HLA-G 3'-UTR SNP and 14-bp deletion polymorphisms in Portuguese and Guinea-Bissau populations.

In the HLA-G locus, the 3'-untranslated region (3'-UTR) begins in the mid exon 6, and ends in exon 8. The occurrence of a 14-bp deletion within exon 8, the only mutation known until now in the 3'-UTR, has been considered a risk factor for disease and allograft rejection. To describe the polymorphism within this region, direct sequencing analysis was performed on 120 DNA samples from Portugal and Guinea-Bissau. Results indicate that exon 8 is less conserved than the coding exons. Nine single nucleotide polymorphisms and the previously described 14-bp deletion were found within exon 8 of both populations. Molecular diversity was higher in the Guinean samples than in the Portuguese; however, little differentiation was found among the populations, suggesting that local selection on exon 8 sequence variation is absent. The screening for sequence motifs suggests that polymorphism on this region may be involved in HLA-G post-transcriptional regulation and, therefore, in phenotype variation.

Potential mosquito vectors of arboviruses in Portugal: species, distribution, abundance and West Nile infection.

Circulation of West Nile virus in Portugal was demonstrated by serological surveys, and the virus was isolated in 1969 from Anopheles maculipennis s.l. A survey of the whole country was carried out (2001-2004) to assess the abundance of mosquito species and to screen them for arbovirus infection. A total of 770 collections yielded 32460 mosquitoes of 15 species. The regions with the highest abundance of mosquitoes were the coastal and estuarine districts of Santarém, Setúbal and Faro. Culex pipiens s.l., An. maculipennis s.l., Cx. theileri and Ochlerotatus caspius were the most abundant and widespread, accounting for 92% of mosquitoes caught. Anopheles maculipennis s.l. and Cx. pipiens s.l. were present all over the country. Culex theileri and. Oc. caspius were more abundant in the southern and coastal areas, respectively. A total of 2355 mosquito pools were screened by RT-PCR for flaviviruses, of which 987 pools were also screened for bunyaviruses. Culex pipiens s.l. and Cx. univittatus collected in 2004 in the southern district of Faro were found to be infected with West Nile virus. The density and proximity of these mosquitoes to the human populations may constitute a public health threat in the case of involvement in arbovirus transmission cycles.

Two distinct introductions of the West Nile virus in Portugal disclosed by phylogenetic analysis of genomic sequences.

In this report, we describe genomic sequencing and analysis of different West Nile virus strains isolated from mosquitoes in the south of Portugal (Alentejo and Algarve) at two different time points (1971 and 2004, respectively). Phylogenetic analysis indicated different origins for the two recorded introductions of WNV in our country, with strains segregating in different sub-clades within lineage 1a. PTRoxo (isolated in 1971) was found to be very similar to an Egyptian WNV strain isolated in 1951, while the viruses isolated in 2004 formed a statistically well-supported group with WNV strains isolated over the last decade in countries lining the Mediterranean (Morocco, Italy, and France). Analyses of the putative amino acid sequences showed all the main expected features described for viral mature proteins except for the absence of the N-glycosylation site in the envelope glycoprotein of PTRoxo, which may be reflected as attenuated neurovirulence and neuroinvasive phenotypes.

Atomic force microscopy and anodic voltammetry characterization of a 49-mer diels-alderase ribozyme.

Atomic force microscopy and differential pulse voltammetry were used to characterize the interaction of small highly structured ribozymes with two carbon electrode surfaces. The ribozymes spontaneously self-assemble in two-dimensional networks that cover the entire HOPG surface uniformly. The full-length ribozyme was adsorbed to a lesser extent than a truncated RNA sequence, presumably due to the formation of a more compact overall structure. All four nucleobases composing the ribozyme could be detected by anodic voltammetry on glassy carbon electrodes, and no signals corresponding to free nucleobases were found, indicating the integrity of the ribozyme molecules. Mg2+ cations significantly reduced the adsorption of ribozymes to the surfaces, in agreement with the stabilization of this ribozyme's compact, stable, and tightly folded tertiary structure by Mg2+ ions that could prevent the hydrophobic bases from interacting with the HOPG surface. Treatment with Pb2+ ions, on the other hand, resulted in an increased adsorption of the RNA due to well-known hydrolytic cleavage. The observed dependence of anodic peak currents on different folding states of RNA may provide an attractive method to electrochemically monitor structural changes associated with RNA folding, binding, and catalysis.

Voltammetric determination of gamma radiation-induced DNA damage.

Homopolydeoxyribonucleotides, poly[dGuo], poly[dAdo], poly[dThd], and poly[dCyd], and calf thymus single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) aqueous solutions previously exposed to gamma radiation doses between 2 and 35 Gy, were studied by differential pulse voltammetry using a glassy carbon electrode. The interpretation of the voltammetric data was also supported by the electrophoretic migration profile obtained for the same ssDNA and dsDNA gamma-irradiated samples by nondenaturing agarose gel electrophoresis. The generation of 8-oxo-7,8-dihydroguanine, 2,8-dihydroxyadenine, 5-formyluracil, base-free sites, and single- and double-stranded breaks in the gamma-irradiated DNA samples was detected voltammetrically, with the amount depending on the irradiation time. It was found that the current peaks obtained for 8-oxoguanine increase linearly with the radiation dose applied to the nucleic acid sample, and values between 8 and 446 8-oxo-7,8-dihydroguanine (8-oxoGua) per 10(6) guanines per Gy were obtained according to the nucleic acid sample. The results showed that voltammetry can be used for monitoring and simultaneously characterizing different kinds of DNA damage caused by gamma radiation exposure.

Electrochemistry of nanoscale DNA surface films on carbon.

A DNA electrochemical biosensor is an integrated receptor-transducer device. The most important step in the development and manufacture of a sensitive DNA-biosensor for the detection of DNA-drug interactions is the immobilization procedure of the nucleic acid probe on the transducer surface. Magnetic A/C Mode atomic force microscopy (MAC Mode AFM) images in air were used to characterize two different procedures for immobilising nanoscale double-stranded DNA (dsDNA) surface films on carbon electrodes. Thin film dsDNA layers presented holes in the dsDNA film that left parts of the electrode surface uncovered while thicker films showed a uniform and complete coverage of the electrode. These two procedures for preparing dsDNA-biosensors were used to study the influence of reactive oxygen species (ROS) in the mechanism of DNA damage by quercetin, a flavonoid, and adriamycin, an anthracycline anticancer drug. The study of quercetin-DNA interactions in the presence of Cu(II) ions indicated that the formation of a quercetin-Cu(II) complex leads to the formation of ROS necessary to react with DNA, disrupting the helix and causing the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Reduced adriamycin radicals are able to directly cause oxidative damage to DNA, generating 8-oxodGuo and ROS are not directly involved in this genomic mutagenic lesion.

Diagnóstico laboratorial pré-natal da infecçao congénita por cytomegalovirus: revisão da experiência de 10 años / Prenatal laboratory diagnosis of cytomegalovirus congenital infection: a ten year review

El virus humano del citomegalovirus (CMV) es el agente más frecuente de infección congénita, afectando aproximadamente al 0,2 a 2,2% de todos los recién nacidos. El Laboratorio de Microbiología del Hospital de Santa Cruz inició su colaboración con varias instituciones obstétricas portuguesas en 1994 en el área del diagnóstico prenatal de laboratorio para el CMV y en el año 2001 el Servicio de Patología Clínica del Centro Hospitalario Cova da Beira inició, igualmente, esta colaboración con algunas instituciones obstétricas del centro y norte del país. Dada la uniformidad de los métodos utilizados por los dos laboratorios, se ha efectuado una revisión conjunta de esta experiencia en esta área. De los 596 líquidos amnióticos estudiados, 30 fueron positivos para CMV. La sensíbilidad, la especificidad, el valor predictivo negativo y valor predictivo positivo del diagnóstico prenatal (evaluados en los casos en que se efectuó la comparación con los métodos de referencia, de la viruria en el recién nacido y la investigación de inclusiones en el feto) fueron, respectivamente del 90, 99, 98 y 95%. Los resultados presentados en esta revisión sugieren que el diagnóstico prenatal virológico para CMV, efectuado siguiendo la metodología que se ha descrito, constituye un arma diagnóstica fiable para la evaluación prenatal de esta infección congénita. La selección de los casos para amniocentesis debe obedecer a las indicaciones de «seroconversión para IgG», «IgM confirmada» (debiendo los métodos de confirmación ser la avidez de las IgG y, en algunos casos, el Western blot para IgM) y las alteraciones eco gráficas de etiología suspechosa para CMV

Human cytomegalovirus (CMV) is the most frequent cause of congenital infection, affecting 0.2 to 2.2% of all live births. In 1994, the Laboratory of Microbiology of the Hospital de Santa Cruz began working in collaboration with several Portuguese Institutions, in the field of prenatal CMV diagnosis. In 2001, the Department of Clinical Pathology of the Centro Hospitalar Cova da Beira also started this collaboration with several Obstetrical Institutions from the north and center regions of Portugal. Since both laboratories have adopted similar experimental methodologies used in CMV prenatal diagnosis, the present work represents a collection of results obtained from both institutions. From the 596 amniotic fluids collected, 30 were found positive for CMV. Sensitivity, specificity, negative predictive and positive predictive values were, 90, 99, 98 and 95%, respectively (when compared with the urine cultures in the newborns or histological examination of the fetuses). The results obtained suggest that virological diagnosis, performed as described above, can be very useful for prenatal diagnosis of CMV congenital infections. Selection for amniocentesis should be restricted to cases with «IgG seroconversion», «confirmed IgM» (by IgG avidity and, in some cases, by IgM Western blot) and ecographic abnormalities

Electrochemical sensing of the behaviour of oligonucleotide lipoplexes at charged interfaces.

Complexes between short oligodeoxynucleotides (ODN) with a variable dG(x)dC(y) base composition and liposomes composed of the cationic lipid DOTAP (ODN lipoplexes) were studied by differential pulse voltammetry at a glassy carbon electrode. Since lipoplexes are spontaneously formed by electrostatic interactions, the objective of the voltammetric study was to investigate their behaviour at the electrode surface/solution interface. It was verified that the peak current in the voltammograms for ODN lipoplexes was due to guanosine oxidation and that it was influenced both by the applied adsorption potential and the lipoplex (+/-) charge ratio used. It was found that for low ODN lipoplexes (+/-) charge ratios the peak current obtained was enhanced when compared to that registered with free ODN for the same concentration. This allowed a higher sensitivity in the determination of ODN by differential pulse voltammetry and a limit of detection of 5.5 ng/mL was achieved. A model that explains the organisation of ODN lipoplexes at the electrode surface/solution interface is proposed. The electrochemical results presented account for a better physicochemical characterisation of lipoplexes at charged interfaces, which can be important for the understanding and development of gene therapy vectors based on ODN lipoplexes.

Voltammetric determination of all DNA nucleotides.

The voltammetric oxidation of all deoxyribonucleic acid (DNA) monophosphate nucleotides is investigated for the first time over a wide pH range by differential pulse voltammetry with a glassy carbon electrode. Experimental conditions such as the electrode size, supporting electrolyte composition, and pH were optimized to obtain the best peak potential separation and higher currents. This enabled the simultaneous voltammetric determination of all four DNA bases in equimolar mixtures and detection limits in the nanomolar range at physiological pH. It was also possible to detect for the first time the oxidation of each of the purine and pyrimidine nucleotides free in solution or as monomers in single-stranded DNA.

Electrochemical and spectroscopic studies of the oxidation mechanism of the herbicide propanil.

Electrochemical oxidation of propanil in deuterated solutions was studied by cyclic, differential pulse, and square wave voltammetry using a glassy carbon microelectrode. The oxidation of propanil in deuterated acid solutions occurs at the nitrogen atom of the amide at a potential of +1.15 V vs Ag/AgCl. It was also found that, under the experimental conditions used, protonation at the oxygen atom of propanil occurs, leading to the appearance of another species in solution which oxidizes at +0.60 V. The anodic peak found at +0.79 V vs Ag/AgCl in deuterated basic solutions is related to the presence of an anionic species in which a negative charge is on the nitrogen atom. The electrochemical data were confirmed by the identification of all the species formed in acidic and basic deuterated solutions by means of NMR spectroscopy. The results are supported by electrochemical and spectroscopic studies of acetanilide in deuterated solutions.

Electrochemical sensing of DNA-adriamycin interactions.

Adriamycin, a cancerostatic anthracycline antibiotic, causes considerable death of tumour cells, together with the induction of breaks in DNA single and double strands. The interaction of this compound with DNA was investigated using an electrochemical DNA-biosensor. Adriamycin intercalation in DNA disrupts the double helix and the detection of guanine and 8-oxoguanine could mimic one possible mechanism for the in vivo adriamycin drug action.

Electrochemical oxidation mechanism of guanine and adenine using a glassy carbon microelectrode.

The electrochemical oxidation mechanism of guanine and adenine was investigated using a glassy carbon microelectrode and cyclic and differential pulse voltammetry. It is pH-dependent and the electron transfer process occurs in consecutive steps with the formation of strongly adsorbed dimers on the electrode surface for both compounds.

Electrochemical detection of in situ adriamycin oxidative damage to DNA.

Adriamycin intercalation and in situ interaction with double helix DNA was investigated using a voltammetric DNA-biosensor. Oxidation and reduction of adriamycin molecules intercalated in double helix DNA were investigated in order to understand the in vivo mechanism of action with this anti-neoplasic drug. The results showed that the interaction of adriamycin with DNA is potential-dependent causing contact between DNA guanine and adenine bases and the electrode surface such that their oxidation is easily detected. A mechanism for adriamycin reduction and oxidation in situ when intercalated in double helix DNA immobilised onto the glassy carbon electrode surface is presented and the formation of the mutagenic 8-oxoguanine explained.

Follow-up study of intrahost HIV type 2 variability reveals discontinuous evolution of C2V3 sequences.

Sequence change dynamics in the highly polymorphic C2V3 region of the envelope glycoprotein gp105 coding sequence of HIV-2 was studied for two HIV-2 seropositive Guinean individuals over a 5-year period. Proviral DNA was amplified by PCR from uncultured peripheral blood mononuclear cells (PBMC), cloned, and sequenced. Amino acid sequence alignment and phylogenetic analysis showed distinct viral populations for the three collected samples (1992, 1995, and 1997) for both individuals. A discontinuous replacement of sequences suggests activation of latent viruses present in reservoirs, a process that has been documented for the HIV-1 infection but unreported so far for HIV-2. The nucleotide sequences presented in this work have been assigned accession numbers AJ400276 to AJ400319.

Genetic analysis of HIV type 2 from Ghana and Guinea-Bissau, West Africa.

The phylogenetic variability of part of the long terminal repeat (LTR) region of HIV-2 strains isolated in 1995 from five individuals residing in Bissau, the capital city of Guinea-Bissau, and collected from seven persons from Kumasi, Ghana in 1996-1997, was analyzed. All Guinean samples and all but one Ghanaian sample clustered with HIV-2 subtype A. One Ghanaian sample (14%) was classified as HIV-2 subtype B. This study adds to previous reports on HIV-2 subtype distribution in West Africa indicating local prevalence of HIV-2 subtype B in Ivory Coast and neighboring Ghana.

Longstanding presence of HIV-2 infection in Guinea-Bissau (West Africa).

We have retrospectively studied the seroprevalence of the human immunodeficiency virus (HIV) in Guinea-Bissau in a sample of sera collected from the whole country in 1980. We tested a total of 1248 individuals and found 11 individuals who were seropositive for HIV-2 but there were no HIV-1 seropositive samples. The mean age of the HIV-2 seropositive people was significantly higher than the age of the seronegative individuals. In the different areas surveyed, the HIV-2 seroprevalence ranged from 0 to 2.5%. A central region of the country, grossly centred in the capital city of Bissau, presented the highest prevalence of HIV-2 seropositivity (>2%), which contrasts with its virtual absence from the more remote rural areas located near the borders with the neighbouring countries. The overall seroprevalence found for HIV-2 in this study is 0.9% (1.8%, when considering the adult seroprevalence only), which proves that the virus was definitely circulating in Guinea-Bissau at the beginning of the 1980s.

Genetic variability of human immunodeficiency virus type 2 C2V3 region within and between individuals from Bissau, Guinea-Bissau, West Africa.

The V3 loop of both HIV-1 and HIV-2 is characterized by a high degree of genetic variation. To investigate the spectrum of HIV-2 variability in nature we have focused on the C2V3 region of Env and analyzed 108 viral sequences obtained from uncultured peripheral blood mononuclear cells obtained from 16 HIV-2-seropositive individuals from Bissau (Guinea-Bissau). The estimated values of genetic divergence between individuals were higher than those calculated from sequence information collected in a single individual. We have also found that the sequences surrounding the V3 loop contribute significantly to the overall genetic diversity of the C2V3 region of HIV-2 gp105, while the V3 loop itself seems to be rather conserved. Phylogenetic analysis demonstrated that all the individuals enrolled in this study were infected with HIV-2 subtype A viruses.

Genetic characterization of HIV type 1 and type 2 from Bissau, Guinea-Bissau (West Africa).

Previous studies from Guinea-Bissau (West Africa) have demonstrated a unique epidemiology with respect to both HIV-1 and HIV-2 infection. In order to evaluate the prevalence and dynamics of HIV-1 and HIV-2 subtypes in Bissau, the capital city of Guinea-Bissau, a cross-sectional study was set up using serological and molecular techniques. Plasma samples from 103 individuals were screened for HIV-1 and HIV-2 antibodies by ELISA and Western-blot. Seropositive results were confirmed by PCR amplification of proviral sequences in primary peripheral blood mononuclear cells (PBMC) with env and LTR primer sets for HIV-2 and env, LTR and pol primers for HIV-1. A total of 38/103 individuals were HIV-seroreactive (four HIV-1, 15 HIV-2, 19 HIV-1/HIV-2). A total of eight out of 19 dually seropositive specimens showed double PCR amplification of HIV-1 and HIV-2 proviral sequences, accounting for 21% of the infected individuals. In the remaining 11 individuals either HIV-2 or HIV-1 sequences were detected, the majority (n=9) amplifying only HIV-2. These screening data demonstrate a high discrepancy between serology and PCR results for dually seroreactive samples, Western-blot giving an overestimation of double infection. Additionally, HIV-1 strains were subtyped by heteroduplex mobility assay (HMA) on the basis of gp120 sequences. Subtyping of HIV-2 was carried out by DNA sequencing and phylogenetic analysis of env V3 molecular clones. For both HIV-1 and HIV-2 strains circulating in Bissau, our results indicate dominance of subtype A.

Improved detection of HIV-2 proviral DNA in dually seroreactive individuals by PCR.

OBJECTIVE: To improve the detection rate of HIV-2 proviral DNA in primary uncultured peripheral blood mononuclear cells (PBMC) of HIV-2-seroreactive and HIV-1-HIV-2 dually seroreactive individuals. MATERIALS AND METHODS: Two newly designed HIV-2 PCR primer pairs in the long terminal repeat (LTR) gag and gag-pol regions and a previously described env and LTR HIV-2 PCR primer pairs were tested on samples from 66 confirmed HIV-2-seropositive individuals (The Gambia, 40; Côte d'Ivoire, 17; Guinea-Bissau, nine), 209 dually seroreactive individuals (The Gambia, 82; Côte d'Ivoire, 127), 24 genetically characterized isolated HIV-1 strains (group M subtypes A-H and group O), one simian immunodeficiency virus (SIV) strain cpz, 10 HIV-2 isolates (subtype A, B and unidentified), two SIVsm isolates, and 10 seronegative samples. RESULTS: All HIV-2 primers evaluated showed 100% specificity since there was no amplification observed with 24 HIV-1, one SIVcpz and 10 seronegative samples. One single copy of the HIV-2 genome could be detected with all outer primer pairs as well as all inner primer pairs on one PCR round used. Sensitivity of primers (at least one of the four primer pairs was positive) to HIV-2-seropositive samples was 100% (all nine) in Guinea-Bissau, 71% (12/17) in Côte d'Ivoire, 100% (all 20) in Gambian AIDS patients, and 85% (17/20) in Gambian pregnant women. Doubling the PBMC of dually seroreactive individuals from 7.5 x 10(4) to 1.5 x 10(5) in the PCR revealed the presence of both HIV-1 and 2 proviral DNA in 72% (92/127) in Côte d'Ivoire and 72% (59/82) in The Gambia. By doubling the number of PBMC, HIV-2 detection in dually seroreactive individuals by PCR was increased from 65 to 77% in Côte d'Ivoire and from 67 to 83% in The Gambia. CONCLUSIONS: The use of 1.5 x 10(5) primary uncultured PBMC and the newly designed HIV-2 primer pairs allowed us to document the highest percentage (72%) ever reported of HIV-1-HIV-2 dual infections amongst HIV-1-HIV-2 dually seroreactive individuals in Côte d'Ivoire and The Gambia. Improved detection of HIV-2 proviral DNA, rather than exposure to both viruses, infection with only one virus, or infection with a unique third virus containing epitopes common to both HIV-1 and HIV-2, contributes to a more accurate monitoring of the prevalence of HIV-1-HIV-2 dual infections.

Active replication of hepatitis B virus (HBV) in HIV type 1 and in HIV type 2 infected patients

A fim de avaliar as consequencias da infeccao por HIV no curso da infeccao por HBV, ou na imunidade anteriormente adquirida, estudamos um grupo de 66 doentes Caucasoides HIV1+ sintomaticos e outro de 38 individuos seropositivos para HIV2 e provenientes da Africa, quanto a marcadores serologicos de infeccao por HBV e quanto a presenca de DNA viral circulante, tomada como sinal de replicacao do virus da hepatite. Os grupos HIV+ foram comparados com controles seronegativos adequados tendo-se verificado que 7,6 por cento dos doentes HIV1+ eram tambem HBV-DNA+ (versus 3,2 por cento nos seronegativos) bem como 2,6 por cento dos HIV2+ (versus 2,9 por cento nos controles seronegativos), nao sendo as diferencas estatisticamente significativas em qualquer um dos casos e nao tendo sido encontrada correlacao entre infeccao por HIV e replicacao ativa de HBV...