Publications by authors named "João Piedade"

22 Publications

  • Page 1 of 1

Evaluating vertical transmission of sexually transmitted infections to newborns.

J Infect Dev Ctries 2021 10 31;15(10):1547-1550. Epub 2021 Oct 31.

Global Health and Tropical Medicine, Institute of Hygiene and Tropical Medicine, NOVA University of Lisbon, Lisbon, Portugal.

Introduction: Sexually transmitted infections are among the most frequent infections affecting pregnant women. We assessed the transmission of hepatitis B virus, human immunodeficiency virus type 1 and Treponema pallidum to newborns from infected parturients.

Methodology: An observational, cross-sectional, analytical facility-based survey was conducted among 57 newborns in Irene Neto Maternity, Lubango city, Huíla province, Angola. Hepatitis B virus DNA molecular identification was done through nested PCR. Human immunodeficiency virus type 1 proviral DNA detection was carried out by two successive nested PCRs. Real-time PCR was performed to examine the presence of T. pallidum DNA. Amplicons from PCR positive samples were sequenced for identity search and genotype assignment.

Results: Hepatitis B virus DNA genotype E was detected in 3/41 (7.3%) newborns from HBsAg (hepatitis B surface antigen) positive mothers. To analyse the association between mothers HBeAg (hepatitis B e antigen) positivity and hepatitis B virus vertical transmission to newborns, a Fisher's exact test was performed, showing a highly significant association (p = 0.006). Human immunodeficiency virus type 1 provirus or T. pallidum DNA was not detected in any newborn.

Conclusions: To prevent hepatitis B virus vertical transmission in Angola it is important to promote universal antenatal screening, expanding hepatitis B virus markers (viral load and/or HBeAg), risk-based infected mothers' antiviral therapy and newborn passive immunoprophylaxis.
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http://dx.doi.org/10.3855/jidc.12731DOI Listing
October 2021

Prevalence of scabies and impetigo in school-age children in Timor-Leste.

Parasit Vectors 2021 Mar 15;14(1):156. Epub 2021 Mar 15.

The Kirby Institute, University of New South Wales, Sydney, Australia.

Background: Scabies and impetigo are endemic in many tropical, low- and middle-income countries. Mass drug administration (MDA) with ivermectin has emerged as a control strategy for these conditions. In 2019, Timor-Leste Ministry of Health planned to implement MDA including ivermectin for the control of lymphatic filariasis, so we undertook a baseline assessment of scabies and impetigo to better understand local epidemiology and contribute to future surveys assessing the impact of MDA.

Methods: A cross-sectional school survey was conducted in April-May 2019 at six primary schools in a semi-urban (Dili) and two rural (Ermera and Manufahi) settings. Children under 19 years of age present at school on survey days were eligible to participate, of whom we enrolled 1183. Trained health workers interviewed and examined 1043 participants to clinically diagnose scabies using the 2020 International Alliance for the Control of Scabies (IACS) diagnostic criteria, as well as impetigo. Prevalence was adjusted for age and sex. Mixed-effects logistic regression models were used to analyse odds of scabies and impetigo infection. All models accounted for clustering at the school level through the use of random effect terms. Population attributable risk of scabies as a cause of impetigo was also estimated.

Results: The overall weighted prevalence of scabies was 30.6%. Children in rural Manufahi were more likely to have scabies than those in semi-urban Dili (53.6% vs 28.2%, adjusted odds ratio [AOR] 3.5). Most cases of scabies were mild (3 to 10 lesions), and lesions were usually distributed on more than one body region. Scabies prevalence was lower among 10 to 14 year olds compared to 5 to 9 year olds. Overall weighted prevalence of impetigo was 11.3%. Relative to Dili, children in rural Ermera and Manufahi were twice as likely to have impetigo. Impetigo was twice as common in children with scabies than in those without, corresponding to an attributable risk of scabies as a cause of impetigo of 22.7%.

Conclusions: Scabies and impetigo prevalence in Timor-Leste is among the highest reported globally, particularly in rural areas. Scabies infestation was strongly associated with impetigo. Comprehensive control strategies are urgently needed in Timor-Leste.
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http://dx.doi.org/10.1186/s13071-021-04645-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962383PMC
March 2021

Molecular epidemiology and host genetics of norovirus and rotavirus infections in Portuguese elderly living in aged care homes.

J Med Virol 2019 06 20;91(6):1014-1021. Epub 2019 Feb 20.

Unidade de Microbiologia Médica, Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade NOVA de Lisboa (UNL), Lisboa, Portugal.

Norovirus (NoV) and rotavirus group A (RVA) are major agents of acute gastroenteritis worldwide. This study aimed to investigate their epidemiological profile in Portuguese elderly living in long-term care facilities and to assess the host genetic factors mediating infection susceptibility. From November 2013 to June 2015, 636 faecal specimens from 169 elderly, mainly asymptomatic, living in nursing homes in Greater Lisbon and Faro district, Portugal, were collected. NoV and RVA were detected by real-time polymerase chain reaction and NoV genotyped by phylogenetic analysis. NoV detection rate was 7.1% (12 of 169). Three GI.3 and one GII.6 strains were genotyped. RVA detection rate was 3.6% (6 of 169), exclusively in asymptomatic individuals. Host genetic factors associated with infection susceptibility were described on 250 samples by saliva-based enzyme-linked immunosorbent assays. The Lewis-negative phenotype was 8.8% (22 of 250) and the rate of nonsecretors was 16.8% (42 of 250). Association to NoV and RVA infection was performed in the subgroup of individuals (n = 147) who delivered both faecal and saliva samples. The majority of NoV- and RVA-positive individuals (90.9% and 83.3%, respectively) were secretor-positive, with Lewis B phenotype. In a subset of individuals, FUT2 and FUT3 genes were genotyped to assess mutations and validate the secretor and Lewis phenotypes. All sequenced nonsecretors were homozygous for FUT2 nonsense mutation G428A. In this study, low detection rates of NoV and RVA infections were found during two winter seasons. However, even in the absence of any outbreak, the importance of finding these infections in a nonepidemic situation in long-term care facilities may have important implications for infection control.
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http://dx.doi.org/10.1002/jmv.25425DOI Listing
June 2019

Spinal Tuberculosis in Young HIV-exposed Infants: Two Cases of Probable Congenital Transmission.

Pediatr Infect Dis J 2019 03;38(3):e48-e50

From the Department of Pediatrics, Hospital Central de Maputo, Maputo, Moçambique.

The presentation of congenital tuberculosis (TB) is varied, with frequent extrapulmonary disease, but congenital spinal TB has been rarely reported. We present 2 cases of spinal TB (1 confirmed, 1 clinical diagnosis) in HIV-exposed infants with likely congenital transmission. Increased vigilance for congenital TB, including uncommon presentations, is needed, particularly in countries with high HIV and TB prevalence.
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http://dx.doi.org/10.1097/INF.0000000000002118DOI Listing
March 2019

Characterization of rotavirus infection in children with acute gastroenteritis in Bengo province, Northwestern Angola, prior to vaccine introduction.

PLoS One 2017 19;12(4):e0176046. Epub 2017 Apr 19.

Global Health and Tropical Medicine (GHTM), Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical (IHMT), Universidade NOVA de Lisboa (UNL), Lisbon, Portugal.

Background: Rotavirus group A (RVA) is considered the leading cause of pediatric diarrhea, responsible for the high burden of diarrheal diseases in sub-Saharan Africa. Despite recent studies, the existent data are scarce for some African countries like Angola, a country with one of the highest RVA-related death estimates. The aim of this study was to determine the RVA detection rate and circulating genotypes in children less than five years of age with acute gastroenteritis attended at the Bengo General Hospital in Caxito, Bengo province, Angola, before vaccine introduction.

Methods: Between September 2012 and December 2013, 342 fecal specimens were collected from children enrolled. Positive samples for RVA by immunochromatographic rapid test were G and P-typed by hemi-nested type-specific multiplex PCR, and subgrouped for the VP6 gene. VP4 and VP7 genes from a subset of samples were sequenced for phylogenetic analysis.

Results: During the study period, a high RVA detection rate was registered (25.1%, 86/342). The age group most affected by RVA infection includes children under 6 months of age (p<0.01). Vomiting was highly associated with RVA infection (72.1%; p<0.001). From the 86 RVA-positive samples, 72 (83.7%) were genotyped. The most prevalent genotype was G1P[8] (34/72; 47.2%), followed by the uncommon G1P[6] (21/72; 29.2%), and G2P[4] (9/72; 12.5%). Only two G-types were found: G1 (60/72; 83.3%) and G2 (11/72; 15.3%). Among the P-genotypes, P[8] was the most prevalent (34/72; 47.2%), followed by P[6] (22/72; 30.6%) and P[4] (9/72; 12.5%). In the phylogenetic trees, the identified G and P-types clustered tightly together and with reference sequences in specific monophyletic groups, with highly significant bootstrap values (≥92%).

Conclusion: This pre-vaccination study revealed, for the first time for Bengo province (Angola), the RVA genotype profile, including phylogenetic relationships, and a high RVA detection rate, supporting the immediate introduction of a RVA vaccine in the national immunization programme.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176046PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397047PMC
April 2017

Negeviruses found in multiple species of mosquitoes from southern Portugal: Isolation, genetic diversity, and replication in insect cell culture.

Virology 2015 Sep 5;483:318-28. Epub 2015 Jun 5.

Unidade de Microbiologia Médica (Grupo de Virologia), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal; Unidade de Parasitologia e Microbiologia Médicas (UPMM), IHMT/UNL, Lisboa, Portugal. Electronic address:

In this report, an RT-PCR approach based on the use of degenerate primers allowed the identification of negeviruses in four different species of mosquitoes (Ochlerotatus caspius, Culex pipiens, Cx. theileri and Cx. univittatus) collected in southern Portugal. The genomes of two of these viruses, sequenced to full completion, were shown to encode all the proteins encoded by previously described negeviruses. One of these viruses induces exuberant cytopathic effect in insect cell culture, with no obvious signs of apoptosis induction, replicating very rapidly and allowing for the detection of viral genomes in the infected culture supernatant as soon as 4h post-infection. This virus was also shown to use a dsRNA intermediate, which was found to be fully formed and active 3h after infection. Phylogenetic analysis of two products encoded by the viral ORF1 placed both viruses among Negev virus cluster, in the recently proposed Nelorpivirus taxon.
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http://dx.doi.org/10.1016/j.virol.2015.04.021DOI Listing
September 2015

High rate of detection of G8P[6] rotavirus in children with acute gastroenteritis in São Tomé and Príncipe.

Arch Virol 2015 Feb 5;160(2):423-8. Epub 2014 Oct 5.

Unidade de Microbiologia Médica, Grupo de Virologia, Centro de Malária e outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008, Lisbon, Portugal,

The burden of rotavirus infections greatly affects the low-income African countries. In the absence of epidemiological data on pediatric diarrhea in São Tomé and Príncipe (STP), a study was conducted from August to December 2011. Rotavirus antigen was detected in 36.7 % of the collected fecal samples (87/237). G8P[6] was identified as the predominant genotype (71.1 % detection rate), while G1P[8] represented only 8.4 %. Phylogenetic analysis of VP7 G8 strains showed clustering within lineage G8d, while VP4 P[6] strains clustered within lineage 1a. Our results represent the first report on rotavirus from STP and show one of the highest detection rates of G8 rotaviruses worldwide.
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http://dx.doi.org/10.1007/s00705-014-2244-7DOI Listing
February 2015

Characterization of an insect-specific flavivirus (OCFVPT) co-isolated from Ochlerotatus caspius collected in southern Portugal along with a putative new Negev-like virus.

Virus Genes 2013 Dec 23;47(3):532-45. Epub 2013 Jul 23.

Unidade de Microbiologia Médica, Grupo de Virologia, Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008, Lisbon, Portugal.

We describe the isolation and characterization of an insect-specific flavivirus (ISF) from Ochlerotatus caspius (Pallas, 1771) mosquitoes collected in southern Portugal. The RNA genome of this virus, tentatively designated OCFVPT, for O. caspius flavivirus from Portugal, encodes a polyprotein showing all the features expected for a flavivirus. As frequently observed for ISF, the viral genomes seems to encode a putative Fairly Interesting Flavivirus ORF (FIFO)-like product, the synthesis of which would occur as a result of a -1 translation frameshift event. OCFVPT was isolated in the C6/36 Stegomyia albopicta (= Aedes albopictus) cell line where it replicates rapidly, but failed to replicate in Vero cells in common with other ISFs. Unlike some of the latter, however, the OCFVPT genome does not seem to be integrated in the mosquito cells we tested. Phylogenetic analyses based on partial ISF NS5 nucleotide sequences placed OCFVPT among recently published viral strains documented from mosquitoes collected in the Iberian Peninsula, while analyses of ORF/E/NS3/or NS5 amino acid sequences cluster OCFVPT with HANKV (Hanko virus), an ISF recently isolated from O. caspius mosquitoes collected in Finland. Taking into account the genetic relatedness with this virus, OCFVPT is not expected to be overtly cytopathic to C6/36 cells. The cytopathic effects associated with its presence in culture supernatants are postulated to be the result of the replication of a co-isolated putative new Negev-like virus.
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http://dx.doi.org/10.1007/s11262-013-0960-9DOI Listing
December 2013

Novel multiregion hybridization assay for the identification of the most prevalent genetic forms of the human immunodeficiency virus type 1 circulating in Portugal.

AIDS Res Hum Retroviruses 2013 Feb 25;29(2):318-28. Epub 2012 Sep 25.

Unidade de Microbiologia Médica-Grupo de Virologia/Unidade de Parasitologia e Microbiologia Médicas, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, P-1349-008 Lisboa, Portugal.

The most efficient method for HIV-1 genetic characterization involves full-genome sequencing, but the associated costs, technical features, and low throughput preclude it from being routinely used for the analysis of large numbers of viral strains. Multiregion hybridization assays (MHA) represent an alternative for a consistent genetic analysis of large numbers of viral strains. Classically, MHA rely on the amplification by real-time PCR of several regions scattered along the HIV-1 genome, and on their characterization with clade-specific TaqMan probes (also known as hydrolysis probes). In this context, the aim of our study was the development of a technical variant of an MHA (vMHA(B/G/02)) for genotyping the most prevalent genetic forms of HIV-1 circulating in Portugal. Different sets of primers were designed for universal and clade-specific amplifications of several sections of the viral genome: gag, pol(Pr), pol(RT), vpu, env(gp120), and env(gp41). vMHA(B/G/02) was implemented using a real-time PCR-based approach, with detection dependent on the use of SYBR Green I. As an alternative, a technically less demanding strategy based on conventional PCR and agarose gel analysis of the reaction products was also developed. This method performed with overall good sensitivity and specificity (>91%) when a convenience sample of 45 plasma-derived HIV-1 strains was analyzed. Apart from the detection of subtype B, G, CRF02_AG, and CRF14_BG viruses, several unique B/G recombinant were also detected. Curiously, recombinant viruses including CRF02_AG sequences were not detected in the group of samples analyzed.
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http://dx.doi.org/10.1089/AID.2012.0025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552165PMC
February 2013

Genetic characterization of an insect-specific flavivirus isolated from Culex theileri mosquitoes collected in southern Portugal.

Virus Res 2012 Aug 28;167(2):152-61. Epub 2012 Apr 28.

Unidade de Microbiologia Médica, Grupo de Virologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisboa, Portugal.

We describe the full genetic characterization of an insect-specific flavivirus (ISF) from Culex theileri (Theobald) mosquitoes collected in Portugal. This represents the first isolation and full characterization of an ISF from Portuguese mosquitoes. The virus, designated CTFV, for Culex theileri flavivirus, was isolated in the C6/36 Stegomyia albopicta (=Aedes albopictus) cell line, and failed to replicate in vertebrate (Vero) cells in common with other ISFs. The CTFV genome encodes a single polyprotein with 3357 residues showing all the features expected for those of flaviviruses. Phylogenetic analyses based on all ISF sequences available to date, place CTFV among Culex-associated flaviviruses, grouping with recently published NS5 partial sequences documented from mosquitoes collected in the Iberian Peninsula, and with Quang Binh virus (isolated in Vietnam) as a close relative. No CTFV sequences were found integrated in their host's genome using a range of specific PCR primers designed to the prM/E, NS3, and NS5 region.
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http://dx.doi.org/10.1016/j.virusres.2012.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919203PMC
August 2012

A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations.

J Biotechnol 2012 Jan 18;157(1):50-63. Epub 2011 Nov 18.

Laboratório de Doenças Infecciosas, CIISA, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.
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http://dx.doi.org/10.1016/j.jbiotec.2011.11.006DOI Listing
January 2012

GB virus C (GBV-C) evolutionary patterns revealed by analyses of reference genomes, E2 and NS5B sequences amplified from viral strains circulating in the Lisbon area (Portugal).

Infect Genet Evol 2012 Jan 25;12(1):86-93. Epub 2011 Oct 25.

Unidade de Microbiologia Médica, Grupo de Virologia, Unidade de Parasitologia e Microbiologia Médicas (UPMM), Instituto de Higiene e Medicina Tropical (IHMT)/Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, P-1349-008 Lisboa, Portugal.

GBV-C is a non-pathogenic virus that is largely dispersed in different human populations. The phylogenetic analysis of the 5'-untranslated region (5'UTR) of the GBV-C genome has led to the segregation of viral strains into six genotypes, but incongruent results are frequently obtained depending on the genome region analyzed. In this report, different phylogenetic approaches and multivariate statistics were combined to disclose evolutionary patterns that contribute to shape GBV-C evolution. The data here presented indicate: (i) that the phylogenetic noise was mostly determined by the size of the analyzed sequence, rather than by its position on the viral genome; (ii) that most genomic segments in the coding sequence seemed to evolve under a similar evolution model, which was different from that which best fits the 5'UTR, with overall large heterogeneity of rate change across the sequence; (iii) that due to saturation of transversions occurring in the 5'UTR at genetic distances <0.10, care should be taken in drawing conclusions about the tree topologies involving the deeper branches, especially when using distance-based methods; (iv) that a non-uniform distribution of InSi and dS occurs over the viral ORF highlighting regions of the viral genome with remarkably low levels of silent substitutions, and implying that the observed differences may contribute to the detected phylogenetic incongruences; and finally (v) that genetic recombination clearly impacts the GBV-C evolution extensively, this being shown for both reference genomes and NS5B GBV-C sequences amplified from Portuguese residents.
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http://dx.doi.org/10.1016/j.meegid.2011.10.011DOI Listing
January 2012

Hepatitis C virus subtypes circulating among intravenous drug users in Lisbon, Portugal.

J Med Virol 2011 Apr;83(4):608-15

Unidade de Virologia/Unidade de Parasitologia e Microbiologia Médicas, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (UNL), Lisboa, Portugal.

Hepatitis C virus (HCV) infects 2-3% of the world population and intravenous drug consumption is the leading cause of transmission in industrialized countries. The unavailability of data on the molecular epidemiology of HCV infection in Portugal prompted the study of HCV subtypes circulating among intravenous drug users residing in the Lisbon metropolitan area and sampled about 10 years apart (1998-2000 and 2008-2009). Partial coding sequences for E1 and/or NS5B were obtained from 124 individuals with HCV viremia, both mono-infected and co-infected with HIV. Phylogenetic analysis showed that, for both time periods, the most prevalent subtypes were 1a and 3a, found, altogether, in 64.9% and 71.6% of the individuals, respectively for the first and the second sampling periods. However, genotype 4 viruses (subtypes 4a and 4d), introduced later, as inferred by comparison of intra-subtype genetic distances, were also relatively frequent even one decade ago (24.6%). This HCV subtype profile for Portuguese intravenous drug users is in agreement with those described for other southern European countries when in association with drug consumption. With the exception of subtype 1b, phylogenetic trees did not show clustering of the Portuguese sequences, but rather phylogenetic mixing of HCV sequences from different geographic origins, as described previously in other Western countries and suggestive of a large international transmission network. Consistent with the low recombination rates reported for HCV, only one sample revealed discordant subtypes for the two regions analyzed (4d in E1 and 4a in NS5B), representing a potential new recombinant that deserves further analysis.
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http://dx.doi.org/10.1002/jmv.21955DOI Listing
April 2011

A new genotype 2 subcluster identified among GBV-C strains circulating in the Lisbon metropolitan area of Portugal.

J Med Virol 2010 Mar;82(3):452-9

Virology Unit/Parasitology and Medical Microbiology Unit, Institute of Hygiene and Tropical Medicine, New Lisbon University (UNL), Lisboa, Portugal.

The rate of infection by the GBV-C virus was investigated in a group of 214 individuals at high risk of infection with parenterally transmitted viruses, and all living in the Lisbon metropolitan area (Portugal). RNA was extracted from plasma samples, and a fragment of the 5'-UTR was amplified by RT-PCR, disclosing a high prevalence of infection (40.7%). Most probably due to similar modes of viral transmission, the majority of GBV-C (+) individuals were found to be coinfected with HIV and/or HCV. A genomic region covering part of the E1/E2 glycoprotein coding sequence was amplified from approximately half of the GBV-C positive samples (44/87). Phylogenetic analysis of nucleotide sequences showed segregation of Portuguese GBV-C strains with genotype 1 (G1, n = 10) and genotype 2 (G2, n = 24) references. Genotype 1 was significantly associated with the African descent of those infected. Curiously, some of the strains assigned to genotype 2 were shown to form a separate cluster (designated G2*) in both neighbor-joining and Bayesian phylogenetic trees, which was confirmed by multivariate principal coordinate analysis. However, analysis of the distribution of intra- and intergenotype genetic distances support the hypothesis that rather than corresponding to a new viral genotype, G2* is a geographical subcluster within the genotype 2 radiation. J. Med. Virol. 82:452-459, 2010. (c) 2010 Wiley-Liss, Inc.
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http://dx.doi.org/10.1002/jmv.21703DOI Listing
March 2010

Natural polymorphisms of HIV type 2 pol sequences from drug-naive individuals.

AIDS Res Hum Retroviruses 2006 Nov;22(11):1178-82

Unidade de Virologia/Unidade de Parasitologia e Microbiologia Médicas, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, P-1349-008 Lisboa, Portugal.

Until today, the susceptibility of human immunodeficiency virus type 2 (HIV-2) to protease and nucleosidic reverse-transcriptase inhibitors (PI and NRTI, respectively) has not been clearly documented. In this report we studied HIV-2 proviral sequences (n = 30) from drug-naive patients. Our results revealed that several amino acid positions in the protease and reverse transcriptase coding sequence harbored residues that have been associated with drug resistance in HIV-1-infected patients. In particular, the M46I substitution in the protease was detected in 90% of the sequences analyzed, which, together with the other substitutions identified, may indicate a reduced susceptibility of HIV-2-infected drug-naive patients to PI. Furthermore, interpretation of genotypic data with four available algorithms, developed for interpretation of HIV-1 sequence data, suggested nonoverlapping profiles of drug resistance.
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http://dx.doi.org/10.1089/aid.2006.22.1178DOI Listing
November 2006

Genetic characterization of human immunodeficiency virus type 1 from Beira, Mozambique.

Microbes Infect 2006 Aug 30;8(9-10):2442-51. Epub 2006 Jun 30.

UEI de Virologia/UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, P-1349-008 Lisbon, Portugal.

In this report, we examined the genetic diversity of HIV-1 strains circulating in the city of Beira, the second largest metropolitan area in Mozambique. A total of 131 blood samples, collected between August and October 2003 from antiretroviral-naïve individuals, were characterized with a combined approach consisting of heteroduplex mobility assay (HMA) subtyping for gag (n=74) and/or env (n=117) genes, and DNA sequence analysis of proviral env (C2V3C3, n=52), LTR (n=30) and/or pol (n=43) genomic regions. Aside from the identification, by bootscanning analysis, of a viral strain with a C/A1 mosaic C2V3C3 structure, classified as subtype A by env HMA, phylogenetic inference studies of the sequence data demonstrated the circulation of genetically diverse subtype C viruses, predominantly of the R5 type. Inspection of the LTR sequences revealed a pattern of structural and regulatory elements typical of subtype C, with 63.3% of the viruses showing three NF-kappaB binding sites. Analysis of the predicted protease sequences enabled us to detect a single primary mutation (I84V, n=1) associated with resistance to protease inhibitors (PI), while secondary mutations were highly prevalent, some of them in combinations which may confer PI resistance. Although an unexpectedly high rate (11.6%) of reverse transcriptase key mutations (V75A, K103N, Y181C, M184I, or P236L) was detected in the sequences analyzed, our data suggest the non-epidemic circulation of resistant viruses, and the absence of multi-class drug resistant viral strains.
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http://dx.doi.org/10.1016/j.micinf.2006.05.005DOI Listing
August 2006

West Nile virus in Southern Portugal, 2004.

Vector Borne Zoonotic Dis 2005 ;5(4):410-3

Unidade de Virologia, Universidade Nova de Lisboa, Lisbon, Portugal.

West Nile virus (WNV) genomic RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in six out of 57 mosquito pools collected in Southern Portugal, during the summer of 2004, yielding an infection rate (IR) of 2.8/1,000 mosquitoes. Phylogenetic analysis of a 217-nucleotide fragment of the NS5 coding region, amplified from Culex pipiens s.l. and Culex univittatus unfed females, demonstrated a close relationship with WNV strains circulating in the Mediterranean basin (Italy, 1998; France, 2000; Morocco, 2003). The data in this short report demonstrate the presence of WNV in mosquitoes in Southern Portugal and the need of permanent surveillance for viral activity within the country.
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http://dx.doi.org/10.1089/vbz.2005.5.410DOI Listing
March 2007

Genetic analysis of human immunodeficiency virus type 1 nef in Portugal: subtyping, identification of mosaic genes, and amino acid sequence variability.

J Med Virol 2005 Sep;77(1):8-16

Unidade de Virologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.

Extending our previous genetic characterization of human immunodeficiency virus type 1 (HIV-1) strains circulating in Portugal, we here report the first phylogenetic and putative amino acid sequence variability analyses of nef accessory gene. Viral sequences (n = 53) were amplified by nested PCR from proviral DNA purified from peripheral blood mononuclear cells of HIV-1 infected individuals (n = 49). Phylogenetic inference analysis demonstrated a distribution of the viral sequences between subtypes A (sub-subtype A1), B, D, F (sub-subtype F1), G, H, and J, with subtypes G and B accounting altogether for more than half of the genotypes found. A significant number of the proviral DNA sequences analyzed (18.4%) were shown to correspond to intragenic nef recombinants, with the majority having the typical CRF02_AG nef structure. In addition, three novel intragenic recombinant structures were found (B/G/B, CRF02_AG/H, and D/G). From phylogenetic analysis, it was concluded that part of the non-recombinant nef genes might have actually been amplified from mosaic viruses: CRF06_cpx, CRF14_BG, and a new envA/nefJ recombinant. While comparing all the putative Nef sequences, significant amino acid sequence variability was observed. However, most of the described nef functional motifs were relatively well conserved in the majority of the sequences analyzed and numerous amino acid changes fell outside these regions. The results presented unambiguously endorse the high level of complexity of HIV-1 epidemics in Portugal.
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http://dx.doi.org/10.1002/jmv.20408DOI Listing
September 2005

Prevalence and partial characterization of genotypes of the human TT virus infecting Portuguese individuals.

Acta Trop 2004 Jul;91(2):197-203

Unidade de Virologia/UPMM, Instituto de Higiene e Medicina Tropical, Rua da Junqueira 96, P-1349-008 Lisboa, Portugal.

The prevalence and genotype distribution of human TT viruses (TTV) were analyzed in 312 Portuguese individuals. Detection of TTV DNA was carried out by polymerase chain reaction (PCR) through the combined use of N22 and UTR-specific primers and revealed a prevalence of infection of 74%. Detection of TTV DNA was not statistically associated to the use of intravenous drugs, infection with HBV, HCV, HIV-1, HIV-1 viral load or CD4 cell count (in HIV-1 infected individuals). Our data suggest that, in the population studied, the prevalence of TTV infection does not seem to be related to intravenous viral transmission. Phylogenetic analysis of 49 plasmid clones harboring N22-specific sequences revealed the circulation of genotypes: 1 (27%, subtype G1a and G1b), 2 (51%, subtype G2b and G2c) and 4 (22%), as well as multiple genotype infections (G1b-G2b and G1a-G4). To our knowledge, this is the first report of TTV detection and partial characterization of TTV genetic variants in Portuguese individuals. Our results show that TTV infection is widespread in Portugal as in other parts of the world.
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http://dx.doi.org/10.1016/j.actatropica.2004.05.009DOI Listing
July 2004

Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon, Portugal.

AIDS Res Hum Retroviruses 2003 Jun;19(6):511-7

Unidade de Virologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal.

We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique.
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http://dx.doi.org/10.1089/088922203766774568DOI Listing
June 2003

Molecular epidemiology of HIV type 1 infection in Portugal: high prevalence of non-B subtypes.

AIDS Res Hum Retroviruses 2002 Mar;18(5):313-25

Unidade de Virologia/Unidade de Parasitologia e Microbiologia Médicas, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, P-1349-008 Lisbon, Portugal.

In this study, we have investigated the diversity of current HIV-1 strains circulating in the metropolitan area of Lisbon, Portugal. A total of 217 HIV-1-positive blood samples, collected between October 1998 and December 2000, was genetically characterized in the gp120 C2V3C3 region (n = 205) or part of the gp41 N-terminal segment (n = 12) by heteroduplex mobility assay (HMA) and/or DNA sequencing. The HMA subtyping efficiency (number of samples unambiguously subtyped by HMA divided by the total number of samples subtyped) was 65.9% (143 of 217), with indeterminate migration patterns of subtype A and G strains contributing significantly to this value. On the overall, subtype B was the most prevalent (50.2%), followed by subtypes G (21.7%), A (17.5%), and F (5.5%), whereas subtypes C, D, H, and J accounted altogether for 5.1% of the infections. Non-B subtypes were responsible for 77.4 and 33.1% of the infections among African immigrants and Portuguese subjects, respectively. Angolan individuals (n = 25) were the only ones infected with all the HIV-1 subtypes documented, probably reflecting a high degree of viral genetic diversification in their country of origin. Phylogenetic analysis showed a predominance of IbNG-like viruses among subtype A sequences and two new major subclusters within subtype G (G(P) and G(P)'). The majority of the Portuguese G sequences described formed a well-defined subcluster (G(P)), supported by bootstrap values >90%, phylogenetically distant from clade G sequences in databases. gag (p24/p7) sequence analysis of these variants confirmed the maintenance of the subtype G subclusters. The multiple subclustering observed for the major clades A, B, D, and G, as well as the variety of subtypes found, indicate a high diversity of HIV-1 variants circulating in Portugal and suggest a need for continuous epidemiologic surveillance.
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http://dx.doi.org/10.1089/088922202753519089DOI Listing
March 2002
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