Agradecemos a David Wang, Anatoly Urisman, Amy Kistler, Kael Fischer, Patrick Tang, e Alexander Greninger para o desenvolvimento inicial e otimização do protocolo Virochip. Este trabalho é apoiado por uma K08 NIH conceder e Prêmio Abbott Discovery (para CC) e do Howard Hughes Medical Institute (para JLD).

As etapas do ensaio Virochip incluem (1) a extração de ácidos nucléicos a partir de amostras clínicas, (2) transcrição reversa do RNA extraído e 2 ª vertente de síntese-cDNA, (3) amplificação por PCR de cDNA preparado de forma aleatória, (3) Cy3 incorporação corante fluorescente (4), a hibridização de material marcado para o microarray Virochip, e (5) de varredura e análise (Figura 1). O protocolo mostrado abaixo irão demonstrar o uso do ensaio Virochip em uma amostra swab nasal de uma criança com uma doença semelhante à gripe. Seqüências Primer Primer A 5&#39;-GTTTCCCAGTCACGATA (N 9) -3 &#39; Primer B 5&#39;-GTTTCCCAGTCACGATA-3 &#39; 1. Extração de ácido nucléico <ol><li> Extração de ácidos nucléicos de amostras clínicas devem ser realizadas sob o Nível de Biossegurança 2 (NB-2) ou em condições mais elevadas, uma vez que estas amostras são potencialmente infecciosos. </li><li> O swab nasal amostra é transportada em meio de holding viral. Alíquota de 200 mL da amostra em tubo de microcentrífuga de 1,5 mL Eppendorf. </li><li> Opcional: pass amostra através de um filtro mM 0,22 (purificar para partículas virais) </li><li> Opcional: amostra pré-tratamento com DNAse (a degradar DNA genômico de host e purificar para protegidas, o ácido nucléico viral encapsidated), utilizando o kit Turbo DNAse (Ambion) por instruções do fabricante </li><li> Extrair a amostra usando o Zymo ZR Viral RNA Extraction Kit ou Kit Qiagen Virus UltraSens acordo com as instruções do fabricante. </li></ol> 2. Transcrição reversa e 2 ª vertente de síntese-CDNA (&quot;Round A&quot;) <ol><li> Adicione 1 uL Primer 40 pmol / mL A a 4 uL extraído RNA. Calor a 65 ° C por 5 min em um termociclador (por exemplo, GeneAmp PCR System 9700) e, em seguida, deixe esfriar em temperatura ambiente x 5 min. </li><li> Adicionar 5 mL de uma mistura principal consiste em 2 mL tampão RT 5X, 1 mL 12,5 mM dNTP, 1 mL de água, 0,5 mL 0,1 M DTT, e 0,5 mL SSIII RT (Invitrogen). Incubar a 42 ° C x 60 minutos. </li><li> Em um termociclador programável, o calor a 94 ° C x 2 min (para abortar a reação da transcriptase reversa), arrefecer até 10 ° C, centrífuga, pulso e, em seguida, adicionar 5 mL Sequenase mix composto por um tampão 5X Sequenase mL, 3,8 mL de água, e 0,15 mL Sequenase (USB Corporation). Para 2 ª vertente de síntese, rampa acima de 10 ° C a 37 ° C por 8 min, segure 8 min, então o calor a 94 ° C min x 2 (para inativar Sequenase) e arrefecer a 10 ° C (espera). </li><li> Opcional: Rd Uma amostra de cDNA podem ser armazenados neste momento a -20 ° C. </li></ol> 3. Amplificação por PCR do CDNA aleatoriamente Primário (&quot;Round B&quot;) <ol><li> Adicionar 5 uL de Rd Uma amostra de 45 mL de uma mistura principal composto de 5 mL tampão PCR 10X, 1 mL 12,5 mM dNTP, 1 mL 100 B cartilha pmol / mL, 1 mL KlenTaq LA (Sigma), e 37 mL de água. </li><li> Executar PCR protocolo da seguinte forma: 94 ° C, 2 min → (25 ciclos de 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 min) → 72 ° C, 5 min → 10 ° C ( espera) </li><li> Verifique o Rd amostra B em um gel de eletroforese de agarose 1,5%. Uma mancha de cerca de 200 - 1000 bp deve ser visualizada (Figura 2A) </li></ol> 4. Cy3 Incorporação corante fluorescente <ol><li> Opcional: outra amostra podem ser rotulados com o corante fluorescente e Cy5 hibridizado ao microarray mesmo. </li><li> Para incorporar aa-dNTP, adicionar 5 uL de amostra Rd B a 45 uL de uma mistura principal, composto por 5 tampão PCR 10X uL, 1 uL 12,5 mM aa-dNTP (Invitrogen), 1 uL 100 pmol / uL cartilha B, 1, 1 uL KlenTaq LA (Sigma) e 37 uL de água. Executar PCR protocolo da seguinte forma: 94 ° C, 2 min → (15 ciclos de 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 min) → 72 ° C, 5 min → 10 ° C ( espera) </li><li> Limpe o Rd amostra C com o DNA Limpo e Concentrador-5 Kit (Zymo Research) por instruções do fabricante. Eluir em 10 mL. </li><li> Adicione 1 uL de bicarbonato de 1M (Sigma) e 1 uL Cy5 (GE Healthcare) para a amostra C Rd. </li><li> Incubar x 1 hora no escuro. </li><li> Limpe a amostra Cy3 marcado com o DNA Limpo e Concentrador-5 Kit (Zymo Research) por instruções do fabricante. Eluir em 12 mL. </li></ol> 5. Hibridização para Virochip Microarray <ol><li> Opcional: usar 1,5 mL para verificar a concentração de cDNA e da quantidade de corante incorporado em um espectrofotômetro Nanodrop (para normalização das amostras) </li><li> Em um tubo de strip PCR adicionar 1 mL de buffer fragmentação 25X, 5 mL tampão 5X bloqueio, 9,5 mL (ou quantidade de normalizar) de Cy3 marcado amostra, e água a um volume total de 25 mL (Agilent) </li><li> Adicionar 25 mL de tampão de hibridização 2X GEX (Agilent). Girar rapidamente para remover as bolhas. </li><li> Carga 40 mL da amostra em slides junta. </li><li> Lugar Agilent-impressos série Virochip (Universidade de California, San Francisco / Agilent) (lado marcado &quot;Agilent&quot; para baixo) em cima do deslize junta e aperte o parafuso firmemente. </li><li> Hibridizar durante a noite em 65 ° C forno, ajuste de velocidade de 10 rpm. </li><li> Para lavar matrizes, pré-aqueça tampão 2 (Agilent) a 37 ° C. Desmonte subaquático junta corrediça / array sanduíche em tampão 1 (Agilent). Lavar em tampão 1 x 1 min. Lavar em tampão pré-aquecido 2 x 1 min. Remover lentamente deslize do buffer 2, tendo o cuidado de evitar bolhas (use kimwipe para espalhar a umidade extra tendo cuidado para evitar tocar directamente lado ativo da matriz) </li><li> Lugar de slides no suporte de slides e inserir em scanner matriz. </li></ol> 6. Virochip Digitalização e Análise <ol><li> Varredura Cy3 marcado matriz em 5 mm ou 2 mm de acordo com o protocolo do instrumento de rastreamento padrão (Figura 2B). </li><li> Analisar microarrays Virochip por inspeção visual, análise de cluster, ou o programa de análise automatizada Virochip, E-Predict 11. Por cada um desses três métodos, o vírus influenza é facilmente detectada na amostra swab nasal (de uma criança com doença semelhante à influenza) (Figura 2C) </li></ol> 7. Resultados representativos: Quando o protocolo é feito corretamente, uma mancha deve ser visto na eletroforese em gel de agarose após a etapa B Rd (Figura 2A). O microarray Virochip na plataforma Agilent será difícil analisar visualmente (Figura 2B), mas se um vírus estiver presente, deve ser facilmente identificado por inspeção visual, análise de cluster, e / ou E-Predict (Figura 1C). As amostras podem ser incrementadas com quantidades medidas de ácido nucléico de um vírus conhecido (por exemplo, vírus do mosaico do tabaco; MS2 bacteriófago) para servir como um controle positivo para o ensaio. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="Figura 1" /> Figura 1. Esquemática do processamento de microarray Virochip e análise. Extraído de ácidos nucléicos a partir de amostras clínicas são amplificados aleatoriamente, marcados com corantes fluorescentes e hibridizada com o microarray Virochip. Microarrays são digitalizadas com resolução de 2 mm e analisados ​​utilizando uma variedade de ferramentas computacionais, incluindo E-Predict. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="Figura 2" /> Figura 2. Passos no ensaio Virochip Após a amplificação por PCR aleatória, uma mancha de 200 -. 1000 bp pode ser visualizado por eletroforese em gel (A) (B) Três microarrays Virochip dos 8 matrizes / lâmina de vidro são mostradas, com uma pequena região. de um microarray blown-up na inserção no canto inferior direito. (C) Automated análise microarray viral usando E-Predict revelando a presença do vírus influenza A na amostra clínica. A pontuação alta similaridade (s = 0,55) corresponde a um p-valor que é próximo de zero, tornando esta uma previsão extremamente significativo.

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

Não há conflitos de interesse declarados.

O protocolo Virochip como descrito aqui é complexo e requer um técnico de pesquisa meticulosa e qualificados. Concentração de reagentes corretos e as condições de PCR, rotulagem, e hibridação são críticos. O protocolo Virochip pode ser facilmente modificado para acomodar a análise de tecidos doentes pelo uso de um método de extração de tecidos, tais como TRIzol (Invitrogen) para a extração de ácidos nucléicos. Sondas podem ser adicionados ou excluídos conforme necessário para obter o espectro desejado e amplitude de cobertura, por exemplo, sondas para detecção de alvos não-virais, tais como bactérias e fungos podem ser projetados e acrescentou &quot;on-the-fly&quot;. Algumas aplicações do ensaio Virochip atualmente em desenvolvimento incluem de amplo espectro de diagnóstico viral em laboratório clínico, investigação de surtos, a triagem pureza de medicamentos e vacinas, e da novela descoberta do patógeno viral.

<table border="1"><tbody><tr><th> Nome do reagente / material </th><th> Nome do reagente / material </th></tr><tr><td> Primera </td><td> 5&#39;-GTTTCCCAGTCACGATA (N9) -3 &#39; </td></tr><tr><td> Primer B </td><td> 5&#39;-GTTTCCCAGTCACGATA-3 &#39; </td></tr><tr><td rowspan="5"> RT Master Mix (5 mL) </td><td> 2 mL tampão 5X RT </td></tr><tr><td> 1 mL 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mL de água </td></tr><tr><td> 0,5 mL 0,1 M DTT </td></tr><tr><td> 0,5 mL SSIII RT (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase Mix (5 mL) </td><td> Um buffer de Sequenase mL 5X </td></tr><tr><td> 3,8 mL de água </td></tr><tr><td> 0,15 mL Sequenase (USB Corporation) </td></tr><tr><td rowspan="5"> º B PCR Master Mix (45 mL) </td><td> 5 mL tampão PCR 10X </td></tr><tr><td> 1 mL 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mL 100 B cartilha pmol / mL </td></tr><tr><td> 1 mL KlenTaq LA (Sigma) </td></tr><tr><td> 37 mL de água </td></tr><tr><td rowspan="5"> º C PCR Master Mix (45 mL) </td><td> 5 uL tampão PCR 10X </td></tr><tr><td> 1 mL 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mL 100 B cartilha pmol / mL </td></tr><tr><td> 1 mL KlenTaq LA (Sigma) </td></tr><tr><td> 37 mL de água </td></tr><tr><td rowspan="4"> Tampão de hibridação (25 mL) </td><td> Um buffer de fragmentação mL 25X (Agilent) </td></tr><tr><td> 5 mL tampão 5X de bloqueio (Agilent) </td></tr><tr><td> 9,5 mL (ou quantidade de normalizar) de Cy3 marcado amostra </td></tr><tr><td> Adicionar água ao volume total de 25 mL </td></tr><tr><td> Agilent Virochip microarray </td><td> licença pendente; disponível a partir de Chiu laboratório, UCSF </td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

O diagnóstico de causas virais de muitas doenças infecciosas é difícil devido à diversidade inerente seqüência de vírus, bem como o surgimento contínuo de novos patógenos virais, como a SARS coronavirus e 2009 pandemia de H1N1 do vírus influenza, que não são detectáveis ​​através de métodos tradicionais. Para enfrentar esses desafios, temos desenvolvido e validado previamente uma plataforma de microarray pan-viral chamado de Virochip com a capacidade para detectar todos os vírus conhecidos, bem como variantes de romance com base na homologia de seqüência conservada 1. Usando o Virochip, identificamos o espectro de vírus associados com infecções respiratórias, incluindo casos de doença grave inexplicável em pacientes hospitalizados, com uma sensibilidade igual ou superior a 2-5 testes clínico convencional. O Virochip também tem sido usado para identificar novos vírus, incluindo o coronavírus SARS 6,7, um clade rinovírus romance 5, XMRV (um retrovírus ligado ao câncer de próstata) 8, bornavirus aviária (a causa de uma doença degenerativa nos papagaios) 9, Cardiovirus e um romance em crianças com doenças respiratórias e diarréicas 10. A versão atual do Virochip foi portado para uma plataforma de microarray Agilent e consiste de aproximadamente 36.000 sondas derivados de ~ 1.500 vírus no GenBank a partir de dezembro de 2009. Aqui demonstramos as etapas envolvidas no processamento de um ensaio Virochip do início ao fim (~ o ​​tempo de resposta 24 horas), incluindo amostras de extração de ácidos nucléicos, amplificação por PCR usando primers aleatórios, incorporação de corantes fluorescentes, e hibridação microarray, digitalização e análise.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

O Virochip é um microarray pan-viral projetado para detectar simultaneamente todos os vírus conhecidos, bem como novos vírus, com base em homologia de seqüência conservada. Aqui demonstramos como executar um ensaio Virochip para analisar amostras clínicas para a presença de vírus conhecidos e desconhecidos.

Usando um ensaio Microarray Pan-Viral (Virochip) para Tela de amostras clínicas para patógenos virais

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

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JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

18.7K visualizações.  University of California, San Francisco. O Virochip é um microarray pan-viral projetado para detectar simultaneamente todos os vírus conhecidos, bem como novos vírus, com base em homologia de seqüência conservada. Aqui demonstramos como executar um ensaio Virochip para analisar amostras clínicas para a presença de vírus conhecidos e desconhecidos.

Vídeo: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral 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 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 ...

Imunologia e Infecção

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Usando Click Chemistry medir o efeito da infecção viral sobre a síntese de RNA do Host-Cell

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilizando Proteínas Fluorescentes para monitorar glicossomo Dynamics do Trypanosoma Africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

A Miniaturized Glycan Microarray Ensaio para a Avaliação de avidez e especificidade do Hemaglutininas vírus da gripe A

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Medição de Inibição da Neuraminidase Influenza títulos de anticorpos por Enzyme-linked Lectina Ensaio

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Utilização de Cápsulas para Coloração Negativa de Amostras Viral dentro de Biocontenimento

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Ensaio de matar Opsonophagocytic para avaliar as respostas imunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Determinação da eficiência do inibidor químico contra o crescimento intracelular toxoplasma Gondii usando um ensaio de crescimento baseado em luciferase

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Medindo anticorpos indutores de fagocitose naturalmente adquiridos para parasitas plasmodium falciparum por um ensaio baseado em citometria de fluxo

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Diagnóstico de Infecção Bacteriana Respiratória Humana Usando Amplificação Isotérmica

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Autor em destaque: Avançando na detecção rápida de patógenos respiratórios usando chip microfluídico 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Detecção rápida de patógenos bacterianos que causam infecções do trato respiratório inferior por meio de amplificação isotérmica mediada por loop baseada em chip microfluídico

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...