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Biomed Res Int ; 2015: 535908, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25961024


Monitoring and control of infections are key parts of surveillance systems and epidemiological risk prevention. In the case of influenza A viruses (IAVs), which show high variability, a wide range of hosts, and a potential of reassortment between different strains, it is essential to study not only people, but also animals living in the immediate surroundings. If understated, the animals might become a source of newly formed infectious strains with a pandemic potential. Special attention should be focused on pigs, because of the receptors specific for virus strains originating from different species, localized in their respiratory tract. Pigs are prone to mixed infections and may constitute a reservoir of potentially dangerous IAV strains resulting from genetic reassortment. It has been reported that a quadruple reassortant, A(H1N1)pdm09, can be easily transmitted from humans to pigs and serve as a donor of genetic segments for new strains capable of infecting humans. Therefore, it is highly desirable to develop a simple, cost-effective, and rapid method for evaluation of IAV genetic variability. We describe a method based on multitemperature single-strand conformational polymorphism (MSSCP), using a fragment of the hemagglutinin (HA) gene, for detection of coinfections and differentiation of genetic variants of the virus, difficult to identify by conventional diagnostic.

Coinfecção/genética , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Infecções por Orthomyxoviridae/genética , Animais , Coinfecção/transmissão , Coinfecção/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/transmissão , Influenza Humana/virologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Polônia , Polimorfismo Conformacional de Fita Simples , Sus scrofa , Suínos
Acta Biochim Pol ; 61(3): 609-14, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25273565


Gram-positive and nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of new, safe systems of heterologous protein expression. Recombinant LAB has been shown to induce specific local and systemic immune response against selected pathogens, and could be a good alternative to classical attenuated carriers. The main goal of our study was to express the avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) in Lactococcus lactis. Results of this study were anticipated to lead to construction of lactococcal strain(s) with potential vaccine properties against the avian influenza A (H5N1) virus. Expression of the cloned H5 gene, its His-tagged variant and chIL-2 gene, under the control of the ptcB gene promoter was attested by RT-PCR on transcriptional level and Western or dot blot analysis on translational level, demonstrating that system can be an attractive solution for production of heterologous proteins. The results of the preliminary animal trial conducted in mice are a promising step toward development of a vaccine against avian bird flu using Lactococcus lactis cells as antigen carriers.

Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/prevenção & controle , Interleucina-2/biossíntese , Lactococcus lactis/genética , Animais , Galinhas , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/química , Vacinas contra Influenza , Interleucina-2/genética , Camundongos , Regiões Promotoras Genéticas
Acta Biochim Pol ; 61(3): 479-83, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25180222


Influenza viruses are the worldwide major causative agents of human and animal acute respiratory infections. Some of the influenza subtypes have caused epidemics and pandemics among humans. The varieties of methods are available for the rapid isolation and identification of influenza viruses in clinical and environmental samples. Since nucleic acids amplification techniques such as RT-PCR have been adapted, fast and sensitive influenza type and subtype determination is possible. However, in some ambiguous cases other, more detailed assay might be desired. The genetic material of influenza virus is highly unstable and constantly mutates. It is known that single nucleotide polymorphisms (SNPs) results in resistance to commercially available anti-viral drugs. The genetic drift of the virus could also result in weakening of immune response to infection. Finally, in a substantial number of patients co-infection with various virus strains or types has been confirmed. Although the detection of co-infection or presence of minor genetic variants within flu-infected patients is not a routine procedure, a rapid and wide spectrum diagnostics of influenza virus infections could reveal an accurate picture of the disease and more importantly, is crucial for choosing the appropriate therapeutics and virus monitoring. Herein we present the evidences that native gel electrophoresis and MSSCP--a method based on multitemperature single strand conformation polymorphism could furnish a useful technique for minor variants, which escape discovery by conventional diagnostic assays.

Eletroforese , Técnicas de Genotipagem , Influenzavirus A/genética , Influenzavirus A/isolamento & purificação , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos , Influenza Humana/diagnóstico , Influenza Humana/virologia , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Polimorfismo Conformacional de Fita Simples , RNA Viral/química
Hum Vaccin Immunother ; 10(3): 577-85, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24407429


Pandemic influenza A(H1N1)pdm09 virus is a global health threat and between 2009-2011 it became the predominant influenza virus subtype circulating in the world. The research describes the MSSCP (Multitemperature Single Strand Conformation Polymorphism) analysis of the hemagglutinin (HA) region encompassing major neutralizing epitope in pandemic influenza isolates from Taiwan. Several genetically distinct changes appeared in isolates obtained in 2010 and 2011. The majority of changes in HA protein did not result in significant modifications, however three modifications were localized in epitope E of H1 and one was part of the interface binding antibodies BH151 and HC45 possibly making the current vaccine less effective.-Taking into account the possibility of the emergence of influenza A with antibody evading potential, the MSSCP method provides an alternative approach for detection of minor variants which escape detection by conventional Sanger sequencing.

Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/virologia , Polimorfismo Conformacional de Fita Simples , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Epitopos/genética , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Taiwan , Adulto Jovem
Arch Microbiol ; 195(10-11): 693-703, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23979561


The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage ϕ24Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and ϕ24Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo-xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo-xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.

Bacteriófago lambda/genética , Toxina Shiga/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Escherichia coli Shiga Toxigênica/virologia , Ativação Viral , Sequência de Aminoácidos , Bacteriófago lambda/fisiologia , Dados de Sequência Molecular , Plasmídeos , Prófagos/genética , Prófagos/fisiologia , Alinhamento de Sequência , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/fisiologia
J Clin Microbiol ; 49(6): 2216-21, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21471335


Mixed infections of a single host with different variants of influenza A virus are the main source of reassortants which may have unpredictable properties when they establish themselves in the human population. In this report we describe a method for rapid detection of mixed influenza virus infections with the seasonal A/H1N1 human strain and the pandemic A/H1N1/v strain which emerged in 2009 in Mexico and the United States. The influenza virus A/H1N1 variants were characterized by the multitemperature single-stranded conformational polymorphism (MSSCP) method. The MSSCP gel patterns of hemagglutinin gene fragments of pandemic A/H1N1/v and different seasonal A/H1N1 strains were easily distinguishable 2 h after completion of reverse transcription-PCR (RT-PCR). Using the MSSCP-based genotyping approach, coinfections with seasonal and pandemic variants of the A/H1N1 subtype were identified in 4 out of 23 primary samples obtained from patients that presented with influenza-like symptoms to hospitals across Poland during the 2009-2010 epidemic season. Pandemic influenza virus strain presence was confirmed in all these primary samples by real-time RT-PCR. The sensitivity level of the MSSCP-based minor genetic variant detection was 0.1%, as determined on a mixture of DNA fragments obtained from amplification of the hemagglutinin gene of seasonal and pandemic strains. The high sensitivity of the method suggests its applicability for characterization of new viral variants long before they become dominant.

Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/virologia , Polimorfismo Conformacional de Fita Simples , Virologia/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Genótipo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Masculino , Pessoa de Meia-Idade , Polônia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Adulto Jovem