Este trabalho foi parcialmente financiado pelo Governo espanhol &quot;Ministerio de Educación y Ciencia&quot; (projeto AGL2008-05275-C01/ALI), pela União Europeia Quadro de Investigação sete projectos financiados VIROBATHE (Contrato n º 513648), VIROCLIME (Contrato n º 243923 ) e pela Agência Catalã da Água, a Agência Catalã de l&#39;Aigua (ACA), Departamento de Controle i Millora dels Ecosistemes Aquático. Durante o estudo desenvolvido Marta Rusiñol era um companheiro do Governo catalão &quot;AGAUR&quot; (FI-DGR).

1. Concentração de partículas virais presentes em amostras de água <ol><li> Coleta e acondicionamento de amostras de água <ol><li> Coletar um mínimo de 2 repetições de 10 L por amostra em recipientes de plástico com fundo plano e uma amostra extra como um controle do processo. Esta última amostra será enriquecida com uma quantidade conhecida de partículas virais e usado como um controle. Nota: É recomendado ter material separado especiais (garrafas, tubos, etc) para amostras fortificadas. </li><li> Verifique a calibragem dos conductimeter e recalibrar se necessário. Prepare um controle negativo usando água da torneira previamente ajustado para a condutividade adequada (ver abaixo de 1,6) em um recipiente plástico de 10 L extras. </li><li> Para amostras fortificadas: Adicionar o volume padrão do vírus de controle (cerca de 10 5 cópias do genoma por 10 L de água) para a amostra. Mix agitando evitando respingos e aerossóis. Controles positivos poderia consistir em uma cepa incomum de adenovírus such como HAdV-35 ou um bacteriófago como MS2. </li><li> Se a amostra apresenta elevada quantidade de material em suspensão (areia ou outros materiais), deixe-o sedimento por 15 minutos. Transferir a água para um novo recipiente. </li><li> Ajustar o pH da amostra de água para 3,5 (± 0,1) pela adição de 1 N HCl. Este passo é importante para a concentração do vírus, por isso certifique-se o pH foi ajustado corretamente. Misture a água por agitação vigorosa ao adicionar o HCl. (Nota: Se o pH é inferior a 3,5 adicione 1 M NaOH). </li><li> Ajustar a condutividade. Se a amostra tem uma condutividade de 1,500 mS / cm ou superior, este passo não é necessário. Se a condutividade é menor do que 1500 mS / cm a formação de material floculado (flocos) não é garantido para ajustar a condutividade a 1500 mS / cm pela adição de sais do mar artificial (Sigma). Misture vigorosamente por meio de agitação durante a adição dos sais do mar. </li><li> Registre o pH das amostras antes e após o condicionamento, bem como ums o volume de HCl utilizado. A condutividade também devem ser registradas após o ajuste do pH. Sempre desinfectar os eletrodos pH metros e conductimeter com uma solução de HCl frescas, elimine o cloro com uma solução de 10% de sódio e, finalmente, tiosulphate enxágüe com água destilada. </li></ol></li><li> Preparação de pré-floculação de leite desnatado 1% (PSM) <ol><li> Verificar a calibração do medidor de pH e os conductimeter e recalibrar se necessário. </li><li> Prepare pré-floculada solução de leite desnatado (1% PSM, w / v), dissolvendo 10 g de leite em pó desnatado (Difco) em 1 L de água do mar artificial (33,3 g dissolve de sais do mar artificial em 1 L de água da torneira sem cloro e autoclave) e cuidadosamente ajustar o pH para 3,5 com HCl 1N. Os flocos devem ser visíveis. Prepare a solução antes de ser utilizado ou armazenar a 4 ° C por 24 h. Para dechlorination use 1 ml de solução tiosulphate 10% por 100 ml de água. </li></ol></li><li> Floculação de partículas virais presentes em amostras de água<ol><li> Adicione 100 ml de PSM 1% para a amostra de água de 10 L. </li><li> Mexa as amostras para 80-10 h para permitir que o vírus para absorver a flocos. Usar um timer para desligamento da agitação após 8-10 h. </li><li> Pare a agitação e deixar o sedimento flocos pela ação da gravidade por 8-10 h. </li></ol></li><li> Coleta e re-dissolução dos flocos. Centrifugação <ol><li> Retire o sobrenadante usando uma bomba peristáltica e uma pipeta de plástico ligado a um tubo de plástico. Para amostras fortificadas o sobrenadante deve ser recolhida em um frasco e desinfectados de acordo com procedimentos internos. Em todos os casos TOME CUIDADO para não recolher o sedimento. </li><li> Recolher os sedimentos com o flocos (aproximadamente 500 ml) em um frasco de centrífuga. </li><li> Equilibrar as panelas pela adição de pH 3,5 PSM. </li><li> Centrifugar as panelas em uma centrífuga de alta velocidade a 8.000 xg por 30 min a 4 ° C. Assim que a centrífuga pára, remova cuidadosamente os vasos centrífuga da centrífuga. </li><li> Muito gcantes deitar fora e desprezar o sobrenadante. Siga as medidas apropriadas para o material infeccioso. </li><li> Adicionar 7 ml de tampão fosfato de dissolver o sedimento em cada frasco de centrífuga. </li><li> Uma vez que os flocos foram dissolvidas, medir e adicionar tampão fosfato para chegar a um volume total de 10 ml. </li><li> Homogeneizar o concentrado viral em vórtice e distribuir os 10 ml em microtubos limpos que devem ser congeladas a -80 ° C até ser necessário, em uma análise mais aprofundada. </li></ol></li></ol> 2. Extração de ácido nucléico <ol><li> Realizar uma extração de ácido nucléico com o Viral RNA Mini Kit QIAamp seguir as instruções do fabricante. Este kit permite o uso de uma plataforma automatizada (como Qiacube, Qiagen). </li></ol> 3. PCR quantitativo de adenovírus humano (HAdV), polyomaviruses JC (JCPyV), adenovírus suína (PAdV) e polyomaviruses bovina (BPyV) <ol><li> Quantificação de cópias do genoma nas amostras <ol><li> Prepararo mix qPCR em uma área limpa em separado usando TaqMan Ambiental PCR Master Mix 2x (Applied Biosystems). A reação ocorre em uma placa de reação de 96 poços ópticos cobertos com capas adesivas óptica. Concentrações do Mix Master, primers e sondas estão descritos na Tabela 1. </li><li> Uma vez que a mistura foi preparada, 15μl alíquota em cada poço, incluindo os controles (ver 3.2). O volume total de uma reação após a adição de destino será 25μl (mix + 15μl de amostra 10μl ou standard). </li><li> Adicionar o ácido nucléico extrações das amostras (10μl) em uma área separada. Executar direta e uma diluição de dez vezes em água purificada de cada amostra em duplicata. Cobrir os poços contendo as amostras com parte de uma cobertura adesiva, manter a outra parte da cobertura para a etapa seguinte. </li><li> Adicionar diluições das suspensões DNA padrão (10μl) de 10 GC/10μl 0-10 6 por triplicado e usando uma micropipeta utilizados exclusivamente para o standard DNA. É aconselhável acrescentar os padrões numa área equipada com radiação UV para a destruição de plasmídeo de DNA e para limpar a micropipeta após cada utilização. Cobrir os poços contendo os padrões com a tampa adesiva pré-cortadas. Nota: Para preparar suspensões padrão a ser utilizado na quantificação de cópias do genoma, a região do DNA alvo deve ser clonado em um plasmídeo e linearizado. No seguinte endereço você vai encontrar um procedimento detalhado de como criar curvas padrão com modelos de plasmídeo de DNA para ser usado em qPCR: <a href="http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf">http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf</a> </li><li> Executar a qPCR em um sistema adequado selecionando os parâmetros adequados (considerando o uso de cobertura adesiva eo volume total em cada poço, etc). Ativação seguintes do Ouro AmpliTaq por 10 min a 95 ° C, 40 ciclos de amplificação são feitas da seguinte forma: 15 s em 95 ° Ce 1 min a 60 ° C para HAdV, JCPyV e BPyV, e 15 s, 95 ° C, 20s a 55 ° C e 20 a 60 ° C para PAdV. </li><li> Uma vez que as reações sejam concluídas, armazenar dados e resultados, conforme descrito no manual do usuário do equipamento utilizado. A quantidade de DNA vai ser definida como a mediana dos dados obtidos após a correção do fator de diluição, quando necessário. </li></ol></li><li> Controles <ol><li> Use os controles positivos e negativos. O ensaio deve incluir mais de um controle não-modelo (NTC) para provar mistura não produz fluorescência. Aconselha-se a executar o controle de processo positivo, a fim de avaliar a inibição enzimática potencial devido aos inibidores presentes nas amostras estudadas. </li><li> Resultados de ensaios de qPCR registro de duas diferentes diluições do DNA padrão e do controle do processo. Utilizar os resultados para preparar gráficos de controle para controle de qualidade (QC) programas relacionados com a sensibilidade e eficiência dos ensaios. </li></ol></li><li> Confirmação daresultados <ol><li> Os resultados positivos podem ser ainda confirmada utilizando sequenciamento nested-PCR e de nucleotídeos dos amplicons, produzindo dados adicionais sobre as sequências de nucleotídeos das estirpes detectadas 1,5,6,7,9,12. </li></ol></li></ol> Seguindo o procedimento descrito, os vírus humanos e animais têm sido detectadas e quantificadas as águas balneares, água do mar e água do rio 2,3. Como um exemplo representativo, amostras de água subterrânea de áreas com altos níveis de nitratos foram avaliados para definir as fontes de contaminação. Amostras de dez litros de água foram coletadas a partir de quatro diferentes poços em áreas rurais de uma região do nordeste da Espanha. Cinco repetições foram coletadas em cada uma réplica bem estar semeada com adenovírus humano 2 utilizadas como controle do processo. As amostras foram processadas de acordo com o protocolo representado na Figura 1. Os quatro repetições analisados ​​em um dos quatro locais estudados apresentaram resultados positivos para PAdV (valor médio 7,74x10 2 GC / L), que estaria relacionada à presença de suspensões de porcos nos arredores do local de amostragem e apoiaria a contaminação fecal de suíno como fonte de nitratos nas águas subterrâneas (Tabela 2). 4. Resultados representativos: <img src="/files/ftp_upload/2820/2820fig1.jpg" alt="Figura 1" /> Figura 1. Procedimento para a detecção e quantificação de vírus na água. <img src="/files/ftp_upload/2820/2820table1.jpg" alt="Tabela 1" /> Tabela 1. Concentração de primers e sondas para ensaios de qPCR. <img src="/files/ftp_upload/2820/2820table3.jpg" alt="Tabela 3" /> Tabela 2. Detecção e quantificação de animais e humanos adenovírus e polyomaviruses em amostras de água do solo. n número de repetições analisadas Porcentagem% de positivos replica (-) Não detectado

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Dois pedidos de patente foram apresentadas em 2009 para proteger a propriedade intelectual de protocolos para a quantificação de PAdV e BPyV.

<html> O procedimento descrito iria cumprir as condições para um método de ajuste para a rotina dos laboratórios de saúde pública e ambiental: reprodutível, confiável, simples e rentável. O protocolo é simples, no entanto ele deve ser seguido com atenção. Baixa condutividade nas amostras sem adicionar a concentração de sais solicitado artificial de água do mar reduziria drasticamente a recuperação de vírus como seria o caso se o tempo de agitação de floculação é significativamente reduzido (menos d...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Centrífuga de alta velocidade (8.000 xg) </td><td> Berckman Coulter </td><td> Avanti J-20XP </td><td></td></tr><tr><td> medidor de pH termômetro, e conductimeter </td><td> Afora </td><td> LPPC3003 </td><td></td></tr><tr><td> Tubos de plástico 100-200 cm de comprimento </td><td> Deltalab </td><td> 350059 </td><td></td></tr><tr><td> Estéril graduado pipetas descartáveis </td><td> Labclinics </td><td> PN10E1 </td><td></td></tr><tr><td> Estéril de tubos de plástico de 1,5 e 10-15 mL (Eppendorf, Falcons, etc) </td><td> Afora </td><td> KA298/00 </td><td></td></tr><tr><td> Potes de centrifugação (500 mL) </td><td> Fisher Scientific </td><td> SE5753512 </td><td></td></tr><tr><td> Agitadores magnéticos e ímãs (um poramostra) </td><td> Fisher Scientific </td><td> 10510 </td><td></td></tr><tr><td> Vidro ou plástico recipientes, com fundos planos para permitir o uso de agitadores magnéticos </td><td> Deltalab </td><td> 191642 </td><td></td></tr><tr><td> A bomba peristáltica para a remoção do sobrenadante (ou uma bomba de vácuo jacto de água) </td><td> Watson-Marlow </td><td> 323E / D </td><td></td></tr><tr><td> Temporizador para switch-off a agitação após 8-10 horas </td><td> Deltalab </td><td> 900400 </td><td></td></tr><tr><td> Ácido clorídrico (0,1 N e 1N) </td><td> Panreac </td><td> 141020.1611 </td><td></td></tr><tr><td> Hidróxido de sódio (1N) </td><td> Panreac </td><td> 131687.1211 </td><td></td></tr><tr><td> Sais do mar artificial de água do mar </td><td> Sigma </td><td> S9883 </td><td></td></tr><tr><td> Leite desnatado (SM) </td><td> Difco </td><td> 232100 </td><td></td></tr><tr><td> Tampão fosfato pH 7, 5 </td><td></td><td></td><td> 1:2 v / v de estéril Na 2 HPO 4 0,2 M e NaH 2 PO 4 0,2 M em pH 7,5 </td></tr><tr><td> Tiossulfato </td><td> Panreac </td><td> 121879.1209 </td><td> Fazer uma solução de 10% em água </td></tr><tr><td> QIAamp Viral RNA Mini Kit </td><td> Qiagen </td><td> 52904 </td><td></td></tr><tr><td> De 96 poços da placa de reacção óptica (500 unidades) </td><td> Applied Biosystems </td><td> 43426659 </td><td></td></tr><tr><td> Óptica cobre adesiva (100 unidades) </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> TaqMan Ambiental PCR Master Mix 2x </td><td> Applied Biosystems </td><td> 4396838 </td><td></td></tr></tbody></table>

cost-effective method for microbial source tracking using specific human and animal viruses

Contaminação microbiana do ambiente representa um risco significativo à saúde. Clássica bacteriana fecal indicadores têm mostrado ter limitações significativas, os vírus são mais resistentes aos processos de inativação muitos e padrão de indicadores de contaminação fecal não informar sobre a fonte de contaminação. O desenvolvimento da relação custo-eficácia dos métodos para a concentração de vírus a partir de água e ensaios moleculares facilita a aplicabilidade de vírus como indicadores de contaminação fecal e como fonte microbiana de rastreamento (MST) ferramentas. Adenovírus e vírus de DNA são polyomaviruses infectar espécies de vertebrados específicas, incluindo os seres humanos e são persistentemente excretados nas fezes e / ou urina em todas as áreas geográficas estudadas. Em estudos anteriores, sugerimos a quantificação do adenovírus humano (HAdV) e polyomaviruses JC (JCPyV) por PCR quantitativo (qPCR) como um índice de contaminação fecal humano. Recentemente, desenvolvemos ensaios qPCR para a quantificação específica de Poradenovírus cine (PAdV) e polyomaviruses bovina (BPyV) como animal de contaminação fecal marcadores com sensibilidades de cópias do genoma 1-10 por tubo de ensaio. Neste estudo, apresentamos o procedimento a ser seguido para identificar a fonte de contaminação em amostras de água utilizando essas ferramentas. Como exemplo de resultados representativos, análise de vírus em águas subterrâneas que apresentem níveis elevados de nitratos é mostrado. Detecção de vírus em baixa ou moderada águas poluídas exige a concentração do vírus, pelo menos, vários litros de água em um volume muito menor, um procedimento que geralmente inclui duas etapas de concentração em série. Este procedimento um pouco pesado ea variabilidade observada em recuperações viral significativamente prejudicar o processamento simultâneo de um grande número de amostras de água. A fim de eliminar o gargalo causado pelos procedimentos de duas etapas que temos aplicado um protocolo um passo desenvolvido em Studie anteriors e aplicável a uma diversidade de matrizes de água. O procedimento inclui: acidificação das amostras de dez litros de água, floculação por leite desnatado, sedimentação por gravidade dos materiais floculados, a coleta do precipitado e centrifugação, ressuspensão do precipitado em 10 ml de tampão fosfato. O concentrado viral é usado para a extração de ácidos nucléicos virais e os adenovírus específicas e polyomaviruses de interesse são quantificados por qPCR. Elevado número de amostras pode ser simultaneamente analisadas por este método de concentração de baixo custo. O procedimento foi aplicado para a análise das águas balneares, água do mar e água do rio e, neste estudo, apresentamos os resultados da análise de amostras de águas subterrâneas. Este método de alto rendimento quantitativo é confiável, simples e rentável.

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Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

O estudo descreve um método de baixo custo para a identificação da fonte de contaminação fecal / urinária ou contaminação por nitratos na água usando qPCR para a quantificação específica de vírus de DNA humano / suínos / bovinos, adenovírus e polyomaviruses, propostos como ferramentas de MST.

Relação custo-benefício Método de Rastreamento Fonte microbiana, utilizando específicas Vírus Humano e Animal

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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Control Charts

SCIENTISTS IN ACTION

12.1K visualizações.  University of Barcelona. O estudo descreve um método de baixo custo para a identificação da fonte de contaminação fecal / urinária ou contaminação por nitratos na água usando qPCR para a quantificação específica de vírus de DNA humano / suínos / bovinos, adenovírus e polyomaviruses, propostos como ferramentas de MST.

Vídeo: Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de Salmonella On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Imunologia e Infecção

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

Monitoramento Laboratório abrangente e Custo Efetivo de HIV / AIDS: um Modelo de Papel Africano

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus usando hemoglobina humana como uma fonte de ferro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Estabelecer uma cultura líquida, coberta de vias respiratórias humanas epiteliais Polarized Calu-3 células para estudar a resposta Cell Host para patógenos respiratórios<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Crescimento Pairwise competição ensaio para determinar a replicação de Fitness de Imunodeficiência vírus humanos

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Bacteriana Artificial Chromosomes: Uma Ferramenta genómica funcional para o Estudo da Positivo-fio vírus RNA

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Medição da gripe neutralizando Antibody Responses to A(H3N2) vírus em soros humanos por Microneutralization ensaios usando as células MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

VirWaTest, um método de ponto de uso para a detecção de vírus em amostras de água

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Quantificação de células T antígeno-específicas humanas proliferating de CD4+ usando o éster do Succinimidyl do carboxyfluorescein

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...