RESUMO
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.
Assuntos
Gastroenterite/epidemiologia , Gastroenterite/virologia , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/virologia , Rotavirus/isolamento & purificação , Adenoviridae/isolamento & purificação , Infecções por Adenoviridae/virologia , Antígenos Virais/genética , Ilhas Atlânticas/epidemiologia , Proteínas do Capsídeo/genética , Pré-Escolar , Diarreia/virologia , Fezes/virologia , Feminino , Técnicas de Genotipagem , Humanos , Lactente , Masculino , Filogenia , RNA Viral/genética , Rotavirus/genéticaRESUMO
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.
Assuntos
Culicidae/virologia , Flavivirus/isolamento & purificação , Aedes , Animais , Flavivirus/classificação , Flavivirus/genética , Flavivirus/fisiologia , Genoma Viral , Dados de Sequência Molecular , Filogenia , Portugal , Especificidade da Espécie , Replicação ViralRESUMO
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.
Assuntos
Culex/virologia , Flavivirus/classificação , Flavivirus/isolamento & purificação , Animais , Linhagem Celular , Análise por Conglomerados , Flavivirus/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Poliproteínas/genética , Portugal , Análise de Sequência de DNA , Proteínas Virais/genética , Cultura de VírusRESUMO
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.
Assuntos
Infecções por Flaviviridae/epidemiologia , Vírus GB C/genética , Genoma Viral , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Regiões 5' não Traduzidas , Evolução Molecular , Infecções por Flaviviridae/virologia , Vírus GB C/classificação , Genótipo , Humanos , Análise Multivariada , Filogenia , Portugal/epidemiologia , RNA Viral/genética , Recombinação Genética , Análise de Sequência de DNARESUMO
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.
Assuntos
Proteínas de Bactérias/biossíntese , Clonagem Molecular/métodos , Vetores Genéticos/genética , Lipoproteínas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Vírus da Febre Suína Africana/genética , Animais , Antígenos Virais/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sequência de Bases , Células Cultivadas , Células Dendríticas/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Lipoproteínas/genética , Lipoproteínas/imunologia , Macrófagos/imunologia , Camundongos , Dados de Sequência Molecular , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/epidemiologia , Hepatite C/virologia , Abuso de Substâncias por Via Intravenosa/complicações , Adulto , Análise por Conglomerados , Usuários de Drogas , Feminino , Hepacivirus/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , Portugal/epidemiologia , RNA Viral/genética , Análise de Sequência de DNA , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
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.