Este trabalho foi financiado pela National Medical Research Council, de Cingapura.

1. Alexa Fluor rotulagem de vírus da dengue <ol><li> Antes da reação de rotulagem, purificar o vírus da dengue com almofada de sacarose e preparar os reagentes e equipamentos necessários, conforme indicado no protocolo. </li><li> Prepare tampão de bicarbonato de sódio 0,2 M fresca, pH 8,5 (tampão de marcação), e 1,5 M tampão hidroxilamina, pH 8.3 (parar de reagente), pouco antes de rotulagem e filtro de esterilizar com filtros de seringa 0,2 Hm. </li><li> Diluir aproximadamente 3x10 8 unidades formadoras de placa (UFP) de vírus da dengue purificada em 1ml de rotulagem de buffer em um tubo de 2 ml. Isto pode ser ampliado proporcionalmente para a rotulagem lote de vírus. </li><li> Reconstituir o liofilizado Alexa Fluor 594 (AF594) ésteres succinimidyl 1mm de rotulagem tampão imediatamente antes da reação rotulagem. Fluorocromos outra do Alexa Fluor corante série pode ser usado de acordo com nossas necessidades. Minimizar a exposição à luz a partir desta etapa. </li><li> Adicionar 100μl do AF594 1mM corante ao vírus diluído agitando suavemente com a ponta da pipeta. </li><li> Incubar a mistura de reacção rotulagem em temperatura ambiente por 1 hora no escuro. Misture por inversão suave a cada 15mins. </li><li> Girar o tubo brevemente em uma centrífuga de mesa e adicionar 100μl do reagente de parada para a mistura de reacção, agitando suavemente com a ponta da pipeta. </li><li> Incubar a temperatura ambiente por uma hora adicional no escuro. Misture por inversão suave a cada 15mins. </li></ol> 2. Purificando Alexa Fluor rotulados vírus da dengue <ol><li> Nesse meio tempo, equilibrar a coluna de purificação com tampão de escolha. Neste experimento, uma coluna PD-10 é equilibrada com 25ml de tampão HNE (Hepes 5mM, 150mm NaCl, 0,1 mM EDTA), pH 7,4, antes de usar. </li><li> Aplicar o vírus rotulados para o topo da coluna e começar a recolher o fluxo através uma vez que o vírus entra no rotulados matriz. Preencher a coluna com tampão HNE uma vez todos os vírus rotulados entrou na matriz. Descartar o primeiro de 2,5 ml de fluxo e recolher o 2ml próxima da fração vírus rotulados. </li><li> Alíquotas e armazenar purificada AF594 vírus da dengue rotulados de -80 ° C, longe da fonte de luz. </li></ol><ol><li> Descongelar uma alíquota e determinar o título do vírus marcado pelo ensaio de placa antes de usar o lote de vírus rotulados. </li><li> Semente de 5x10 4 por bem de células Vero, cultivadas em M-199 meio de crescimento, em uma placa de quatro poços com uma lamela na parte inferior do bem um dia antes da infecção. </li><li> Retire o sobrenadante da cultura em bem e infectar as células com multiplicidade de infecção de um vírus da rotulados em volume 100μl (diluído em M-199 meio de manutenção, conforme necessário) para 10min a 37 ° C. </li><li> Remover o inóculo e lavar as lamelas duas vezes em 1xPBS. </li><li> Fixar as células em paraformalydehyde 3% para 30 minutos. </li><li> Lavar as lamelas 3 vezes em 1xPBS. </li><li> Permeabilizar as células com uma solução de permeabilização contendo saponina 0,1% e BSA 5% em 1xPBS para 30 minutos. </li><li> Incubar as células com puro-centrifugação sobrenadante clarificado da cultura hibridoma anticorpo monoclonal 3H5 para 1h em câmara úmida, protegido da luz. </li><li> Lavar as lamelas 3 vezes em tampão de lavagem (1xPBS contendo 1mM de cloreto de cálcio, cloreto de magnésio 1 mM e 0,1% saponina). </li><li> Incubar as células com anticorpos IgG AF488 anti-mouse, diluída em solução 1:100 permeabilização, por 45mins em câmara úmida, protegido da luz. </li><li> Lavar as lamelas 3 vezes em tampão de lavagem e enxágüe de vez em água deionizada. </li><li> Dab a borda da lamínula contra uma toalha de papel para drenar o excesso de água e monte sobre a lâmina de vidro com 8μl Mowiol 4-88 Dabco contendo 2,5%. </li><li> Permitir que a solução de montagem para definir durante a noite a 4 ° C antes de visualizar usando um microscópio confocal Zeiss. Aquisições seqüenciais devem ser realizadas fluoróforo uma emocionante ao mesmo tempo e alternar entre os detectores concomitantemente. </li><li> As imagens são então analisados ​​para co-localização da coloração de anticorpos E proteínas com o vírus rotulados usando o software LSM Zeiss Zen para estimar o grau de rotulagem. </li></ol> 3. Resultados representante Um exemplo da produção de vírus da dengue marcado com corante AF594 é mostrado na Figura 2. Normalmente, menos de 10 vezes caem do título inicial deve ser observado após a rotulagem de sucesso. No entanto, deve-se notar que todos os buffers tem que estar preparado para a nova rotulagem para ser bem sucedido e os ésteres Alexa Fluor succinimidyl deve ser utilizado imediatamente após a reconstituição como eles hidrolisar em nonreactive ácidos livres em soluções aquosas 8. Em seguida, o vírus rotulados tem que ser verificada para fluorescência suficiente antes do uso em experimentos. Um simples teste de imunofluorescência foi feito em células Vero e do grau de rotulagem pode ser estimada a partir da co-localização do vírus marcados com anti-E coloração do anticorpo da proteína. Cortaral células foram examinadas e uma imagem típica confocal é mostrado na Figura 3. Co-localização análise das imagens utilizando o software LSM Zen demonstraram sobreposição coeficientes que variam de 0,65-0,8, sugerindo que cerca de 65 a 80% do virions foram marcados com o corante. <img src="/files/ftp_upload/3168/3168fig1.jpg" alt="Figura 1" /> Figura esquema 1.Overall retratando o Alexa Fluor rotulagem tintura de dengue procedimento vírus. Primeiro, os buffers relevantes e vírus da dengue purificada estão preparados. O Alexa Fluor corante é reconstituído e adicionado ao vírus da dengue diluído em rotulagem buffer. A reação é então interrompida uma hora depois, com a adição do reagente de parar. Posteriormente, o vírus rotulados é purificado através de uma coluna de exclusão de tamanho para remover tinta livre. Finalmente, o vírus é rotulado re-titulada por ensaio de placa e testado para fluorescência. <img src="/files/ftp_upload/3168/3168fig2.jpg" alt="Figura 2" /> Figura 2. O número médio de virions viáveis ​​(UFP / ml), conforme determinado em um ensaio de placa antes e depois da AF594 rotulagem. Uma alíquota do AF594 vírus da dengue é rotulado descongeladas e re-titulada por ensaio de placa e ele mostra tipicamente menos de 10 vezes queda de o título de partida. Barras de erro indicam o desvio padrão de duplicatas. <img src="/files/ftp_upload/3168/3168fig3.jpg" alt="Figura 3" /> Figura 3. Co-localização de AF594 etiquetas com proteínas do vírus da dengue E em células Vero. Células cultivadas em lamínulas no dia anterior estavam infectadas com dengue AF594 rotulada a MOI de 1 por 10 minutos a 37 ° C. As células foram fixadas e marcadas com anti-E de anticorpos, e examinadas para co-localização da proteína E (verde) e AF594 rotulagem (vermelho). Sinais fluorescentes foram visualizados sob a ampliação 63X usando Zeiss LSM 710 microscópio confocal. Barra de escala é 10μm. Amarelas indicam áreas de co-localização, como mostrado no encarte.

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Embora AF594 corante foi usado neste relatório, uma ampla gama de fluoróforos na succinimidyl Alexa Fluor ésteres série está disponível com a química rotulagem similar. Isso poderia estender a aplicação rotulagem além de imagens. Citometria de fluxo pode ser usado como uma alternativa para microscopia confocal para estimar o grau de rotulagem para os fluoróforos que pode ser animado e detectado pela máquina FACS. Corantes Alexa Fluor são pequenas moléculas que reagem com grupos...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Bicarbonato de sódio </td><td> Sigma-Aldrich </td><td> S6297 </td><td></td></tr><tr><td> Hidroxilamina </td><td> Sigma-Aldrich </td><td> 159417 </td><td></td></tr><tr><td> Hidróxido de sódio </td><td> Merck </td><td> 106498 </td><td></td></tr><tr><td> AF594 ésteres succinimidyl </td><td> Sondas moleculares, Invitrogen </td><td> A20004 </td><td></td></tr><tr><td> PD-10 coluna </td><td> GE Healthcare </td><td> 17-0851-01 </td><td></td></tr><tr><td> Hepes </td><td> Sigma-Aldrich </td><td> H6147 </td><td></td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S3014 </td><td></td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E9884 </td><td></td></tr><tr><td> M-199 </td><td> Invitrogen </td><td> 11150 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070.03 </td><td></td></tr><tr><td> 4 bem-placa </td><td> Nunc </td><td> 176740 </td><td></td></tr><tr><td> Lamínulas </td><td> Einst </td><td> 0111520 </td><td></td></tr><tr><td> Lâmina de microscópio </td><td> Vela Marca </td><td> 7105 </td><td></td></tr><tr><td> 3H5 hibridoma </td><td> ATCC </td><td> HB46 </td><td></td></tr><tr><td> PBS 10x </td><td> 1 Base de Dados </td><td> BUF-2040-10X1L </td><td></td></tr><tr><td> Saponina </td><td> Sigma-Aldrich </td><td> S4521 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A7906 </td><td></td></tr><tr><td> Cloreto de magnésio </td><td> Sigma-Aldrich </td><td> M2670 </td><td></td></tr><tr><td> Cloreto de cálcio </td><td> Sigma-Aldrich </td><td> C3306 </td><td></td></tr><tr><td> Paraformaldehye </td><td> Sigma-Aldrich </td><td> 15,812-7 </td><td></td></tr><tr><td> Mowiol 4-88 </td><td> Calbiochem </td><td> 475904 </td><td></td></tr><tr><td> Dabco </td><td> Sigma-Aldrich </td><td> D27802 </td><td></td></tr><tr><td> Centrífuga de mesa </td><td> Eppendorf </td><td> 5424 </td><td></td></tr><tr><td> Microscópio confocal </td><td> Zeiss </td><td> LSM 710 </td><td></td></tr></tbody></table> Para preparar M-199 meio de crescimento, adicionar 50ml de FBS, 5 ml de piruvato de sódio e 5 ml de não-aminoácidos essenciais para 500ml de M-199, filtro estéril. Para preparar M-199 meio de manutenção, adicionar 15ml de FBS, 5 ml de piruvato de sódio e 5 ml de não-aminoácidos essenciais para 500ml de M-199, filtro estéril.

visualizing dengue virus through alexa fluor labeling

Os eventos iniciais na interação entre vírus e células podem ter profunda influência sobre o resultado da infecção. Determinar os fatores que influenciam essa interação pode levar a uma melhor compreensão da patogênese da doença e assim influenciar a vacina ou o projeto terapêutico. Assim, o desenvolvimento de métodos para investigar essa interação seria útil. Os recentes avanços na fluoróforos desenvolvimento 1-3 e tecnologia de imagem 4 pode ser explorada para melhorar o nosso conhecimento atual sobre a patogênese da dengue e assim pavimentar o caminho para reduzir os milhões de infecções por dengue ocorrem anualmente. O vírus da dengue envolvido tem um andaime externo composto de 90 glicoproteína do envelope (E) dímeros que protejam o reservatório nucleocapsídeo, que contém uma fita única de RNA positivo genoma 5. As subunidades idênticas de proteína na superfície do vírus pode assim ser rotulados com um corante reativo de amina e visualizados através de microscopia de imunofluorescência. Aqui, apresentamos um método simples de rotulagem do vírus da dengue com Alexa Fluor succinimidyl éster corante dissolvido diretamente em um buffer de bicarbonato de sódio que rendeu vírus altamente viável após rotulagem. Não existe um procedimento padronizado para a rotulagem de vírus vivo e protocolo do fabricante existente para a rotulagem de proteínas geralmente requer a reconstituição do corante em dimetilsulfóxido. A presença de dimetilsulfóxido, mesmo em quantidades mínimas, podem bloquear a infecção produtiva de vírus e também induzir citotoxicidade de 6 células. A exclusão do uso de dimetil sulfóxido neste protocolo, assim, reduzida essa possibilidade. Corantes Alexa Fluor têm fotoestabilidade superior e são menos sensíveis ao pH do que as tinturas comuns, como a fluoresceína e rodamina 2, tornando-as ideais para estudos sobre a absorção celular e transporte endossomal do vírus. A conjugação de Alexa Fluor tintura não afeta o reconhecimento do vírus da dengue rotulado por vírus específicos de anticorpos e seus supostos receptores em células hospedeiras 7. Esse método pode ter aplicações úteis em estudos virológicos.

Watch this Scientific Journal Video about Visualizing Dengue Virus through Alexa Fluor Labeling at JoVE.com

Visualizing Dengue Virus through Alexa Fluor Labeling

Aproveitando os avanços na fluoróforo desenvolvimento e tecnologia de imagem, um método simples de Alexa Fluor rotulagem de vírus da dengue foi planejado para visualizar a interação precoce entre vírus e células.

Visualizando Vírus da Dengue por meio Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

visualizing-dengue-virus-through-alexa-fluor-labeling

Research

JoVE Journal

Immunology and Infection

13.7K visualizações.  DSO National Laboratories. Aproveitando os avanços na fluoróforo desenvolvimento e tecnologia de imagem, um método simples de Alexa Fluor rotulagem de vírus da dengue foi planejado para visualizar a interação precoce entre vírus e células.

Vídeo: Visualizing Dengue Virus through Alexa Fluor Labeling

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Imunologia e Infecção

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Avaliação da eficácia e toxicidade de RNAs Segmentação HIV-1 Produção para uso em Gene ou Quimioterapia

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Salvamento e Caracterização do Vírus Recombinante de um Clone Infeccioso do Vírus do Novo Mundo Zika

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualizando o macrófago armadilhas extracelular usando microscopia Confocal

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

A citometria de fluxo simples com base em ensaio para determinar In Vitro anticorpo dependente do realce do vírus da Dengue, usando soro convalescente vírus Zika

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extração rápida, segura e simples Manual cabeceira de ácidos nucleicos para a detecção de vírus em amostras de sangue total

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

Dengue vírus RNA no sobrenadante de cultura de células infectadas por reação em cadeia da polimerase em tempo real de quantitativos de medição

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificação de codificação e Classes de RNA não-codificante expressa em suínos, sangue total

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Um modelo murino da doença da encefalite viral aguda induzida pelo vírus da dengue

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Teste in vitro ELISA para avaliar a potência da vacina antirrábica

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...