This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

Todos os suínos utilizados na coleta dos seguintes dados estavam protegidos sob O Comitê Animal Care Institucional e Use Ohio State University (uso de animais protocolo número 2009A0134-R1). 1. Preparação de Transporte Viral Médio e Coleta de Amostras Vials <ol><li> Adicionar 37 g de Brain Heart Infusion a 900 ml de água purificada e misturar cuidadosamente com uma vareta de agitação e um agitador magnético, enquanto se aquece a 70 ° C para dissolver completamente o pó. </li><li> Autoclave o caldo a 121 ° C durante 15 min. Legal na bancada até que o caldo atingir a temperatura ambiente. Refrigerar a 4 ° C durante a noite se necessário. </li><li> Dissolve-se, por mistura com uma vareta de agitação magnética, 6,02 g de sal de sódio da penicilina G e 10 g de sulfato de estreptomicina em 50 ml de água purificada, à temperatura ambiente. Adiciona-se água purificada adicional para levar o volume total a 100 ml. Filtrar a solução através de uma membrana de polietersulfona de 0,22 um para um frasco estéril. </li><li> Adicionar assepticamente a penicilina filtrada e estreptomicina solução para o resfriado Brain Heart Infusion caldo e misture bem (barra de agitação ou redemoinho frasco à mão) para fazer o meio de transporte viral. </li><li> Assepticamente testar o pH do meio de transporte viral; se necessário, ajustar o pH para 7,4 ± 0,2 com HCl ou NaOH. </li><li> Realizar testes de controlo de qualidade sobre o meio de transporte viral por meio de testes para influenza A proteína da matriz do vírus por TRr-PCR, tal como descrito por Zhang e Harmon. 22 </li><li> Dispensar 5 ml do meio de transporte viral em frascos de polietileno de alta densidade estéreis com a capacidade de 8 ml. Rotular os tubos de ensaio para utilização no campo. </li><li> Armazenar os frascos prontos para uso em caixas padrão crio a -20 ° C até a coleta de amostras. </li></ol> 2. Recolha de Nasal Wipes de suínos <ol><li> Descongele o número apropriado de frascos imediatamente antes da coleta da amostra; será necessário um frasco para injectáveis ​​por suíno amostrados. Descongelamento na salatemperatura leva aproximadamente 30-45 min. Manter os tubos descongelados arrefecer em blocos de gelo até o uso. </li><li> Don equipamento de proteção individual como ditadas pela situação (por exemplo, fato-macaco, capas para botas, máscara, protetor auricular, etc.). </li><li> Entre na área de animal. Se necessário, limitar o porco a uma pequena área mas não conter. Não despertar descansando animais. </li><li> Coloque em um par de luvas de exame descartáveis. Tome cuidado para não contaminar as luvas por porcos tocando, pessoas ou objetos inanimados. </li><li> Use uma mão enluvada para remover um estéreis 5,08 cm x 5,08 cm (2 pol. X 2 pol.) Gaze de algodão de sua embalagem. Compressas de gaze estéreis deve ser usada sempre que possível. Compressas de gaze embrulhados individualmente são mais convenientes de usar do que aqueles envolvidos com duas almofadas de gaze por pacote. </li><li> Segure a gaze de algodão com as pontas dos dedos de uma mão expor tanto da almofada possível para amostragem. </li><li> Limpe a gaze em todo o focinho do porco, tendocuidado extra para entrar nas narinas externas, se possível. Recolhe secreções nasais visíveis (aproximadamente 1 ml) com a mesma almofada de gaze. </li><li> Usando o mesmo lado, dobre a compressa de gaze sobre si mesmo para facilitar a colocação no frasco contendo meio de transporte viral. </li><li> Utilizando outro lado, remover a tampa do frasco e colocar a almofada de gaze dobrada para dentro do frasco. Recapitular o frasco e agite o frasco para garantir a mistura do compressa de gaze e meio de transporte viral. </li><li> Retire as luvas e coloque em um recipiente apropriado. Mudar de luvas entre cada porco. </li><li> Verificar o identificador do frasco. Número recorde de identificação porco, idade, sexo, sinais clínicos, e outras notas consideradas necessárias ao investigador. </li><li> Refrigere as amostras imediatamente após a coleta. Considere fazer alíquotas da amostra antes de congelar para evitar múltiplos ciclos de congelamento e descongelamento. Tão rapidamente quanto possível após a colheita, colocar as amostras em gelo seco durante o transporte para o laboratório. Armazenar os tubos a -80° C até que o teste é iniciado. </li></ol> 3. Detecção do vírus influenza A Ácido Nucleico <ol><li> Descongelar as amostras num banho de grânulo seco 37 ° C durante 5 min e em seguida terminar o descongelamento das amostras no cimo da bancada à temperatura ambiente durante 20-30 min. A adição simultânea de vários frascos para os grânulos irá fazer com que o banho ao calor; recomenda-se precaução, para não superaquecer os frascos. </li><li> Retirar 100 ml de meio de transporte viral para extração de RNA. <ol><li> Usar um de alto rendimento (96 formato de placa de poço) plataforma esférulas magnéticas para a extracção de ARN a partir de um grande número de amostras. 31 </li></ol></li><li> Use em tempo real PCR transcriptase reversa para a influenza A detecção rápida do vírus. 31 </li></ol> 4. O isolamento do vírus influenza A partir nasais Wipes <ol><li> Descongelar as amostras tal como descrito acima na Secção 3.1 </li><li> Trate amostras descongeladas com sulfato de gentamicina (1000ug / ml), anfotericina B (22,5 ug / ml), e sulfato de canamicina (325 ng / mL). </li><li> Inocular em Madin-Darby de rim canino (MDCK) células epiteliais como descrito anteriormente. 32 </li></ol>

<ol>
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The authors have nothing to disclose.

Coleta de amostras de suínos utilizando swab nasal com ponta de poliéster tem se mostrado útil para conduzir a vigilância IAV; no entanto, a utilização do procedimento de swab nasal dificulta os esforços de vigilância devido ao uso obrigatório de um laço à contenção. Toalhetes nasais representam um refinamento de técnicas de amostragem suína atuais para minimizar o estresse sobre as pessoas e suínos durante a coleta de amostra. Enquanto o método tem sido desenvolvido e validado para exposição em ambie...

<html> Os vírus influenza A (IAV) causam doenças respiratórias em muitas espécies, incluindo aves domésticas, suínos e seres humanos. Devido ao rearranjo da evolução viral rápida segmentado genoma IAV pode ocorrer e novas cepas IAV frequentemente surgem. Suína são uma espécie que pode servir como um recipiente de mistura para rearranjo de iAVS a partir de múltiplas espécies hospedeiras. 1 Atualmente, existem três principais subtipos de IAV normalmente circulam entre os porcos da América do Norte (H1N1, H1N2, H3N2), mas várias introduções IAV de seres humanos têm levou a extensa diversidade IAV dentro desses subtipos. 2 evolução rápida da iAVS infectam suínos tem sido evidente desde 1998, quando um recombinante triplo IAV contendo segmentos de genes de vírus humanos, aviários e suínos 3 tornou-se difundido entre os porcos nos Estados Unidos. 4 O gene interno segmentos de que IAV recombinado triplo permanecem altamente prevalente entre iAVS atualmente infectando porcos. 5 &quot;&gt; Em todo o mundo, IAV é uma importante causa de doenças respiratórias em suínos em que os sinais clínicos típicos incluem febre, anorexia, letargia, tosse, dificuldade para respirar, espirros, corrimento nasal e pouco ganho de peso. IAV pode ser particularmente caro para semear fazendas onde reprodutiva falha devido a febre IAV induzida e leitões fracos-nascido foram documentados 6,7. Nos Estados Unidos, IAV é comumente detectada em rebanhos suínos comerciais e extensa diversidade antigênica e genômica e evolução contínua entre IAV suína infectar tem dificultado o controle deste vírus 8-11. Preocupações de saúde pública sobre a emergência de uma estirpe IAV pandemia resultante do rearranjo em suínos foram realizados em 2009, quando um IAV suína-linhagem contendo segmentos de genes da linhagem suína norte-americana triple-reassortant e da linhagem suína aviária do tipo Eurasian causou uma pandemia mundial em humanos 12. A pandemia do vírus (A (H1N1) pdm09), desde então,rearranjado com suínos endêmica IAV estirpes 13,14 e algumas destas estirpes recentemente rearranjado foram transmitidos de volta para os seres humanos. 15 A freqüência de eventos de rearranjo e surgimento de novas cepas IAV com potencial pandêmico faz a vigilância ativa de vírus circulando IAV em suínos imperativo, especialmente na interface suína-humano. A interface suína-humano é importante para a transmissão bi-direcional interespécies do IAV. Humano-a-suína transmissão que ocorrem na produção suína comercial é responsável por uma grande quantidade de diversidade IAV actualmente presente na população de porcos. Feiras agrícolas são os maiores configurações para o comingling de pessoas e suína nos Estados Unidos e são locais para a transmissão zoonótica do IAV 15-21. Em 2012 conhecido, durante o surto de uma variante H3N2 IAV, 93% dos casos relatados no atendimento uma feira agrícola nos dias 15 Análise Genômica antes do início da doença.dos isolados virais de suínos de exposição em comparação com isolados humanos confirmaram transmissão zoonótica. suína 21 Exposição infectado com IAV muitas vezes não mostram sinais clínicos da doença, 21-23, indicando a necessidade de testes de diagnóstico diretos. Amostragem de suínos visivelmente doente por si só não irá identificar com sucesso prevalência IAV em suínos e não pode ser invocado para identificar novas cepas de IAV emergentes entre suína. A vigilância ativa é absolutamente necessário para a detecção de estirpes emergentes da IAV nos suínos e para avaliar sua ameaça tanto para suínos e de saúde pública. A maioria das actividades de fiscalização do IAV são voluntárias e, portanto, são necessários métodos minimamente perturbadoras. Os três principais procedimentos de coleta de amostra ante-mortem para IAV infectar suínos são: swab nasal, fluidos orais e nasais toalhetes. As actuais recomendações para a amostragem suína indivíduo para detectar a inserção lista IAV-de fibra sintética com ponta cotonetes nas narinas como o método preferidopara recolher secreções nasais e células epiteliais. 24,25 Porque os porcos podem tentar evitar este procedimento, uma equipe de pessoal treinado deve conter suínos manualmente ou com um laço, dependendo do tamanho do animal. 26 O processo de contenção é trabalhoso para o pessoal e estressante para os porcos. Além disso, exposição suína estão freqüentemente envolvidos em várias competições em uma feira de modo a percepção de stress em um animal de competição pode fazer proprietários resistente aos esforços de vigilância. Com probabilidades de detecção IAV que variam de 80-100% em IAV rebanhos infectados, fluidos orais tornaram-se uma alternativa popular para nasal swabs para a detecção molecular do IAV em populações de suínos. 27,28 Além disso, líquidos orais podem fornecer uma janela mais ampla de IAV detecção de swab nasal após a infecção inicial. No entanto, IAV isolamento a partir de fluidos orais tem sido problemática com apenas 50% das tentativas de isolamento de vírus, resultando em IAV recuperação. 29 Usando toalhetes nasais em vez de swab nasal durante a vigilância IAV em suínos supera as limitações descritas acima. Toalhetes nasais não requerem o uso de uma armadilha de restrição e pode ser realizada sem perturbar os animais testemunhas ou do procedimento. Treinamento técnico mínimo é necessário para coletar limpezas nasais, que permite que os profissionais não-veterinários, incluindo os proprietários de suínos, para recolher as amostras de vigilância. Toalhetes nasais foram comparadas anteriormente para esfregaços nasais para a detecção e isolamento de vírus influenza A e 30 do protocolo detalhado para este método não-invasivo de amostragem é descrito abaixo.

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			<td style="width:360px;">MP Biomedicals, LLC</td>
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			<td>50 mg/ml</td>
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			<td style="width:360px;">Thermo Scientific</td>
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			<td height="26" style="height:26px;width:216px;">Dermacea Gauze Sponge, 8 ply</td>
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			<td>5.08 cm &#215; 5.08 cm (2 in. &#215; 2 in)&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Nitrile Exam Gloves</td>
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nasal wipes for influenza a virus detection and isolation from swine

Vigilância de vírus da gripe A em suínos é fundamental para a saúde humana e animal, porque o vírus influenza A evolui rapidamente em populações de suínos e novas estirpes estão surgindo continuamente. Suínos são capazes de ser infectado por diversas linhagens do vírus influenza A, tornando-os anfitriões importantes para o surgimento e manutenção da nova gripe A estirpes de vírus. Amostragem suínos em diversas configurações, como granjas comerciais de suínos, feiras e mercados agrícolas, de animais vivos é importante para fornecer uma visão abrangente das cepas que circulam atualmente IAV. O ante-mortem técnica de amostragem padrão-ouro atual (ou seja, coleta de swab nasal) é trabalhoso porque exige contenção física dos porcos. Toalhetes nasais envolvem esfregando um pedaço de tecido em todo o focinho do porco com o mínimo de retenção do animal. A nasal limpe procedimento é simples de executar e não requer pessoal com formação profissional ou veterinária manipulação animal. While ligeiramente menos sensível do que swab nasal, as taxas de detecção e isolamento do vírus são suficientes para fazer nasal limpa uma alternativa viável para a amostragem de suínos individuais quando são necessários métodos de amostragem de baixa tensão. O protocolo de processo descreve as etapas necessárias para coletar uma nasal viável limpe a partir de um porco individual.

A utilização bem sucedida deste método produz TRr-PCR resulta que, acompanhada com a utilização de um controlo interno durante a extracção de ARN e TRr-PCR, mostram amostras não contêm inibidores de PCR a partir de quaisquer detritos apanhados ambiente durante a amostragem. Após a inoculação de exemplo, os poços de isolamento de vírus deve ser livre de detritos ambientais visível a partir da amostra. Há um consenso razoável entre os resultados rRT-PCR e os resultados de isolamento de vírus com o entend...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.3K visualizações.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

Vídeo: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

High-throughput Detection Method for Influenza Virus

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

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

Alta capacidade método de detecção de vírus Influenza

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

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Purificação por afinidade de Complexos de Influenza Virus ribonucleoproteína da cromatina de células infectadas

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-imunoprecipitação da proteína do rato Mx1 com a nucleoproteína do vírus da gripe A

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

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 ...

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 ...

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.