cmdtla.org - Saúde Tropical

Research Groups

Virology

VIROLOGY
Rotavirus
EuroHIVResistance
HIV Disease progression
HIV-1 Drug Resistance
HIV-2 Drug Resistance
Replication of the hepatitis delta virus
Proteome of liver cells
Nucleocytoplasmic transport of HDV

VIROLOGY

Celso Cunha

Perpétua Gomes

Ricardo Camacho

Claudia Istrate

Keywords: [Hepatitis D virus] [Proteome] [Nucleocytoplasmic transport] [HIV] [Molecular epidemiology] [Resistance]··

More information at Group Report

Rotavirus

rotavirus

Rotavirus and viruses causing gastroenteritis (epidemiological approach):

Current research projects concern epidemiological studies of rotavirus and furthermore viruses causing gastroenteritis in Portugal and PALOP countries.

New approach towards rotavirus vaccines

Another topic of interest represents the development of a rotavirus vaccine candidate using the recombinant protein technology more precisely development and validation in the mouse model of a subunit vaccine using rotavirus virus-like particles produced using the insect cell-baculovirus expression system.

Main collaborations:

Centre for Infectious Diseases (London, UK), Department of Infectious Diseases or Laboratory of Virology (INSA-Lisbon, Portugal), Department of Molecular Virology (University of Linköping, Linköping, Sweden), Faculty of Veterinary Medicine (UTL- Lisbon, Portugal)

EuroHIVResistance

[Ricardo Camacho & col]: EuroHIVResistance (EC funded project): transmission of drug resistan viruses is a serious heath care problem, because of its potencial to jeopardize therapy options and thus having a serious impact on the prognosis of HIV infection. EuroHIVresistance is an European multicenter study to monitor the transmission of HIV-1 resistant viruses, as well as the changes in molecular epidemiology accross Europe.

HIV Disease progression

Evaluation of the disease progression in patients with a known date of seroconversion

[Ricardo Camacho & col] Evaluation of the disease progression in patients with a known date of seroconversion (EC funded project CASCADE): in this project we monitor prospectively the evolution of the HIV-1 infection in patients with a known date of seroconversion. This approach allows us to relate any event with the date of infection, and thus to gain insight of the natural history of the infection and its changes over time.

HIV-1 Drug Resistance

HIV-1 Drug Resistance:[Ricardo Camacho & col]: most of the knowledge we have about HIV-1 drug resistance are based on the study of subtype B viruses, which are predominant in the USA and European epidemic. Less is know about therapy response and molecular markers for HIV-1 drug resistance in the HIV-1 Group M subtypes. In Portugal, subtype B dominated the epidemics in the beginning; somewhere during the last decade, subtype G established a sub-epidemic on its own, followed by CRF02_AG and, to a lesser extent, other non-B subtypes. It is therefore important for us to study the these HIV-1 genetic variants. We are studying the changes in the viral genome of subtypes G, CRF_AG, C and F selected by therapy and their impact on drug efficacy, as well as establishing rules for the interpretation of resistance tests. This project is integrated in a European multicenter study (CHAIN), in the final stages of approval by the EC.

HIV-2 Drug Resistance

HIV-2 Drug Resistance [Perpétua Gomes &col]: clinical failure of anti-retroviral therapy in patients infected by both HIV-1 and HIV-2 has been reported since long. The knowledge of the pathogenesis of drug resistance, including its molecular markers, has since been developed for HIV-1 subtype B and, to a lesser extent, to some of the non-B subtypes. But for HIV-2, that knowledge is still lacking. There are some reports in the literature; however, the number of patients in these studies is small and their approach is not systematic. We are sequencing the pol gene (including all the protease and the first 275 positions of Reverse Transcriptase) from all the HIV-2 infected patients from 5 hospitals in the Lisbon area. In a first approach, we plan to identify the mutations selected under therapy; then we plan to assess the impact on drug efficacy of each one of them using site-directed mutagenesis. With this knowledge, we plan to determine the optimal approach for HIV-2 therapy.

Replication of the hepatitis delta virus

A yeast to hybrid approach to study the mechanisms of replication of the hepatitis delta virus

This is a recent project which intends to search for cellular proteins that interact with the HDAg and clarify the potential role of the identified proteins in the HDV replication cycle. 112 positive clones were obtained using a GAL4 based system and after screening of human liver cDNA library. These clones were sequenced and analysed. We are currently testing 10 of the putative HDAg interacting proteins using blot overlay and immunoprecipitation assays. This project is a collaboration with the Department of Biology of the University of Aveiro.

Proteome of liver cells

Analysis of the proteome of liver cells expressing the hepatitis delta virus (POCI/SAU-IMI/55112/2004)

This project is aimed to identify the changes in the proteome of human liver cells induced by the expression of HDV. Using 2DE and MALDI-TOF MS we were able to identify 32 proteins differentially expressed in transiently transfected Huh7 cells when compared with the corresponding mock transfected cell line (Mota et al., Journal of Proteomics, in press). Additionally, comparison of an HDV RNP stably expressing cell line (Huh7-D12) with Huh7 cells allowed us to identify 23 differentially expressed proteins of which 6 were also found in transiently transfected Huh7 cells (Mota et al., submitted).

Nucleocytoplasmic transport of HDV

Nucleocytoplasmic transport of the hepatitis delta virus (POCTI/ESP/37786/2001)

[Celso Cunha] This line of investigation aims to understand the mechanisms and factors involved in the nucleocytoplasmic shuttling of HDV RNPs. Concerning nuclear import of delta antigens (HDAgs), a nuclear localization signal has been identified in aa 66-75 which is essential for import of the native virus protein (Alves et al.,Virology, 370: 12-21). The export oHDV RNPs to the cytoplasm iswas previously found to be mediated by the virus RNA. In this context we searched for a motif in the virus genomic and antigenomic RNAs capable of promoting the export of heterologous RNAs using a CAT based assay and Real time PCR. The obtained results allowed us to identify a 150 nt sequence in the antigenomic RNA that functions as a constitutive transport element (CTE; Freitas et al., paper in preparation).