RESUMO
Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Ensaio de Imunoadsorção Enzimática , Sensibilidade e Especificidade , Animais , Ensaio de Imunoadsorção Enzimática/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Bovinos , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/sangue , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Chile , Genótipo , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Antígenos Virais/imunologia , Cricetulus , Células CHO , Anticorpos Antivirais/sangue , Proteínas Recombinantes/imunologiaRESUMO
Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.
Assuntos
Anti-Inflamatórios/farmacologia , Endotoxinas/antagonistas & inibidores , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Peptídeos/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Barreira Hematoencefálica/metabolismo , Células CHO , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Expressão Gênica , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Modelos Biológicos , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas/métodos , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Introducción: en infecciones por Streptococcus beta hemolíticos los del grupo A de Lancefield son el principal causante de faringitis en niños, y entre los no A los del Grupo C ocupan un lugar importante.Objetivo: tipificar molecularmente las cepas que participaron en un brote de faringitis en niños y demostrar la utilidad de la técnica de electroforesis de campos pulsantes en la identificación de las cepas circulantes.Métodos: se caracterizaron mediante electroforesis de campos pulsantes 12 aislados de Streptococcusbeta hemolíticos pertenecientes a niños atendidos en el Hospital Juan Manuel Márquez durante un brote de faringitis aguda en los meses de enero a marzo de 2008.Resultados: mediante el test de seroagrupamiento se encontró que 6 de los aislados, correspondiente al primer periodo del brote, eran Streptococcus del grupo C y los otros 6 aislados clasificaron como Streptococcuspyogenes, con una mayor presencia en la segunda etapa del brote. La subtipificación mediante la macrorrestriccion con SmaI y electroforesis de campos pulsantes mostró la existencia de dos poblaciones clonales consecutivas durante el brote.Conclusiones: los resultados obtenidos demuestran la utilidad que pudiera tener la subtipificación de aislados mediante electroforesis de campos pulsantes durante un brote o una reemergencia facilitando el control epidemiológico, la localización de la fuente y la toma de decisiones cuando esta fuera necesaria(AU)
Assuntos
Humanos , Eletroforese em Gel de Campo Pulsado/métodos , Faringite/terapia , Streptococcus agalactiae , Streptococcus/fisiologia , Técnicas de Tipagem Bacteriana/métodos , Bactérias/classificaçãoRESUMO
Introducción: en infecciones por Streptococcus beta hemolíticos los del grupo A de Lancefield son el principal causante de faringitis en niños, y entre los no A los del Grupo C ocupan un lugar importante. Objetivo: tipificar molecularmente las cepas que participaron en un brote de faringitis en niños y demostrar la utilidad de la técnica de electroforesis de campos pulsantes en la identificación de las cepas circulantes. Métodos: se caracterizaron mediante electroforesis de campos pulsantes 12 aislados de Streptococcusbeta hemolíticos pertenecientes a niños atendidos en el Hospital Juan Manuel Márquez durante un brote de faringitis aguda en los meses de enero a marzo de 2008. Resultados: mediante el test de seroagrupamiento se encontró que 6 de los aislados, correspondiente al primer periodo del brote, eran Streptococcus del grupo C y los otros 6 aislados clasificaron como Streptococcuspyogenes, con una mayor presencia en la segunda etapa del brote. La subtipificación mediante la macrorrestriccion con SmaI y electroforesis de campos pulsantes mostró la existencia de dos poblaciones clonales consecutivas durante el brote. Conclusiones: los resultados obtenidos demuestran la utilidad que pudiera tener la subtipificación de aislados mediante electroforesis de campos pulsantes durante un brote o una reemergencia facilitando el control epidemiológico, la localización de la fuente y la toma de decisiones cuando esta fuera necesaria(AU)
Introduction: in the context of infection by beta hemolytic Streptococci, Lancefield group A is the main cause of pharyngitis in children, whereas Streptococci C play an important role in the non group A. Aims: the purpose of the study was to molecularly typify the strains involved in a pharyngitis outbreak in children, and show the usefulness of pulsed field gel electrophoresis technique for identification of circulating strains. Methods: twelve beta hemolytic Streptococcus isolates from children cared for at Juan Manual Márquez hospital were characterized by pulsed field gel electrophoresis during an acute pharyngitis outbreak from January to March 2008. Results: the serogrouping test found that six of the isolates, corresponding to the first stage of the outbreak, were group C Streptococci, whereas the other six classified as Streptococcus pyogenes, with a greater presence in the second stage. Subtyping by Sma I macrorestriction and pulsed field gel electrophoresis revealed the presence of two consecutive clonal populations during the outbreak. Conclusions: results show the potential usefulness of subtyping isolates with pulsed field gel electrophoresis during an outbreak or an instance of re-emergence, thus facilitating epidemiological control, location of the source, and decision making when required(AU)