Instituto de Ciências Translacional UTMB-NIH concessão 1UL1RR029876-01 e Prather Alisha por sua assistência com edição de vídeo para este manuscrito.

1. Preparação animal <ol><li> Chegada Animal: Na chegada ao nível de biossegurança animal 2 (ABSL2) instalações, permitir que os animais de um mínimo de dois dias para se aclimatar ao seu novo ambiente. Após este período de repouso, o controlo dos animais para avaliar sua saúde e aparência física geral. <ol><li> Quando se utiliza repórteres fluorescentes, é importante colocar todos os sujeitos animais com uma dieta livre de alfafa para limitar a quantidade de GI autofluorescência e sinal de fundo. </li></ol></li><li> Shave animais: Para melhorar o sinal bioluminescente detectado da região craniana durante o exame; raspar as cabeças de todos os animais. Anestesiar os animais com uma mistura de gás de oxigénio e vaporizado isoflurano. Uma vez que os animais são totalmente anestesiado, use cortadores elétricos para raspar a cabeça. Terminado o barbear, os animais regressar às suas gaiolas e observar a recuperação completa dos efeitos da anestesia. </li><li> Bio Medic DadosSistemas de inserção de transponder (BMDs) (a realizar na sequência de corte dos ratinhos enquanto que os animais são completamente anestesiados): Para um meio menos invasivos para controlar a temperatura de um implante pré-programado BMDs transponder sem fios via subcutânea na região dorsal de cada rato. Estes transponders permitir a identificação sem fio e gravação de temperatura. A seguir à implantação do transponder, aplicar um adesivo de tecido de grau veterinária para a ferida. </li><li> Coleta basal: Antes de infecção, gravar uma temperatura basal e peso para todos os animais. Coleta de sangue para análise de teste de placa de neutralização de redução (PRNT) através de retro-orbitais (RO) sangrar enquanto os ratos estão sob anestesia. Após a coleta de sangue, aplique pomada veterinária antibiótico para reduzir infecção secundária potencial bacteriana. Esta colecção de base deverá ser completado com consideração para o número de vezes que os animais são colocados sob anestesia devido ao stress em ratos. Futuras coleções ro should ser concluída após imagem, enquanto o mouse ainda está sob anestesia. O sangue coletado de um máximo de RO deve ser em torno de 200 ul. </li><li> Infecção: ratinhos lugar sob anestesia, como descrito anteriormente. Inocular por via intranasal com uma dose de 5x10 6 a 5x10 7 pfu TC83-Luciferase em um volume total de 40 ul de diluição de fosfato salino tamponado (PBS) por meio de infecção intranasal. Após a infecção, coloque os ratos dentro de sua gaiola habitação e observar a recuperação. </li></ol> 2. Imagens de animais Disclaimer: Mantenha ratos dentro de sua unidade habitacional, o gabinete de segurança biológica (CSB), ou a caixa de contenção XIC em todos os momentos. Os BSCs aprovados para este protocolo são uma classe B1 ou B2 Tipo II. <ol><li> Injectar todos os animais com 10 ul por grama de peso do corpo do intraperitoneal (IP), a luciferina, a uma concentração de 15 mg / ml. <ol><li> Os ratinhos que receberam Ampligen são para ser injected IP a uma dose de 12,5 mg de peso corporal / kg a infecção -4 hr pós ou 48 hr após a infecção com base no seu grupo. </li></ol></li><li> Antes da injecção com a luciferina, separar os ratos em grupos de três para garantir um bom ajuste dentro da caixa de contenção XIC-3. <ol><li> Para todas as imagens, a utilizar uma pomada ocular / lubrificante para evitar a secagem da córnea durante todo o procedimento. </li></ol></li><li> Abra o software LivingImage e pressione Inicializar para preparar a caixa de imagem; economia auto set, tempo de exposição ao automóvel (tempo de exposição em auto é um novo recurso do software LivingImage 3.2 e mais recente), ver campo para C, e filtro para abrir; campo de vista é baseada no número de murganhos que você está imagiologia e de filtro pode ser optimizada, conforme necessário para outros projectos de imagiologia. </li><li> Confirmar que os filtros de carvão de ar não expiraram, o tanque de oxigênio está cheio, o reservatório e isoflurano é preenchido. Coloque pequenas tiras de fita isolante no XIC-3 icaixa solation que ajuda com a redução da circulação de animais durante o exame. </li><li> Injectar os ratos com IP luciferina, como descrito na seção 2.1 e lugar dentro da câmara de indução isoflurano (com a tampa aberta) por 3-5 min. </li><li> Após 5 min, fechar a tampa da câmara de indução e iniciar o fluxo de isoflurano. O anestésico é operado a cerca de 3-5% com o conjunto de taxa de fluxo de oxigénio em cerca de 2,0 L / min. Depois de completamente inconsciente esperar um adicional de 30 seg e transferir os animais para a caixa de isolamento XIC-3. Verifique se o gabinete de biossegurança a ser utilizada permite a segura eliminação de isoflurano como esse procedimento irá envolver perda de isoflurano da câmara de indução (a unidade IVIS contém suas próprias passivos latas Fair e da caixa de contenção XIC-3 conecta diretamente à entrada / saída soquetes para garantir o fluxo contido anestésico). <ol><li> Transfira os ratos com base em chip / número de grupo na caixa de isolamento XIC-3 com o conector de isoflurano em anexo umd abrir. Posicione os ratos para que eles são dispostos em decúbito esternal com a cabeça suavemente descansando sobre as tiras de fita. </li></ol></li><li> Limpe a caixa de contenção XIC com as mãos de etanol, spray e fundo da XIC-3 com cavicide, e transferir para a câmara de imagem IVIS. Reconectar a anestesia para a caixa de contenção, uma vez dentro da unidade de imagem. </li><li> Depois de 10 min após a injecção de luciferina, a imagem dos ratinhos através do software de imagem viva. </li><li> Seguindo imagem inicial com um filtro aberto, iniciar uma imagem 3-Dimensional DLIT utilizando a análise de seqüência e configuração Assistente de imagem para bioluminescente da luciferase firefly. Este processo de imagem terá 4-5 minutos e deve ser completada em um campo de visão relevante para o número de ratinhos desejados. <ol><li> Ex vivo-análise é possível após necropsia e coleta do cérebro. Coloque o cérebro em uma placa de Petri estéril, escorrer 50-100 ul de estoque luciferina em cima, e lugar dentro da imaging caixa. </li></ol></li><li> Finalize uma imagem aberta de segunda filtro em 15-17 de infecção pós min após a conclusão da imagem DLIT. Assegurar a análise de sequência é desligado eo campo de visão e configurações de filtro são retornados às configurações anteriores. </li></ol> 3. Ratos de retorno e análise de dados <ol><li> Desligue o vaporizador isoflurano e transferir a caixa de contenção XIC de volta para o armário de biossegurança. </li><li> Coloque os ratos de volta dentro de sua unidade habitacional, perto do topo, borrife o exterior com desinfetante apropriada e lugar de volta na prateleira habitação. <ol><li> Garantir ratos se recuperaram totalmente da anestesia. </li></ol></li><li> Repetir o procedimento acima, como a experiência requer até imagiologia foi concluída. </li><li> Desinfectar o BSC, desligue o equipamento, e transferir todos os dados para um local fora do laboratório para análise posterior. </li><li> Analisar os dados e gerar três imagens tridimensionais baseadas em resultados IVIS UTIlisador o software LivingImage. <ol><li> Garantir que a imagem é devidamente orientada e iluminação / cor equilíbrio está definido. Dentro da paleta de ferramentas as opções incluem Ajuste de imagem, Correções, Informação, Imagem e Ferramentas de ROI. </li><li> Utilize formas de ROI para identificar os pontos fortes de sinal específicos baseados em localização. </li><li> Reconstruir a topografia da superfície para realçar o corpo do mouse, inicializar reconstrução DLIT 3D, e usar ajuste linear para definir o mapa de órgãos, para a recomposição topográfica. </li></ol></li><li> Análise de titulação mais viral pode ser concluído através de coleta de sangue e de órgãos. Titulação neste estudo foi concluído através de homogeneização do tecido cerebral em modificado águias médio (MEM). O homogenato foi diluído em série e que infectou uma placa de 6 poços de células Vero para 1 hr. As células foram cobertas com uma camada agarose/2xMEM 1,5% e incubadas durante 2 dias a 37 graus. As células foram fixadas com formalina a 10% durante 30 minutos e coradas comvioleta de cristal. </li></ol>

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Embora este protocolo abrange os aspectos de imagem para análise in vivo, é importante reconhecer o vector bioluminescente como factor-chave para estudos futuros. Nossa utilização de TC83, uma estirpe de vacina atenuada de VEEV, como um vector para a expressão de luciferase assegura que grandes quantidades da enzima estão a ser produzidos, devido à elevada taxa de replicação do vírus no SNC como previamente descritos 1-4. Enquanto a adição de um segundo promotor subgenómico e os...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> D-luciferina </td><td></td><td></td><td></td></tr><tr><td> Isoflurano </td><td></td><td></td><td></td></tr><tr><td> Xenogen IVIS Sistema (Spectrum) </td><td> Caliper Life Sciences </td><td></td><td></td></tr><tr><td> XGI-8-gás Sistema de Anestesia </td><td> Caliper Life Sciences </td><td></td><td></td></tr><tr><td> XIC-3 caixa de contenção </td><td> Caliper Life Sciences </td><td></td><td></td></tr><tr><td> LivingImage 4,0 Software </td><td> Caliper Life Sciences </td><td></td><td></td></tr><tr><td> Telemetria / identificação fichas </td><td> Bio Medic Data Systems </td><td> IPTT-300 </td><td> Identificação animal e TemperATUREZA </td></tr><tr><td> BD seringa TB Integra 1ml com 26 gx 3/8 agulha &quot; </td><td> Fisher Scientific </td><td> 305279 </td><td></td></tr><tr><td> Vet adesivo de tecido de Bond </td><td> Fisher Scientific </td><td> NC9259532 </td><td></td></tr><tr><td> Vetropolycin Pomada oftálmica </td><td> Produtos Veterinários Webster </td><td> 78444656 </td><td></td></tr><tr><td> Fosfato de Dulbecco Buffered Saline 1X </td><td> Invitrogen </td><td> 14190-144 </td><td></td></tr><tr><td> BMDs Chip Leitor </td><td> Bio Medic Data Systems </td><td> DAS-7007S </td><td></td></tr><tr><td> DAS-HOST Software </td><td> Bio Medic Data Systems </td><td></td><td> Usado para fazer download de informações da sonda </td></tr></tbody></table>

in vivo imaging systems (ivis) detection of a neuro-invasive encephalitic virus

Os avanços modernos em tecnologia de imagem estimular o desenvolvimento eo aperfeiçoamento da forma de pesquisa viral é realizado. Inicialmente proposto por Russel e Burch em 3Rs Hume (substituição, redução, refinamento), a utilização de modelos animais na pesquisa científica está sob constante pressão para identificar novas metodologias para reduzir o uso de animais, melhorando a precisão científica ea velocidade. Um grande desafio para diretores de Hume no entanto, é a forma de garantir os estudos são estatisticamente preciso, reduzindo a morbidade das doenças animais e números totais. Estudos de eficácia de vacinas actualmente requerem um grande número de animais, a fim de ser considerado estatisticamente significativo e muitas vezes resultam em morbidade e mortalidade terminais para a identificação de protecção imunitária. Utilizamos sistemas de imagem in vivo (IVIS) em conjunto com uma enzima bioluminescente pirilampo para controlar progressivamente a invasão do sistema nervoso central (SNC) através de uma ciaphalitic vírus em um modelo murino. Tipicamente, a doença progride de forma relativamente lenta, no entanto a replicação do vírus é rápida, especialmente dentro do sistema nervoso central, e pode levar a um resultado, muitas vezes, fatal. Após a infecção intranasal de murganhos com TC83-Luc, uma estirpe de vírus atenuado da encefalite equina venezuelana modificado para expressão de um gene da luciferase, somos capazes de visualizar a replicação do vírus no cérebro, pelo menos, três dias antes do desenvolvimento de sintomas clínicos de doença. Utilizando invasão do SNC como objectivo o desenvolvimento de doença encefálica chave que são capazes de identificar rapidamente terapêutico e proteção da vacina contra TC83-Luc infecção antes de desenvolver os sintomas clínicos. Com a tecnologia IVIS somos capazes de demonstrar o teste rápido e preciso de drogas e vacinas terapêuticas, reduzindo o número de animais e da morbilidade.

Com um vírus geneticamente modificado, TC83-Luciferase, vimos um aumento na intensidade do sinal bioluminescente como a replicação do vírus se move da região nasal para o SNC central (Figura 1). Devido à elevada taxa de replicação viral, esperamos ver altos níveis de sinal de bioluminescência (Figura 2A) que dependem do vector e a resposta dos animais imunes ao vector. Esperamos que este aumento de sinal para prosseguir até atingir um pico, entre 5-7 dias após a infecção, ...

Watch this Scientific Journal Video about In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus at JoVE.com

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizando luciferase e sistemas de imagem in vivo (IVIS) como um novo meio de identificar objectivos de doença antes de desenvolvimentos clínicos ocorrer. IVIS nos permitiu visualizar em tempo real a invasão de vírus encefalíticas durante vários dias, fornecendo um modelo de doença mais precisa para o estudo futuro. Também permitiu-nos identificar as características potenciais de proteção de antivirais e de vacinas mais rápido do que os modelos animais atualmente utilizados. A capacidade de utilizar animais individuais sobre múltiplos pontos temporais garante condições de polícia reduzidos, custos e morbidade global para os animais utilizados garantir um meio mais humanas e mais científica do estudo da doença.

In Vivo Imaging Systems Detecção (IVIS) de um vírus de Neuro-invasiva encefalítico

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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JoVE Journal

Immunology and Infection

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

20.5K visualizações.  University of Texas Medical Branch. Utilizando luciferase e sistemas de imagem in vivo (IVIS) como um novo meio de identificar objectivos de doença antes de desenvolvimentos clínicos ocorrer. IVIS nos permitiu visualizar em tempo real a invasão de vírus encefalíticas durante vários dias, fornecendo um modelo de doença mais precisa para o estudo futuro. Também permitiu-nos identificar as características potenciais de proteção de antivirais e de vacinas mais rápido do que os modelos animais atualm...

Vídeo: In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

In vivo de imagens de parasitas Leishmania transgênicas em um host ao vivo

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Imunologia e Infecção

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Não-invasiva por imagem do Homing leucócitos e Migração In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

Estimar as taxas de produção de vírus em Sistemas Aquáticos

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Usando imagens Bioluminescent para Investigar sinergismo entre Streptococcus pneumoniae E Vírus da Influenza A em Ratos infantil

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Alta capacidade método de detecção de vírus Influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Nasal Wipes para Influenza A detecção de vírus e Isolamento de suínos

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

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 ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

Deteção e remoção de contaminação de Nuclease durante a purificação do protótipo recombinante vírus espumoso Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

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 ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Uma plataforma de alto rendimento para o rastreio de Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...