBV positive sera (~1000) stored in our lab. Moreover, the investigation of the mechanisms of HDV RNA-directed RNA transcription will continue, also as a collaborative project with John Taylor’s lab in Philadelphia.
HIV - The future planned research activities of the HIV team include:
Association with the European network EURESIST.
Building of an European database for the study of antiretroviral resistance (EURESIST).
Continue ongoing research in HIV-1 and HIV-2 drug resistance, with novel targets (integrase, env).
Identification of resistance pathways in HIV-2.
Antiretroviral therapy in resource-poor settings.
Identification of the main problems and potential solutions for the treatment of HIV infections in resource poor settings, with a special focus of laboratory monitoring.
New research line:
Recently, a post-doc, Claudia Istrate, joined our group in order to implement a new rotavirus research line. Two main objectives are intended to be achieved: 1- Characterization of the circulating rotavirus strains in Portugal, Angola and Mozambique. This involves the participation of the Portuguese Paediatrics Society and the EuroRotaNet, the European rotavirus research network which we recently joined. Ultimately, we aim to understand the potential efficacy of the two commercial vaccines based on attenuated virus strains. We further aim to be able to identify possible genetic rearrangements between strains phenomena that already occurred and was described in Europe. This project involves also the collaboration of the London School of Tropical Medicine where the coordination of EuroRotaNet is currently based. 2- Investigation and development of a new vaccine based on virus-like particles. This project, started by CI during the post-doc period in Lijkoping University, Sweden, will continue now in Lisbon with the collaboration of the Swedish group.
Funding, source, dates (indicate in full including amount of current and pending funding)
Currently, the hepatitis D research is financed by FCT. However, the project ends by august 2008. We also have some funding available from bench fees of PhD and masters students, as well as those who attend the post-graduate courses we organize. Nevertheless, we urgently need to apply for funding. The last FCT call for projects opened in the beginning of 2007.
The HIV related activities are financed by the EU, National government and private foundations, and the pharmaceutical industry. We will continue to search for new funding for the planned activities preparing applications for the forthcoming competitive calls.
Concerning the rotavirus research, we intend to apply as soon as new FCT calls open. The EuroRotaNet we recently joined will finance 50% of the budget for the research.
Previous publications in the area (5 in the last 5 years. If available you must indicate at the end of the citation, impact factor of the journal (IF=) and number of citations (n° C=). Give title and full citation in original language)
Alves, C., Freitas, N., Cunha, C.. Characterization of the nuclear localization signal of the hepatitis delta virus antigen. Virology 370 (1): 12-21.
Mota, S., Mendes, M., Penque, D., Coelho, A. V., Cunha, C. Changes in the proteome of Huh7 cells induced by transient expression of hepatitis D virus RNA and antigens. Journal of Proteomics 71; 71-79.
Claudia Istrate, Iyadh Douagi, Annie Charpillienne, Åsa Hidmark, Kari Johansen, Marie Larsson, Karl-Eric Magnusson, Didier Poncet, Lennart Svensson and Jorma Hinkula. Bone marrow dendritic cells internalize live RF-81 bovine rotavirus and rotavirus-like particles (RF 2/6-GFP-VLPs and RF 8*2/6/7-VLPs) but are only activated by live bovine rotavirus. Journal of Scandinavian Immunology, 2007, Jun; 65(6), 494-502
Claudia Istrate, Jorma Hinkula, Lennart Hammarström and Lennart Svensson. Individuals with selective IgA deficiency resolve rotavirus disease and develop higher antibody titers (IgG, IgG1) than IgA competent individuals. Journal of Medical Virology 80:531–535 (2008)
Elin Johansson, Claudia Istrate, Annie Charpillienne, Jean Cohen, Jorma Hinkula, Didier Poncet, Lennart Svensson and Kari Johansen. Amount of maternal rotavirus-specific antibodies influence the outcome of rotavirus vaccination of newborn mice with virus-like particles. Vaccine (2008) 26, 778—785
Abecasis A, Paraskevis D, Epalanga M, Fonseca M, Burity F, Bartolomeu J, Carvalho AP, Gomes P, Vandamme AM, Camacho RJ. HIV-1 genetic variants circulation in the North of Angola. Infection, Genetics and Evolution 5 (3), 231–237, 2005
Special Requirements (equipment, facilities, staff or other special needs essential to carry out the future research program)
Equipment: In the last FCT call for new equipment the CMDT-LA proposal was not granted. A large number of equipment in our laboratory is more then 20 years old. No new heavy equipment was acquired in the last 10 years. We need new laminar air flows, a CO2 incubator, a thermocycler, and electrophoresis tanks, to substitute the old items. Additionally, new microscopes are needed: an inverted light microscope, a fluorescence microscope and a confocal.
Human resources: Hiring a post-doc and a database manager to work on HIV research. Hiring a post-doc to work on HDV research.
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