Este trabalho é apoiado em parte NIH concede R01 A1064826-01, Sub-19 AI062627-01, NO1-HHSN26600500032C (para SM). Agradecemos a nossos membros de laboratório para discussão e ajuda técnica, Tina Heil, pela revisão crítica do manuscrito. Gostaríamos de agradecer a David Bina, Milwaukee Departamento de Saúde Laboratório de vírus cultura e PCR.

<ol>
	<li>Hirst, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+agglutination+of+red+cells+by+allantoic+fluid+of+chick+embryos+infected+with+influenza+virus.">The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus.</a> Science. 94, 22-23 (1941).</li><li>Jonsson, N., Gullberg, M., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. Immunol. 53, 149-154 (2009).</li><li>Kalter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinating+Behavior+of+Mouse+and+Egg-adapted+Type+A+(PR8)+Influenza+Virus.">Hemagglutinating Behavior of Mouse and Egg-adapted Type A (PR8) Influenza Virus.</a> Science. 110, 184-184 (1949).</li><li>Dulbecco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Plaques+in+Monolayer+Tissue+Cultures+by+Single+Particles+of+an+Animal+Virus.">Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus.</a> Proc. Natl. Acad. Sci. U. S. A. 38, 747-752 (1952).</li><li>Deyde, V. M., Nguyen, T., Bright, R. A., Balish, A., Shu, B., Lindstrom, S., Klimov, A. I., Gubareva, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+molecular+markers+of+antiviral+resistance+in+influenza+A+(H5N1)+viruses+using+a+pyrosequencing+method.">Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method.</a> Antimicrob. Agents Chemother. 53, 1039-1047 (2009).</li><li>Jonsson, N., Gullberg, M., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. Immunol. 53, 149-154 (2009).</li><li>Rahman, M., Kieke, B. A., Vandermause, M. F., Mitchell, P. D., Greenlee, R. T., Belongia, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+Directigen+flu+A+B+enzyme+immunoassay+and+direct+fluorescent+assay+for+detection+of+influenza+infection+during+the+2004-2005+season.">Performance of Directigen flu A+B enzyme immunoassay and direct fluorescent assay for detection of influenza infection during the 2004-2005 season.</a> Diagnostic Microbiology and Infectious Disease. 58, 413-418 (2007).</li><li>Landry, M. L., Ferguson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suboptimal+detection+of+influenza+virus+in+adults+by+the+Directigen+Flu+A+B+enzyme+immunoassay+and+correlation+of+results+with+the+number+of+antigen-positive+cells+detected+by+cytospin+immunofluorescence.">Suboptimal detection of influenza virus in adults by the Directigen Flu A+B enzyme immunoassay and correlation of results with the number of antigen-positive cells detected by cytospin immunofluorescence.</a> J. Clin. Microbiol. 41, 3407-3409 (2003).</li><li>Herrmann, B., Larsson, C., Zweygberg, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+typing+of+influenza+viruses+A+and+B+by+a+nested+reverse+transcription-PCR:+comparison+to+virus+isolation+and+antigen+detection+by+immunofluorescence+and+optical+immunoassay+(FLU+OIA).">Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (FLU OIA).</a> J. Clin. Microbiol. 39, 134-138 (2001).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> Am. J. Epidemiol. 27, 493-497 (1938).</li><li>Doerr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins:+into+the+infrared.">Fluorescent proteins: into the infrared.</a> Nat. Meth. 6, 482-483 (2009).</li><li>Shu, B., Wu, K. H., Emery, S., Villanueva, J., Johnson, R., Guthrie, E., Berman, L., Warnes, C., Barnes, N., Klimov, A., Lindstrom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+the+CDC+real-time+reverse+transcriptase+PCR+swine+flu+panel+for+detection+of+2009+A+(H1N1)+pandemic+influenza+virus.">Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.</a> J. Clin. Microbiol. 49, 2614-2619 (2011).</li></ol>

Não temos nada a divulgar.

O surto recente epidemia de gripe aviária no Sudeste Asiático e um medo global da pandemia de gripe suína impor enormes preocupações para a saúde e segurança públicas. Enfoque crítico tem se dedicado na geração de vacinas para as estirpes existentes e mais recentes do vírus da gripe. Disponibilidade de rápidas tecnologias de alta capacidade para a detecção precisa e precisos cálculos título viral será tremendamente melhorar o tratamento. As técnicas mais comumente usados ​​empregadas para calcular título de vírus incluem ensaios de placa formadoras e influenza de hemaglutinação. O ensaio de placas formando foi desenvolvido para estimar os títulos de bacteriófagos e foi aprovado por Renato Dulbecco para calcular animais títulos virais 4. Para executar este ensaio, 10-de dobragem diluições de estoque de influenza ou espécimes animais, tais como líquido de LBA são preparadas e alíquotas de 0,1 ml são inoculadas em a monocamada de células MDCK em 6-bem placas. O vírus é permitido para adsorver sobre as células MDCK seguidos por o anúnciodição de um meio nutriente e de agarose. Durante a incubação, as células originais infectados liberar progênie viral que se espalhou apenas às células vizinhas. O efeito citopatogénico da infecção por influenza viral produz uma zona circular de células infectadas chamado de uma placa bacteriana. Cada placa bacteriana é, presumivelmente, formado após infecção de uma única célula com uma única partícula do vírus seguido pela replicação do vírus. Contraste entre as células vivas e das placas de pode ser reforçada por corantes, tais como violeta de cristal. Uma grande desvantagem deste ensaio é a falta de reprodutibilidade e enormes variações dentro de experimentos. Outro ensaio usado para determinar o título de influenza viral é a ensaio de hemaglutinação. A proteína presente hemaglutinina na superfície do vírus da gripe de forma eficiente liga-se a N-acetilneuramínico-ácido contendo proteínas sobre de mamíferos e de aviária células vermelhas do sangue 1. Esta interação entre o vírus da gripe e os eritrócitos leva a aglutinação sob a forma de uma treliça. O lEvel de aglutinação é usado para estimar o título de o vírus da gripe em amostras de teste. Ensaio de aglutinação eritrocitária é utilizada apenas para determinar a titulação de um vírus conhecido e não puder ser usadas para propósitos identificação da estirpe. Uma vez que este ensaio não faz diferença entre o ao vivo e os vírus de mortas, é difícil para se obter um cálculo exacto da os títulos virais. Atuais diagnósticos clínicos de infecções de gripe são baseados em testes sorológicos, a reação em cadeia da polimerase, imunofluorescência direta e amostra de cultura de células 1,6. O novo método descrito aqui também tem clínica e laboratorial potencial de diagnóstico. Influenza detecção kits Directigen FLU-A. (BD Biosciences, San Jose, CA) e BinaxNow (Binax, Inc., Scarborough, ME) ensaio de imunocromatografia uso para detectar o antígeno viral, como nucleoproteína 7 Usando estes kits, a detecção de vírus pode ser alcançada dentro de 15 min; no entanto, todas as amostras negativas tem que ser s ainda maiscreened pelo método de cultura de células. Vários estudos também relataram baixa sensibilidade desses kits, particularmente, quando a infecção foi leve ou baixo 8. Em nosso método, nós consistentemente detectar preço tão baixo quanto 100 PFU de vírus. Isto sugere a nossa técnica é útil na detecção de vírus H1N1 em amostras clínicas, mesmo quando a infecção é leve. Um método de fluorescência-Based para detectar antígenos virais tem sido relatada, onde ou tomas de aspirado de secreção nasofaríngea foram diretamente analisados ​​ou foram inoculados em uma monocamada de células suscetíveis para a multiplicação do vírus e análises subseqüentes 9. No entanto, estes métodos estão limitados para fins de diagnóstico e não são adequados para os cálculos de título de vírus. Q-PCR ajuda a identificar a presença de RNA viral e não consegue estimar o grau de infecciosidade do vírus vivos. Comparado a esses ensaios, IR sistema de ensaio à base de corantes vai ajudar na detecção e enumeração título viral em amostras clínicas e laboratoriais. Em caso de surto de influenza, écrítico para diagnosticar milhares de amostras em um período de tempo curto. Nosso método baseado em uma placa de 384 poços e scanner IR pode digitalizar 6 placas em um tempo (&gt; 2.000 amostras), portanto, oferecendo uma maior vantagem sobre outros métodos diretos fluorescentes. Viabilidade de processamento rápido de milhares de amostras clínicas poderiam reduzir a variabilidade teste comprimento, de tempo de ensaio e os custos. O uso de um anticorpo M2-específico oferece a vantagem de ser independente diferenças de estirpes em proteínas H e N. A proteína M2-é relativamente conservada em cepas de a maioria dos de influenza que infectam os seres humanos. Se os anticorpos específicos para as manchas que circulam são combinados com o nosso sistema de uma pode medir tanto o título de o de vírus e de identificar a estirpe. No entanto, uma vez que M2 é o alvo para os fármacos que Amantadine-like, os mutantes pode surgir que poderia alterar o sítio de ligação para o mAb usados ​​aqui. Isto representa uma limitação potencial para este sistema em indivíduos que estão sendo tratados por essas drogas. Múltiploensaios são usados ​​para enumerar os títulos de influenza em amostras de teste. Para calcular vírus da titulação, a dose de cultura 50% de tecido infecciosa (TCID 50) e de ovos alantóide fluido baseados em ensaios de são amplamente utilizados 1,10. Contudo, as necessidades de vários dias fazer estes métodos menos confiáveis. Além disso, em estes métodos de cálculos baseados sobre a alteração de 50% no número de placas de fornecer teóricas, mas, não precisas titulações virais. Placa formando ensaios podem ser realizados dentro de 96 horas, no entanto, a falta de reprodutibilidade é uma grande preocupação em cálculos título viral. A presente técnica descrita aqui tem várias vantagens distintas. Primeiro, é uma técnica altamente reprodutível e com resultados consistentes. Em segundo lugar, cálculos título viral são baseados em quantificação simples de intensidades de fluorescência que são comparados contra definidas curvas padrão. Em terceiro lugar, dois ou mais anticorpos primários pode ser usado para diagnosticar múltiplos sorotipos em uma dada amostra. Além disso, IR dye-conjugados anticorpos têm minimal autofluorescência e têm sido utilizados para a célula de imagem 11. UV ou visual de radiação (300-600 nM) resulta em autofluorescência celular elevada devido à presença de altos quânticos rendendo fluoróforos endógenos. Estes incluem aminoácidos aromáticos, Lipo-pigmentos, nucleotídeos piridínicos (NADPH), elastina, colágeno e coenzimas flavina. Esta propriedade intrínseca de células faz com que seja difícil de utilizar estas fontes de radiação como sondas para quantificar a expressão de proteínas específicas. Em contraste, quase IR corantes excitar e emitem entre 670-1000 nm. Moléculas hospedeiras Muitos celulares e hidrocarbonetos poliaromáticos não excitar menos este comprimento de onda e devido a espalhamento de luz reduzida, emitem fluorescência de fundo extremamente baixa. Além disso, a janela entre o plano de fundo e de detecção específico é vastamente melhorada devido a um coeficiente de extinção elevado de os fluoróforos perto de-IR. Fotoestabilidade, relação sinal-para-ruído superior, é extremamente relevante na quantificação dos títulos da reação virais. Use de microflcâmaras uidic irá automatizar o processo de seleção por meio de controles robóticos, e que pode nos permitir testar altamente contagiosas amostras clínicas com facilidade. Assim, a utilização de perto de-IR corantes nestes métodos é o ideal e altamente confiável para enumerar os títulos virais em amostras de tecido.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Albumina de soro bovino </td><td> Atlanta Biologicals </td><td> S11750 </td><td></td></tr><tr><td> PR8 vírus </td><td></td><td></td><td> De Dr. Thomas Moran M </td></tr><tr><td> Goat anti-mouse IRDye @ 800 </td><td> LI-COR Biosciences </td><td> 926-32210 </td><td> 1:200 de diluição </td></tr><tr><td> LI-COR Odyssey IR scanner </td><td> LI-COR-biociências </td><td> 9201-01 </td><td></td></tr><tr><td> 384-poços de placa de fundo plano </td><td> Nalge Nunc internacional </td><td> 164730 </td><td></td></tr><tr><td> 96-poços de placa de fundo plana </td><td> Nalge Nunc internacional </td><td> 165305 </td><td></td></tr><tr><td> Meio DMEM </td><td> InvitrOgen </td><td> 11965126 </td><td></td></tr><tr><td> Meio RPMI </td><td> Invitrogen </td><td> 11875135 </td><td></td></tr><tr><td> O bicarbonato de sódio </td><td> Invitrogen </td><td> 25080 </td><td></td></tr><tr><td> L-glutamina-100x </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Trypson-EDTA </td><td> Invitrogen </td><td> R001100 </td><td></td></tr><tr><td> Piruvato de sódio </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> Anti-M-2 anticorpo </td><td></td><td></td><td> De Dr. Thomas Moran M </td></tr><tr><td> Anti-NP mAb </td><td> CDC </td><td> V2S2208 </td><td> Obtido com um MTA </td></tr></tbody></table>

high-throughput detection method for influenza virus

Vírus influenza é um patógeno respiratório que faz com que um elevado grau de morbidade e mortalidade a cada ano em múltiplas partes do mundo. Portanto, o diagnóstico preciso da cepa infectante e high-throughput screening rápido de um grande número de amostras clínicas é fundamental para controlar a propagação de infecções pandemia. Atuais diagnósticos clínicos de infecções de gripe são baseados em testes sorológicos, a reação em cadeia da polimerase, imunofluorescência direta e 1,2 espécime de cultura de células. Aqui, nós relatamos o desenvolvimento de uma técnica de romance de diagnóstico usado para detectar os vírus da gripe ao vivo. Nós utilizado o humano do mouse-adaptado A/PR/8/34 (PR8, H1N1) vírus 3 para testar a eficácia dessa técnica usando células MDCK 4. Células MDCK (10 4 ou 5 x 10 3 por poço) foram cultivados em 96 - ou placas de 384-bem, infectadas com PR8 e proteínas virais foram detectadas utilizando anti-M2 seguido por um IR dye-conjugada seganticorpo secundário. M2 5 e hemaglutinina 1 são duas proteínas marcadoras principais usados ​​em muitas diferentes ensaios de diagnóstico. Que empregam IR-dye-conjugados anticorpos secundários minimizou a autofluorescência associada com outras corantes fluorescentes. O uso de anticorpo anti-M2 permitiu-nos para usar a intensidade antígeno-específica de fluorescência como uma métrica direta de quantidade viral. Para enumerar a intensidade de fluorescência, foi utilizado o LI-COR Odyssey baseada scanner IR. Este sistema usa dois de canal detecções de baseados em laser IR para identificar fluoróforos e diferenciá-los a partir de ruído de fundo. O canal de primeira excita em 680 nm e emite menos 700 nm, para a ajudar a quantificar o plano de fundo. O segundo canal detecta fluoróforos que excitam a 780 nm e emissão a 800 nm. Digitalização de PR8-infectados células MDCK em o scanner IR indicado um de fluorescência título de-dependente viral brilhante. Uma correlação positiva da intensidade de fluorescência para título de vírus de partida a partir de 10 2 -10 5 PFU poderia ser conconsistentemente observada. Positividade Minimal, mas detectável consistentemente visto com 10 2 -10 3 PFU PR8 os títulos virais demonstraram a alta sensibilidade das tinturas de perto de-IR. A relação sinal-para-ruído foi determinada por comparando-se os mock-infectados ou isotipo de anticorpo-tratados com células MDCK. Usando as intensidades de fluorescência a partir de 96 - ou formatos de placa 384-bem, nós construídas curvas de titulação padrão. Nestes cálculos, a primeira variável é o título viral enquanto a variável segundo é a intensidade de fluorescência. Portanto, nós utilizamos a distribuição exponencial para gerar uma curva de fit-para determinar a relação polinomial entre os títulos virais e intensidades de fluorescência. Colectivamente, podemos concluir que IR à base de corantes sistema de detecção de proteína pode ajudar a diagnosticar infecção cepas virais e enumerar precisamente o título dos patógenos infectantes.

Watch this Scientific Journal Video about High-throughput Detection Method for Influenza Virus at JoVE.com

High-throughput Detection Method for Influenza Virus

Este método descreve o uso de sistema de imagem de Infravermelhos corante baseada em para a detecção de A H1N1 no bronchioalveolar o lavado de fluido (BAL) de camundongos infectados em uma alta sensibilidade. Esta metodologia pode ser realizado em um 96 - ou 384-bem prato, requer &lt;volume de 10 uL de material de teste e tem o potencial para a triagem concomitante de agentes patogénicos múltiplos.

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, ...

high-throughput-detection-method-for-influenza-virus

Research

JoVE Journal

Immunology and Infection

26.0K visualizações.  Blood Research Institute. Este método descreve o uso de sistema de imagem de Infravermelhos corante baseada em para a detecção de A H1N1 no bronchioalveolar o lavado de fluido (BAL) de camundongos infectados em uma alta sensibilidade. Esta metodologia pode ser realizado em um 96 - ou 384-bem prato, requer 

Vídeo: High-throughput Detection Method for Influenza Virus

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Geração de recombinante do vírus Influenza de DNA plasmidial

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Imunologia e Infecção

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 ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX Detecção colorimétrica em um Microarray de DNA para Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Propagação do vírus Influenza em ovos de galinha embrionados

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

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 ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In Vitro A desmontagem do vírus influenza A capsï¿½eos por centrifugação em gradiente

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Detecção de alto rendimento de patógenos respiratórios em espécimes animais por Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Estudos do Vírus Influenza A em um Modelo de Infecção de Camundongos

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Geração de variantes de fuga de neutralizar os anticorpos monoclonais de vírus da gripe

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

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 ...