Os autores gostariam de agradecer Nada Naffakh e Marie-Anne Rameix-Welti (Institut Pasteur) para o vírus rWSN-PB2-Strep.

<html> Um fluxograma esquemático do protocolo é mostrado na fig. 1 e uma tabela de reagentes é apresentada abaixo. 1. Infecção (16 - 24 h) <ol><li> Semente para fora células HeLa em 5 de 150 mm pratos de plástico de células de cultura em meio Dulbecco modificado por Eagle (DMEM)-elevado médio de glicose, de modo que eles atingem cerca de 80% de confluência no dia seguinte (aproximadamente 5 x 10 células 8). É importante que as células não chegar a 100% de confluência. </li><li> No dia seguinte, diluir a Strep-etiquetados com estoque de vírus influenza em salino tamponado com fosfato (PBS) contendo 0,3% de albumina sérica bovina (BSA) a uma multiplicidade de infecção de 3. </li><li> Remover o meio de crescimento a partir das células, lavar uma vez com PBS, e adicionar 10 ml de PBS contendo o vírus às células. Incubar durante 1 h à temperatura ambiente. </li><li> Substituir o meio infecção com DMEM de alto glicose, e continuar a incubação a 37 ° C. </li></ol>&quot;&gt; O comprimento de incubação depende da fase da infecção a ser estudado (ver discussão). 2. Fraccionamento subcelular (3 h) <ol><li> Lave as células uma vez com PBS frio e, em seguida, suspender as células em PBS frio, com um raspador de borracha e transferência para um tubo cónico de 50 ml. </li><li> As células da pelota em uma centrífuga de bancada durante 5 min a 1000 rpm e, em seguida, aspirar PBS. </li><li> Ressuspender o sedimento de células em 10 ml de tampão de sacarose a frio (10 mM HEPES pH 7,9, 10 mM de KCl, 2 mg de acetato de mM (MgOAc), 3 mM de CaCl2, 340 mM de sacarose, 1 mM de ditiotreitol (DTT), mistura de 1% inibidor de protease) . Após a suspensão, passar as células através de uma ponta de pipeta 200 uL ligado a um 10 ml pipeta de plástico de soro de perturbar aglomerados de células. </li><li> Incubar 10 min em gelo. Inverta suavemente periodicamente para ressuspender as células. </li><li> Adicionar Nonidet P-40 (NP-40) até uma concentração final de 0,5% e vórtice a alta velocidade durante 15 seg. Imediatamente centrifugar durante 10 min a 3.500 xg, a 4 ° C em uma centrifuga de mesage. </li><li> Remover o sobrenadante e armazenar a 4 ° C. Esta é a fracção citoplasmática. O sedimento deve ser menor e mais branco do que a original sedimento celular. Todas as fracções pode ser armazenado durante pelo menos 4 horas a 4 ° C. </li><li> Lavar o sedimento em 5 ml de tampão frio de sacarose, re-centrífuga como no passo 2,5 e descartar o sobrenadante. </li><li> Ressuspender o sedimento (contendo núcleos) em 1,5 ml de tampão de extracção a frio nucleoplasmic (HEPES 50 mM pH 7,9, 150 mM de KOAc, 1,5 mM de MgCl2, 0,1% de NP-40, 1 mM de DTT, mistura inibidor de protease de 1%). </li><li> Transfira para uma gelada, todo em vidro 4 ml Dounce homogeneizador com um pilão apertada e homogeneizar os núcleos com 20 golpes. Evitar a formação de espuma durante a homogeneização. A resistência deve aumentar depois de aproximadamente 10 pancadas. Homogeneização eficaz pode ser confirmado sob um microscópio de contraste de fase. </li><li> Transferir os núcleos homogeneizada para um tubo de microcentrífuga de 1,5 ml e incubar 20 min a uma roda giratória a 4 °C. </li><li> Centrifugar a 16.000 x g numa microcentrífuga a 4 ° C durante 10 min. </li><li> Remover o sobrenadante e armazenar a 4 ° C. Esta é a fracção nucleoplasmic. </li><li> Ressuspender o sedimento em 1,5 ml de tampão de digestão (50 mM de HEPES pH 7,9, 10 mM de NaCl, 1,5 mM de MgCl2, 1 mM de DTT, 1% de mistura inibidor de protease, 100 U / ml Benzonase (para análise de RNA, substituto com 20 U / ml de RNase livre de DNase I)) pré-aquecida a 37 ° C, e incubar 10 min a 37 ° C. </li><li> Adicionar 42 ul de NaCl 5 M (para uma concentração final de 150 mM de NaCl) e incubar 20 min mais a 4 ° C. </li><li> Centrifugar a 16.000 x g numa microcentrífuga a 4 ° C durante 10 min. </li><li> Remover o sobrenadante e armazenar a 4 ° C. Esta é a fracção CH150. </li><li> Ressuspender o sedimento em 1,5 ml de tampão de sal de alta frio (50 mM HEPES pH 7,9, 500 mM de NaCl, 1,5 mM de MgCl2, 0,1% de NP-40, 1 mM de DTT, mistura inibidor de protease de 1%) e incubar 20 min em gelo. </li><li> Centrifugar a 16.000 x g &lt;/ Em&gt; numa microcentrífuga a 4 ° C durante 10 min. </li><li> Remover o sobrenadante e armazenar a 4 ° C. Esta é a fracção ch500. </li></ol> 3. Strep-tag Purificação (2 h) <ol><li> Adicionar 300 ul de NaCl 5 M para a fracção citoplasmática (para uma concentração final de 150 mM de NaCl). </li><li> Alíquota de 100 uL de concentrado contas Strep-Tactin Sepharose por fração em um tubo cônico de 15 ml. </li><li> Adicionar 10 volumes de Strep tampão de lavagem (20 mM HEPES pH 7,9, 150 mM de NaCl (500 mM de NaCl durante ch500 amostras), EDTA 1 mM) a esferas, inverter o tubo de misturar, e centrifugar 5 min a 1000 x g em um tampo de mesa centrifugar. </li><li> Descartar o sobrenadante, repetir o passo 3,3 duas vezes e, em seguida, ressuspender o sedimento grânulo em um volume igual de tampão de lavagem Strep. </li><li> Adicionar 200 ul de suspensão lavada grânulo Strep-Tactin para cada fracção (em tubos de 1,5 ml de microcentrífuga de nuceloplasmic e CH150, e ch500 fracções e um tubo de 15 ml cónica para fracção citoplasmática). </li><li> Rodartubos 1h a 4 ° C em uma roda rotativa. </li><li> Centrifugar os tubos 1 min a 1000 x G a 4 ° C. Elimine o sobrenadante. </li><li> Adicionar 1 ml de tampão de lavagem a frio Strep a cada tubo. Transferir grânulos a partir de 15 mL de tubo (fracção citoplasmática) a 1,5 mL tubo. Lavar todos os tubos 1 min a 4 ° C, centrifugando depois como na etapa 3,7. Repetir este passo de lavagem duas vezes. </li><li> Após o passo de última lavagem, remover todos os vestígios de tampão de lavagem, utilizando uma pipeta de ponta plana. Adicionar 100 uL de tampão de eluição (tampão de lavagem Strep contendo 2,5 mM desthiobiotin) e misturar suavemente. </li><li> Incubar 15 min em gelo. Misture suavemente contas ocasionalmente tocando no fundo dos tubos. </li><li> Tubos de centrífuga 1 min a 1000 x G a 4 ° C em microcentrífuga. Recolher o sobrenadante contendo eluídas Strep-tag vRNPs. </li></ol> 4. Os resultados representativos A eficiência do fraccionamento é melhor julgado por análise de Western blot dos lisados ​​fraccionados usando antibodies específicos para marcadores subcelulares (Fig. 2). Especificamente, o fraccionamento subnuclear bem sucedida deveria mostrar pouca ou nenhuma celular RNA polimerase II (Pol II), na nucleoplasmic, ou fracção nuclear solúvel 4, e mais deve ser extraída com 150 mM de NaCl 5. Uma degradação de Pol II é também reprodutivelmente observada em células infectadas por vírus de influenza em comparação com células não infectadas, consistentes com a literatura 6. A purificação do vRNPs é melhor julgado por prata ou coloração com Coomassie, como mostrado para um eluato citoplasmática na fig. 3. Na nossa experiência, a maioria Strep-PB2 é purificado como parte de um vRNP totalmente formado, isto é, polimerase pouco solúvel é capturado. Isso se reflete na alta NP: PA/PB1/PB2 proporção observada pela coloração de prata ou Coomassie. Uma relação mais baixa sugere a contaminação com tampão de RNases, como vários RNases ubíqua (tais como a RNase A) são conhecidos por RNA clivam virais, de tal forma a dissocicomi a polimerase de NP multímeros 7. Curiosamente, o tratamento com Benzonase sozinho, enquanto suficiente para digerir RNA viral, parece ter nenhum efeito sobre os rácios estequiométricas de proteínas vRNP. Embora não possamos explicar esse fenômeno, observou-se ruptura de vRNPs após digestão com RNases outros (dados não mostrados), sugerindo que Benzonase pode deixar certas regiões estruturalmente importantes RNA intacto. Após purificação do vRNPs a partir do citoplasma e nucleoplasma (realizada sem digestão com nuclease), 8 bandas fracas mas discreta de elevados pesos moleculares pode ser observada por coloração com prata, que correspondem aos pesos moleculares das preditos 8 segmentos do genoma de influenza (ver ref. 3 ). As quantidades relativas de vRNPs purificadas a partir de cada fracção em 9 h após a infecção são mostrados na fig. 4. A distribuição de vRNPs varia ao longo do curso da infecção, com forte acumulação na fracção CH150 em pontos de tempo iniciais, eacumulação aumentada no nucleoplasma e citoplasma em pontos de tempo tardios. <img src="/files/ftp_upload/4028/4028fig1.jpg" alt="A Figura 1"/> Figura Fluxograma 1. Do fracionamento subcelular. Adaptado da ref. 3. <img src="/files/ftp_upload/4028/4028fig2.jpg" alt="A Figura 2"/> Figura 2. Ocidental análise de mancha de fracções subcelulares de influenza células infectadas por vírus. 10 9 células HeLa foram infectados com influenza WSN estirpe do vírus durante 9 h antes do fraccionamento subcelular. Quantidades iguais de proteína total foram carregados em cada pista e analisados ​​utilizando os anticorpos indicados. RNA polimerase II (Pol II) foi detectada utilizando o clone 8WG16, que reconhece todas as formas do domínio C-terminal (a banda hypophosphorylated é o mais proeminente). Adaptado da ref. 3. <img src="/files/ftp_upload/4028/4028fig3.jpg" alt="A Figura 3"/> Figura 3.Análise de mancha de prata purificada vRNPs. As células HeLa foram infectadas com rWSN (como um controlo negativo) ou rWSN-PB2-Strep durante 9 horas, seguido de purificação Strep a partir da fracção citoplasmática. Asterisk denota bandas que não são visíveis após a digestão RNase. Adaptado da ref. 3. <img src="/files/ftp_upload/4028/4028fig4.jpg" alt="A Figura 4"/> Figura 4. Análise de manchas de Prata da vRNPs purificadas de diferentes frações celulares. 10 9 células HeLa foram infectadas com rWSN-PB2-Strep ou rWSN para 9 h, seguido por fraccionamento subcelular e purificação Strep. Quantidades iguais de eluato a partir de cada fracção foram carregados. Adaptado da ref. 3.

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Enquanto muitos estudos foram recentemente identificadas proteínas individuais ou redes celulares envolvidos na infecção por vírus influenza virus 8, o significado funcional da maioria destas interacções permanece obscura. Dada a dependência absoluta de cromatina funções baseadas para a síntese de RNA vírus influenza e da natureza complexa biofísicas e bioquímicas do núcleo 9, as novas técnicas serão necessários para elucidar estas funções. O fraccionamento subnuclear apresentamo...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> DMEM de alto glicose </td><td> Gibco </td><td> 11965-092 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A9418 </td><td></td></tr><tr><td> Inibidor da protease da mistura G </td><td> Serva </td><td> 39101 </td><td></td></tr><tr><td> Benzonase Nuclease </td><td> Novagen </td><td> 71206 </td><td> 25 U / uL </td></tr><tr><td> DNase I, livre de RNase </td><td> ThermoScientific </td><td> EN0523 </td><td> 50 U / uL </td></tr><tr><td> Dounce homogeneizador </td><td> Wheaton </td><td> 432-1271 </td><td> Use tipo &quot;B&quot; pilão </td></tr><tr><td> Strep-Tactin Sepharose </td><td> IBA GmbH </td><td> 2-1201-025 </td><td> Formato de coluna de 50% de suspensão também pode ser usado </td></tr><tr><td> Desthiobiotin </td><td> IBAGmbH </td><td> 2-1000-002 </td><td></td></tr></tbody></table>

affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

Como todos os vírus de RNA fita negativa-, o genoma dos vírus da gripe é embalado na forma de complexos de ribonucleoproteína virais (vRNP), em que o genoma de cadeia simples é encapsidado pelo nucleoproteína (NP), e associada com o complexo trimérico polimerase consistindo da PA, PB1, PB2 e subunidades. No entanto, em contraste com a maioria dos vírus de ARN, os vírus influenza realizar a síntese de RNA viral nos núcleos das células infectadas. Curiosamente, a síntese de mRNA virai utiliza celulares pré-mRNAs como iniciadores, e tem sido proposto que este processo tem lugar em cromatina 1. As interacções entre a polimerase virai e do hospedeiro a RNA polimerase II, bem como entre NP e nucleossomas hospedeiras foram também caracterizados 1,2. Recentemente, a geração do vírus da influenza recombinantes que codificam um One-Strep-Tag geneticamente fundido com o terminal C da subunidade PB2 da polimerase virai (rWSN-PB2-Strep 3), seren descrito. Estes vírus recombinantes permitem a purificação de PB2 contendo complexos, incluindo vRNPs, a partir de células infectadas. Para obter purificado vRNPs, culturas de células são infectadas, e vRNPs são purificados por afinidade a partir de lisados ​​derivados a partir destas células. Contudo, os procedimentos de lise utilizadas até à data têm sido baseados em um-passo de lise de detergente, o qual, apesar da presença de uma nuclease geral, muitas vezes extrair cromatina-bound único material de forma ineficiente. O nosso trabalho preliminar sugeriu que uma grande porção de vRNPs nucleares não foram extraídos durante a lise das células tradicional, e, portanto, não poderia ser purificado por afinidade. Para aumentar este eficiência de extracção, e para separar cromatina-ligado a partir de não-ligados a cromatina vRNPs nucleares, adaptado um passo a passo do protocolo de extracção subcelular para influenza células infectadas por vírus. Resumidamente, este procedimento primeiro separa os núcleos a partir da célula e, em seguida, extrai solúveis proteínas nucleares (aqui denominado &quot;a fracção nucleoplasmic&quot;).O material insolúvel remanescente nuclear é então digerido com Benzonase, um DNA inespecífica / RNA nuclease, seguido de dois passos de extracção de sal: em primeiro lugar utilizando 150 mM de NaCl (denominado &quot;CH150&quot;), em seguida, NaCl 500 mM (&quot;ch500&quot;) (fig. 1 ). Estes passos de extracção de sal foram escolhidos com base na nossa observação de que 500 mM de NaCl foi suficiente para solubilizar mais de 85% de vRNPs nucleares e ainda assim permitir a ligação de vRNPs etiquetados para a matriz de afinidade. Após fraccionamento subcelular das células infectadas, é possível purificar afinidade vRNPs pB2-marcados a partir de cada fracção individual e analisar a sua proteína e os componentes de RNA utilizando Western Blot e extensão do iniciador, respectivamente. Recentemente, utilizou este método para descobrir que vRNP exportação formar complexos durante pontos finais após a infecção sobre a fracção de cromatina extraiu-se com 500 mM de NaCl (ch500) 3.

Watch this Scientific Journal Video about Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells at JoVE.com

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

Os vírus da gripe replicar seu genoma de RNA em associação com a célula hospedeira da cromatina. Aqui, apresentamos um método para purificar intactos os complexos de ribonucleoproteínas virais da cromatina das células infectadas. Purificou complexos virais podem ser analisados ​​por Western blot e tanto de extensão do primer de proteína e do conteúdo de ARN, respectivamente.

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 ...

affinity-purification-influenza-virus-ribonucleoprotein-complexes

Research

JoVE Journal

Immunology and Infection

13.0K visualizações.  Universitätsklinikum Freiburg. Os vírus da gripe replicar seu genoma de RNA em associação com a célula hospedeira da cromatina. Aqui, apresentamos um método para purificar intactos os complexos de ribonucleoproteínas virais da cromatina das células infectadas. Purificou complexos virais podem ser analisados ​​por Western blot e tanto de extensão do primer de proteína e do conteúdo de ARN, respectivamente.

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

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

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, ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

Purificação de vacúolos patógeno de<em&gt; Legionella</em&gt; Infectados Fagócitos

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

Expressão da Funcionais recombinante hemaglutinina ea neuraminidase Proteínas do vírus Influenza H7N9 Novel usando a expressão Sistema de Baculovirus

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

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 ...

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 ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

A purificação a partir de ARN intracelularmente Grown<em&gt; Listeria monocytogenes</em&gt; Em macrófagos

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Fenotípica e análise funcional de células reguladoras T ativadas isolados de ratinhos infectados com o vírus da coriomeningite linfocítica crônica

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Determinação do biofilme Iniciação em células infectadas por vírus por bactérias e fungos

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

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

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