RESUMO
Classical swine fever (CSF) causes severe disease in pigs, characterized by hemorrhage, fever, and leucopenia. A primary target of the virus is endothelial cells, where a pro-inflammatory and pro-coagulant response occurs with downregulation of gap junctional communication; these changes establish a basis for haemostatic imbalance. The aim of this study was to gain an understanding of the effect of classical swine fever virus (CSFV) on endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability. Porcine aortic endothelial cells (PAECs) were infected with CSFV at different multiplicity of infection (M.O.I.) for 48 h. Downregulation of the transcription and translation levels of eNOS was detected by semi-quantitative RT-PCR, immunoconfocal microscopy, and western blotting. This was accompanied by a reduction in NO bioavailability and attenuation of angiogenesis. Without influence from the progeny virus titer, the decrease in eNOS protein was reversed by an ERK inhibitor (PD98059) and two PI3/Akt inhibitors (LY294002 and wortmannin). In addition, we found that the transcription factors AP1, Sp1, and GATA1/2 may be involved in the downregulation of eNOS promoter activity. In conclusion, infection of PAECs with CSFV attenuated the expression of eNOS and reduced NO bioavailability through activation of the ERK and PI3/Akt pathways.
Assuntos
Aorta/metabolismo , Vírus da Febre Suína Clássica/fisiologia , Peste Suína Clássica/metabolismo , Células Endoteliais/metabolismo , Óxido Nítrico/metabolismo , Animais , Aorta/citologia , Aorta/enzimologia , Aorta/virologia , Células Cultivadas , Peste Suína Clássica/enzimologia , Peste Suína Clássica/genética , Peste Suína Clássica/virologia , Regulação para Baixo , Células Endoteliais/enzimologia , Células Endoteliais/virologia , Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , SuínosRESUMO
Infectious bursal disease (IBD), an immunosuppressive disease that affects all ages of chickens, results in significant losses in the poultry industry. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with a chromatographic lateral flow dipstick (LFD) for the detection of infectious bursal disease virus (IBDV) was developed. The whole process of testing can be completed in less than 70 min using biotin-labeled primers, an FITC-labeled DNA probe, and the LFD. The detection limits for IBDV using RT-LAMP and RT-LAMP-LFD were the same at 10(-1)plaque forming units (PFU). When other unrelated viruses and cells were tested, no false positive results were observed. In addition, the amplification efficiency of RT-LAMP was enhanced when a loop primer was used. The RT-LAMP-LFD product started to be detected after 40 min. Clinical samples were used to compare assays using RT-PCR, nested RT-PCR, RT-LAMP, and RT-LAMP-LFD and the positive rates were 16%, 40%, 40%, and 40%, respectively. In conclusion, this assay is an easy, rapid, accurate, and sensitive method for the detection of IBDV and will improve the screening of field samples, especially when veterinarians have limited resources.
Assuntos
Infecções por Birnaviridae/veterinária , Cromatografia/métodos , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Aves Domésticas/diagnóstico , Virologia/métodos , Animais , Infecções por Birnaviridae/virologia , Galinhas , Técnicas de Laboratório Clínico/métodos , Primers do DNA/genética , Sondas de Oligonucleotídeos/genética , Doenças das Aves Domésticas/virologia , Sensibilidade e Especificidade , Fatores de Tempo , Medicina Veterinária/métodosRESUMO
Avian influenza virus (AIV) is an enveloped virus with segmented RNA that belongs to the Orthomyxoviridae. Recently, avian influenza virus isolates have not only posed a significant threat to the poultry industry but also serious public health concerns. The full-length viral hemagglutinin (HA), neuraminidase (NA) or both genes were inserted into the yeast Kluyveromyces lactis genome to allow for secreted expression. Both hemagglutinin and neuraminidase activities were demonstrated for the expressed proteins. Based on PNGase F digestion and immunoassays, N-glycosidically linked high mannose or hybrid-type carbohydrate chains on the HA protein are predominant. It is noteworthy that when co-expression of the HA and NA proteins was carried out, the NA protein was able to react with the HA protein, resulting in deglycosylation in a manner similar to PNGase F digestion. Such post-translational modifications in the HA and NA proteins of AIV are described for the first time.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H5N2/genética , Kluyveromyces/metabolismo , Neuraminidase/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Virais/metabolismo , Expressão Gênica , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H5N2/isolamento & purificação , Kluyveromyces/genética , Neuraminidase/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/genéticaRESUMO
A loop-mediated isothermal amplification (LAMP) assay using six primers targeting a highly conserved region of the B2L gene has been developed to diagnose orf virus. The assay produces a ladder-like pattern of products on an agarose gel that can be specifically digested with BsrGI enzyme. The sensitivity of the LAMP assay, which was determined to be a single copy of the standard plasmid, was 100 fold and 10 fold higher than PCR and nested PCR, respectively; furthermore, no cross-reactivity was founded with the other tested viruses. By staining the products directly in the tube with PicoGreen or ethidium bromide, the products can be visualized with a similar sensitivity as by gel electrophoresis. Clinical samples were tested using PCR, nested PCR and LAMP assay and the positive rates were 60%, 70% and 70%, respectively. The LAMP assay allows easy, rapid, accurate and sensitive detection of infection with orf virus and is especially applicable in a resource-limited situation.