RESUMO
Significant progress has been made in the past 10 years in unraveling the molecular biology of highly pathogenic arenaviruses that are endemic in several West African countries (Lassa fever virus) and in some regions of South America (Argentine and Bolivian hemorrhagic fever viruses). While this has resulted in proof-of-concept studies of novel vaccine candidates in non-human primates and in the discovery of several novel antiviral small molecule drug candidates, none of them has been tested in the clinic to date. The recent Ebola outbreak in West Africa has demonstrated very clearly that there is an urgent need to develop the prophylactic and therapeutic armamentarium against viral hemorrhagic fever viruses as part of a global preparedness for future epidemics. As it pertains to this goal, the present article summarizes the current knowledge of highly pathogenic arenaviruses and identifies opportunities for translational research.
Assuntos
Antivirais/uso terapêutico , Infecções por Arenaviridae/epidemiologia , Infecções por Arenaviridae/terapia , Pesquisa Biomédica , Febre Lassa/epidemiologia , Febre Lassa/terapia , Vacinas Virais , África Ocidental/epidemiologia , Animais , Arenavirus/patogenicidade , Argentina/epidemiologia , Bolívia/epidemiologia , Epidemias/prevenção & controle , Febres Hemorrágicas Virais/diagnóstico , Febres Hemorrágicas Virais/epidemiologia , Febres Hemorrágicas Virais/terapia , Humanos , Febre Lassa/diagnóstico , Vírus Lassa/patogenicidadeRESUMO
Fluoroquinolone resistance is a growing problem that has only recently emerged in S. agalactiae. Between 2005-2007, WHONET--Argentina network evaluated levofloxacin susceptibility in 1128 clinical S. agalactiae isolates, 10 (0.9%) of which proved to be resistant. Nine of them had come from 5 hospitals (in Buenos Aires City and 4 Argentinean provinces) and recovered from urine (n=7) and vaginal screening cultures (n=2). Three strains were also resistant to macrolides, lincosamides and B streptogramins due to the ermA gene. All nine fluoroquinolone-resistant isolates bore the same two mutations, Ser79Phe in ParC and Ser81Leu in GyrA proteins. Genetic relationships were analyzed by Apal-PFGE and two clones were determined, A (n=6) and B (n=3). To our knowledge, these are the first fluoroquinolone-resistant S. agalactiae isolates detected in Latin America.
Assuntos
Antibacterianos/farmacologia , Fluoroquinolonas/farmacologia , Streptococcus agalactiae/efeitos dos fármacos , Argentina , Farmacorresistência Bacteriana , Humanos , Streptococcus agalactiae/isolamento & purificaçãoRESUMO
OBJECTIVE: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/beta-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli beta-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). DESIGN: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/beta-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. METHODS: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for beta-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. RESULTS: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/beta-gal showed widespread expression of the beta-galactosidase transgene around the injection areas. CONCLUSIONS: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.
Assuntos
Técnicas de Transferência de Genes , Adeno-Hipófise/fisiologia , Hormônios Hipofisários/metabolismo , Animais , Células Cultivadas , Feminino , Expressão Gênica , Vetores Genéticos , Hormônio do Crescimento/metabolismo , Herpesvirus Humano 1/genética , Hiperplasia , Mutação , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Adeno-Hipófise/patologia , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Prolactina/sangue , Prolactina/metabolismo , Prolactinoma/genética , Prolactinoma/metabolismo , Prolactinoma/patologia , Ratos , Ratos Sprague-Dawley , Valores de Referência , Transgenes/fisiologiaRESUMO
A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) expressing beta-galactosidase under the control of the polyhedrin promoter was generated in our laboratory. To this end, we cloned the AgMNPV-2D genomic DNA fragment containing the polh gene and subcloned and sequenced the polyhedrin gene and its flanking regions. Based on this sequence information, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites. The transfer vector (pAgPHZ) was constructed by the consecutive cloning of these PCR fragments flanking the Escherichia coli LacZ gene, in place of the polh gene. pAgPHZ was used for cotransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the required recombinant, generated by homologous recombination with the polh locus, was identified by its polh(-)/LacZ+ plaque phenotype. Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses. The kinetics and levels of expression of beta-galactosidase in UFL-AG-286 cells infected with the recombinant were tested by SDS-PAGE and enzymatic activity assays.
Assuntos
Lepidópteros/virologia , Nucleopoliedrovírus/genética , Regiões Promotoras Genéticas , Proteínas Virais/genética , Animais , Sequência de Bases , Southern Blotting , Linhagem Celular , Clonagem Molecular , DNA Viral , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Óperon Lac , Dados de Sequência Molecular , Proteínas de Matriz de Corpos de Inclusão , Reação em Cadeia da Polimerase , Recombinação Genética , Proteínas Estruturais ViraisRESUMO
A granulovirus (GV) isolated from Epinotia aporema (Lepidoptera: Tortricidae)-a major soybean pest-was studied in terms of its main morphological, biochemical, and biological properties. The ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly described granulovirus (EpapGV) indicate that it is indeed a good candidate for the biological control of this pest.
Assuntos
Baculoviridae/classificação , Baculoviridae/isolamento & purificação , Lepidópteros/virologia , Controle Biológico de Vetores/métodos , Sequência de Aminoácidos , Animais , Baculoviridae/patogenicidade , Genoma Viral , Larva/virologia , Microscopia Eletrônica , Dados de Sequência Molecular , Organelas , Glycine max , Proteínas Virais/genética , VirulênciaRESUMO
RNA polymerase pausing and transcriptional antitermination regulates gene activity in several systems. In arenavirus infected cells the switch from transcription to replication is subjected to a hairpin-dependent termination and requires protein synthesis to bypass this signal. The transcriptional antitermination control by Junín virus nucleocapsid protein N, has been demonstrated in vivo by infecting BHK-21 cells expressing this viral protein in the presence of translation inhibitors. This is the first demonstration in vivo of a transcriptional antitermination control in arenavirus-infected cells.
Assuntos
Arenavirus/fisiologia , Células Eucarióticas/virologia , Proteínas do Nucleocapsídeo/fisiologia , Animais , Arenavirus/genética , Arenavirus/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular , Cricetinae , Vírus Junin/química , Vírus Junin/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Viral/análise , RNA Viral/genética , Transcrição Gênica , Ativação Transcricional , Transfecção , Replicação Viral/genéticaRESUMO
DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.
Assuntos
Impressões Digitais de DNA/métodos , Lactobacillus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Streptococcus/isolamento & purificação , Animais , Sequência de Bases , DNA Bacteriano , Lactobacillus/classificação , Lactobacillus/genética , Leite/microbiologia , Dados de Sequência Molecular , Fenótipo , Sequências Repetitivas de Ácido Nucleico , Streptococcus/classificação , Streptococcus/genética , Iogurte/microbiologiaRESUMO
Arenaviridae is a worldwide distributed family, of enveloped, single stranded, RNA viruses. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as the geographic distribution. In this study the phylogenetic relationship among the members of the Arenaviridae was examined, using the reported genomic sequences. The comparison of the aligned nucleotide sequences of the S RNA and the predicted amino acid sequences of the GPC and N proteins, together with the phylogenetic analysis, strongly suggest a possible kinship of Pichindé and Oliveros viruses, with the Old World arenavirus group. This analysis points at the evolutive relationships between the arenaviruses of the Americas and can be used to evaluate the different hypotheses about their origin.
Assuntos
Arenavirus/genética , Filogenia , Arenavirus/classificação , Sequência de Bases , Evolução Molecular , Genes Virais , Vírus Pichinde/genética , Análise de Sequência de RNA , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
The Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper we report the nucleotide sequences of S RNA of Candid #1 and its more virulent ancestors XJ#44 and XJ (prototype). Their relationship to Junin virus wild-type MC2 strain and other closely and distantly related arenaviruses was also examined. Comparisons of the nucleotide and amino acid sequences of N and GPC genes from Candid #1 and its progenitor strains revealed some changes that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype.
Assuntos
Vírus Junin/genética , Vacinas Virais/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Humanos , Dados de Sequência Molecular , Nucleocapsídeo/genética , RNA Viral , Homologia de Sequência de Aminoácidos , Vacinas Atenuadas/genética , Proteínas do Envelope Viral/genéticaRESUMO
Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.