This work was funded by both the&#160;Canadian Institutes of Health Research&#160;and&#160;Natural Sciences and the Engineering Research Council of Canada. M. Bisaillon is a Chercheur Boursier Senior from the Fonds de Recherche en Sant&#233; du Qu&#233;bec and also a member of the Centre de Recherche Clinique du Centre Hospitalier Universitaire de Sherbrooke. We thank Dr. Ralf Bartenschlager for the generous gift of the HCV replicon system. We also thank Dr. Charles Rice and Dr. Daniel Lamarre for kindly providing the hepatic cell line. We also want to thank Guillaume Tremblay for technical assistance.

1. Células e Preparação de tratamento <ol><li> Numa placa de 24 poços, colocar uma tampa de vidro no fundo dos poços. Lavar cada poço com tampão fosfato salino (PBS). </li><li> Semente células Huh-7 que suportam o replicão de VHC subgenï¿½ico em cada poço a uma densidade de 5 x 10 4 células / poço. Se não houver tratamento a fazer, as células podem ser semeadas a uma densidade mais elevada. O meio de cultura é de Dulbecco Modified Eagle Médium (DMEM) suplementado com 10% de Soro Fetal Bovino (FBS), L-glutamina 2mM, piruvato de sódio 1 mM e 250 mg / mL de G-418 (para manter o replicão). </li><li> Incubar a 37 ° C e 5% de CO 2. No dia seguinte, as células com o tratamento de 3 ^ M da hLF purificado a partir de leite humano. </li></ol> 2. O paraformaldeído / Sacarose Preparação (12% de PAF e 12% de sacarose) <ol><li> Coloque 500 ml de PBS num recipiente e aquecê-la compreendida entre 20 ° C e 30 ° C. Dissolve-se 60 g de sacarose em PBS. Dissolve-se 60 g de paraformaldehyde (PAF) na / solução de sacarose PBS. Adiciona-se lentamente NaOH 2 N (3 a 7 ml) até que uma solução clara é obtida. </li><li> Ajustar o pH para 7,4 com HCl. Completar o volume a 500 ml com PBS. Filtrar por gravidade sobre filtro Whatman. Antes do uso, dilui-se a solução com PBS até uma concentração final de 4% de PAF e 4% de sacarose (de armazenamento de 4 ° C durante 1 semana no máximo). </li></ol> 3. Imunofluorescência <ol><li> No momento desejado (0 horas, 2 horas ou 24 horas de tratamento), lavar células duas vezes (1 minuto), com PBS e fixar com PBS / 4% de PAF / 4% de sacarose durante 20 min à TA. As células são tipicamente a 30-40% de confluência e 5 1,5x10 células são utilizadas. </li><li> Permeabilizar as células com 0,15% de Triton X-100 em PBS durante 5 min à temperatura ambiente e em bloco de 10% de soro de cabra normal (NGS) em PBS durante 20 min. </li><li> Dilui-se o anticorpo primário da proteína de interesse em 10% NGS (exemplo: rato primária anticorpo anti-hLF diluída 1: 1.000). Aplicar o anticorpo primário às células. </li><li> Após 4 horas à temperatura ambiente ou O /N a 4 ° C, lavar as células três vezes durante 5 min com 10% de NGS. Corar as células com o anticorpo secundário marcado com fluorocromo diluído em 10% NGS (exemplo: Fluorocromo com um comprimento de onda de excitação de 488 nm ligada a um anticorpo anti-rato de 1: 1000) durante 1 h à TA no escuro. </li><li> Lavar as células uma vez durante 5 min com PBS e as células com manchar 4 &#39;, 6-diamidino-2-fenilindole (DAPI) (1 ug / ml) durante 15 min à temperatura ambiente no escuro. </li><li> Lavar as células duas vezes durante 5 min com PBS. Mount cobrir vidros no SlowFade meio de montagem antes de visualização através da microscopia. As células estão agora prontos para análise por epifluorescência ou microscopia confocal. </li><li> Use diferentes controlos para assegurar a especificidade da detecção. Por exemplo, todos os anticorpos podem ser omitidos a fim de detectar a autofluorescência. O anticorpo primário pode ser omitido para determinar a coloração não específica por o anticorpo secundário. Finalmente, se possível, controlar as células ou tecidos que não fazerexpressar a molécula de interesse, também pode ser usado. </li><li> Visualize as amostras que usam o microscópio de fluorescência apropriado (epifluorescência ou confocal) e filtros adequados para a etiqueta fluorescente utilizado. As lâminas podem também ser armazenadas numa caixa de corrediça a &lt;-20 ° C para análise posterior. </li></ol> 4. Microscopia de Fluorescência <ol><li> Manter as amostras no escuro para evitar a fotodegradação. Ligue a fonte de luz do microscópio escolhido (epifluorescência ou confocal). </li><li> Ligue o microscópio. Ligue a câmera para o microscópio. Coloque o slide no microscópio para visualização. Adicionar o óleo de imersão sobre a corrediça para aumentar a abertura numérica da lente objectiva. </li><li> Selecione os filtros apropriados. Ajuste o foco e objetivo adequado. Ajuste as persianas, a fim de detectar o fluorocromo adequado, mas evitar a fotodegradação. Mantenha fora luzes do teto para minimizar fundo claro quando se olha para amostra. </li><li> Alterar thOs filtros para visualizar vários fluorocromos (por exemplo DAPI e para um comprimento de onda de excitação de 488 nm). Tire fotos com a câmera. </li></ol>

<ol>
	<li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+techniques.">Immunofluorescence techniques.</a> J Invest Derm. 133 (1), e4 (2013).</li><li>Portugal, J., Waring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignment+of+DNA+binding+sites+for+4&#8217;,6-diamidine-2-phenylindole+and+bisbenzimide+(Hoechst+33258).+A+comparative+footprinting+study.">Assignment of DNA binding sites for 4&#8217;,6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study.</a> Biochim Biophys Acta. 949 (2), 158-168 (1988).</li><li>Lichtman, J. W., Conchello, J. -. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nat Methods. 2 (12), 910-919 (2005).</li><li>St Croix, C. M., Shand, S. H., Watkins, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+comparisons,+applications,+and+problems.">Confocal microscopy: comparisons, applications, and problems.</a> BioTechniques. 39, S2-S5 (2005).</li><li>Yi, M., Kaneko, S., Yu, D. Y., Murakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+envelope+proteins+bind+lactoferrin.">Hepatitis C virus envelope proteins bind lactoferrin.</a> Journal of virology. 71, 5997-6002 (1997).</li><li>Wakabayashi, H., Oda, H., Yamauchi, K., Abe, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrin+for+prevention+of+common+viral+infections.">Lactoferrin for prevention of common viral infections.</a> J Inf Chem. 20 (11), 666-671 (2014).</li><li>Picard-Jean, F., Bouchard, S., Lariv&#233;e, G., Bisaillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+inhibition+of+HCV+replication+represents+a+novel+mechanism+of+action+by+the+innate+immune+Lactoferrin+protein.">The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein.</a> Antiviral research. 111, 13-22 (2014).</li><li>Johnson, G. D., Davidson, R. S., McNamee, K. C., Russell, G., Goodwin, D., Holborow, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fading+of+immunofluorescence+during+microscopy:+a+study+of+the+phenomenon+and+its+remedy.">Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy.</a> J Immunol Methods. 55 (2), 231-242 (1982).</li><li>Green Remington, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein:+a+perspective.">fluorescent protein: a perspective.</a> Protein Sci. 20 (9), 1509-1519 (2011).</li></ol>

The authors declare that they have no competing financial interests.

A epidemia de HCV permanece uma ameaça global, com 80% dos pacientes recém-infectadas desenvolvem uma infecção crônica, colocando-os em risco de cirrose, insuficiência hepática ou carcinoma hepatocelular. Por acção directa agentes antivirais combatem a replicação do HCV e maturação representam agentes anti-HCV de primeira linha, como recentemente demonstrado pela aprovação regulamentar de dois NS3 N-terminal inibidores da protease (boceprevir e telaprevir). A actividade anti-VHC hLF é actualmente acredita-se que se comportam como um inibidor de entrada viral, ligando circulam viriões e impedindo-os de entrar no seu alvo hepatocelular. A nossa pesquisa sobre a hLF apresenta um novo mecanismo intracelular anti-HCV de acção (distinta da neutralização virião extracelular) 7. Imunofluorescência pode ser eficientemente usado para visualizar a distribuição e / ou a localização de uma molécula-alvo numa amostra biológica. Conforme encontrado em muitas outras técnicas de fluorescência, um dos pró principalblemas associados com a imunofluorescência é fotobranqueamento (destruição fotoquímica do fluorocromo) 8. Essa perda de actividade causada por fotobranqueamento pode ser contornado, quer através do emprego de fluorocromos mais robustas que são menos propensas a fotobranqueamento ou através da redução da intensidade ou do tempo de exposição do vão de luz. Além disso, uma vez que os anticorpos não podem atravessar a membrana celular, a imunofluorescência é geralmente apenas limitado às células fixadas. Abordagens alternativas para monitorar a distribuição / localização de proteínas em células vivas, por conseguinte, em geral contam com a expressão de proteínas contendo domínios de proteínas fluorescentes, tais como a proteína fluorescente verde (GFP) 9. No entanto, a adição de tais epitopos GFP está por vezes associada a uma modificação na localização e actividade das proteínas. Imunofluorescência também pode ser utilizado num número de diferentes amostras de tecido, incluindo secções de linhas de células cultivadas ou de células individuais, é também frequentemente utilizada para monitorizara localização / distribuição de proteínas, glicanos, e pequenas moléculas. As principais vantagens desta técnica reside no seu simplicidade e o facto de que pode ser usado em combinação com outros métodos, que não anticorpos de coloração fluorescente (por exemplo, coloração de ADN). Além disso, os anticorpos secundários produzidos comercialmente são relativamente barato e disponível em uma vasta gama de cores. No estudo actual, a imunofluorescência de células hepáticas de apoio a um replicão de VHC subgenï¿½ico revelou uma redução qualitativa na intensidade de coloração do VHC após tratamento com hLF. Esta é uma observação interessante como este sistema replicão subgenómico que falta o HCV E1 e E2 glicoproteínas do envelope, que são os alvos farmacológicos geralmente aceites da hLF. Ao contrário da actividade de neutralização do virião extracelular, a hLF teriam de ser interiorizada pelos hepatócitos infectados com o VHC, a fim de impedir a replicação viral. O uso de microscopia fuorescence nos permitiu avacomeu essa possibilidade. Após a pré-incubação de hepatócitos infectados pelo HCV, a hLF exógeno foi internalizado com sucesso, e mostrado para impedir a replicação viral intracelular. A compreensão deste romance atividade antiviral hLF constitui um passo em frente na compreensão do mecanismo (pleiotropic e provável) mundial de inibição dessa proteína imunidade inata.

Immunofluorescence is a technique that uses a fluorescence microscope to visualize the distribution and/or localization of a target molecule in a biological sample. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell1. It is typically used on tissue sections and cultured cell lines in order to analyze the distribution/localization of various biological molecules such as proteins, nucleic acids and glycans. It should be noted that immunofluorescence is often used in combination with other non-antibody methods of fluorescent staining such as the 4',6-diamidino-2-phenylindole (DAPI) stains which are typically used to label DNA2. Moreover, this technique involves fixation of the cells which allows the analysis of cells at a specific time.
Different types of microscopes can be used to analyze immunofluorescence samples. The simplest is the epifluorescence microscope (Figure 1) for which excitation of the fluorochrome and detection of the fluorescence are done through the same light path3. Because most of the excitation light is transmitted through the sample, only reflected excitatory light can reach the objective together with the emitted light. This approach unfortunately leadsto a frequent high signal to noise ratio.In contrast, confocal microscopy (Figure 2) offers a distinct advantage for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light4. This approach allows the reconstruction of three-dimensional structures from the obtained images. However, since an important fraction of the light from the sample is blocked at the pinhole, long exposures are often required.
There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect). Direct immunofluorescence involves a primary antibody linked with a fluorochrome (Figure 3). This method is less frequently used because it provides lower signal, involves higher cost and less flexibility1. Moreover, such antibodies are generally harder to find commercially. On the other hand, the direct attachment of the fluorochrome to the antibody significantly reduces the number of steps in the procedure, saving time and frequently reducing non-specific background signal. This also limits the possibility of antibody cross-reactivty.
Indirect immunofluorescence involves a primary unlabelled antibody which is specific for the epitope of interest1. A secondary antibody which carries the fluorochrome then recognizes the primary antibody and binds to it (Figure 3). Although indirect immunofluorescence is more complex and time consuming than direct immunofluorescence, it is frequently used because of its high sensitivity and it also provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In addition, a vast array of commercial secondary antibodies is available at affordable prices.
Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion5. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. This manuscript presents a study in which immunofluorescence assays were performed to monitor the internalization of hLF, an important component of the immune system6, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus (HCV).

<table><tbody><tr><td>DMEM</td><td>Wisent</td><td>319-005-CL</td><td></td></tr><tr><td>PAF</td><td>BioShop</td><td>PAR070.1</td><td>Flammable solid, skin irritant, lungs and eyes&amp;nbsp;</td></tr><tr><td>PBS</td><td>Wisent</td><td>311-425-CL</td><td>Without Ca&lt;sup&gt;2+ &lt;/sup&gt;&amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>NGS</td><td>Wisent</td><td>053-150</td><td></td></tr><tr><td>AlexaFluor 488-labeled anti-mouse</td><td>Invitrogen</td><td>A11017</td><td></td></tr><tr><td>AlexaFluor 568-labeled anti-rabbit</td><td>Invitrogeb</td><td>A21069</td><td></td></tr><tr><td>Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA)</td><td>Invitrogen</td><td>W11261</td><td>Potentially mutagenic</td></tr><tr><td>Anti-NS5A rabbit</td><td>Abcam</td><td>ab2594</td><td></td></tr><tr><td>Anti-hLF mouse</td><td>Abcam</td><td>ab10110</td><td></td></tr><tr><td>SlowFade</td><td>Invitrogen</td><td>S36937</td><td></td></tr><tr><td>Hoechst stain</td><td>Life Techn.</td><td>H1399</td><td>Potentially&amp;nbsp;mutagenic&amp;nbsp;
and carcinogenic</td></tr><tr><td>hLF</td><td>Sigma</td><td>L0520</td><td></td></tr><tr><td>Nikon Eclipse visible/epifluorescence Microscope</td><td>Nikon</td><td>TE2000-E</td><td></td></tr><tr><td>epifluorescence/confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr></tbody></table>

immunofluorescence to monitor the cellular uptake of human lactoferrin and its associated antiviral activity against the hepatitis c virus

Imunofluorescência é uma técnica de laboratório vulgarmente usados ​​para estudar vários aspectos da biologia. É tipicamente usado para visualizar a distribuição e / ou a localização de uma molécula alvo em células e tecidos. Imunofluorescência baseia-se na especificidade de anticorpos fluorescentes marcado contra os seus antigénios correspondentes no interior de uma célula. Ambas abordagens directas e indirectas de imunofluorescência pode ser usado que se baseiam no uso de anticorpos ligados a um fluorocromo. A imunofluorescência direta é menos freqüentemente usado porque fornece sinal inferior, envolve custos mais elevados e menos flexibilidade. Em contraste, imunofluorescência indireta é mais comumente utilizado devido à sua alta sensibilidade e fornece um sinal amplificado desde mais de um anticorpo secundário pode anexar a cada anticorpo primário. Neste texto, tanto microscopia de epifluorescência e microscopia confocal foi utilizada para monitorizar a internalização da lactoferrina humana, um componente importante da resposta imunesistema, em células hepáticas. Além disso, nós monitorizado o potencial inibitório da hLF na replicação intracelular do vírus da hepatite C por meio de imunofluorescência. Ambas as vantagens e desvantagens associadas a estes métodos são discutidos.

A capacidade do Huh-7 linha celular hepática para internalizar exogenamente administrada hLF foi monitorizada utilizando imunofluorescência em um miscroscope confocal. hLF foi adicionado ao meio de cultura e a internalização foi deixada por 24 horas, após o que, a hLF extracelular foi lavada com PBS e a membrana ligada a hLF residual foi degradada com um tratamento com tripsina 5 min (1 mL). As células foram re-semeadas e deixou-se voltar a aderir durante 18 h antes de coloração por imunofluorescência. A fim de delinear os limites citoplasmáticos, as membranas de células foram coradas com aglutinina de gérmen de trigo (WGA) fluorocromo de um comprimento de onda de excitação de 488 nm conjugado. A coloração foi conseguido hLF utilizando um anticorpo primário anti-hLF e um fluorocromo com um comprimento de onda de 633 nm, ligado ao anticorpo secundário. De modo a determinar a localização precisa da hLF, células foram fotografadas seguinte sequencial de 0,5 um corte transversal horizontal através de microscopia confocal. A Figura 4 apresenta um optsecção transversal-ical correspondente à espessura de célula médio. A seguir à adição de exógeno hLF, uma quantidade significativa da hLF foi detectado no interior do citoplasma de hepatócitos. Sem hLF foi observada no interior das células não tratadas quando usando as configurações instrumentais idênticas. Em conjunto, estes dados confirmam que os hepatócitos têm a capacidade de adquirir a hLF do seu ambiente extracelular. A capacidade da hLF para inibir a replicação intracelular do VHC foi próxima monitorizada utilizando microscopia de epifluorescência. Células Huh-7 que suportam o replicão de VHC subgenómico (expressando HCV NS3, NS4A, NS4B, NS5A, NS5B proteínas) replicação foram tratados com 3 uM da hLF para 0, 2 ou 24 horas. A coloração dupla imunofluorescência para proteínas hLF e NS5A do HCV foi analisada por microscopia de epifluorescência. Estes resultados indicam que a coloração de HCV subgenï¿½ico replicão (conforme medido por imunofluorescência do HCV não estrutural 5A proteína) foi reduzida nas células tratadas com a hLF. Figura 5 </s<html> Trong&gt; revela uma redução qualitativa de aproximadamente 50% na intensidade de coloração do VHC após 24 horas de tratamento da hLF. Esta é uma observação interessante como este sistema replicão subgenómico que falta o HCV E1 e E2 glicoproteínas do envelope, que são os alvos farmacológicos geralmente aceites da hLF. Concomitante com o desaparecimento da coloração do HCV, a acumulação da hLF começou a ser detectada após 2 horas, e a acumulação da hLF forte foi observada após 24 h. <img alt="figura 1" src="/files/ftp_upload/53053/53053fig1.jpg" /> Figura 1. Princípio de microscopia de epifluorescência. A este tipo de microscopia, filtros de excitação seleccionar o comprimento de onda específico, que excita o espécime (Tissus anticorpos ou células que albergam marcado com fluorescência). Além disso, os filtros de emissão são escolhidas para corresponder às características espectrais de emissão do fluoróforo utilizado para marcar o espécime.carga / 53053 / &quot;target =&quot; _ blank 53053fig1large.jpg &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 2" src="/files/ftp_upload/53053/53053fig2.jpg" /> Figura 2. Princípio da microscopia confocal. Este tipo de microscopia usa uma pinhole emissão para eliminar out-of-focus sinal de luz. Uma vez que apenas luz produzida por fluorescência muito perto do plano focal pode ser detectada, a resolução óptica da imagem é muito melhor do que a dos microscópios epifluorescência. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53053/53053fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53053/53053fig3.jpg" /> Figura 3. Esquema da imunofluorescência indireta e direta. A imunofluorescência direta envolveapenas um tipo de anticorpo marcado com um fluorocromo que é específico para a molécula de interesse (anticorpo primário). Em contraste, imunofluorescência indirecta envolve anticorpos secundários marcados com fluorocromos que são específicos para as espécies de o anticorpo primário não marcado. Mais de um anticorpo secundário pode anexar a cada anticorpo primário que aumenta o sinal de fluorescência. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53053/53053fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53053/53053fig4.jpg" /> Figura 4. hepatocelular absorção da hLF. Exógenos células Huh-7 foram tratados com 0 uM ou 3 uM da hLF, durante 24 h, lavou-se duas vezes, submetida a um tratamento com tripsina 5 min, deixou-se voltar a aderir durante 18 horas e submetidos a imunofluorescência. Os núcleos foram coradas wom DAPI (azul canal), membranas plasmáticas foram marcadas com WGA (canal verde) e hLF foi corado com um anticorpo anti-hLF (canal vermelho). As imagens foram adquiridas por microscopia confocal a um aumento original de 60x. As secções transversais ópticos horizontais representam a média de espessura das células. Barra de escala, 10 um. Modificado de (11). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53053/53053fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figure5" src="/files/ftp_upload/53053/53053fig5.jpg" /> Figura 5. Tratamento hLF reduz coloração VHC. Células Huh-7 que suportam a replicação de HCV replicão subgenï¿½ico foram tratados com 3 uM da hLF para 0, 2, ou 24 h e submetida a imunofluorescência. Os núcleos foram coradas com DAPI (azul canal), o HCV foi revelada por um anticorpo anti-NS5A (canal vermelho) e hLF foi corado com um anticorpo anti-hLF umntibody (canal verde). As imagens foram adquiridas por microscopia de epifluorescência a um aumento original de 60x. Barra de escala, 10 um. Modificado de (11). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53053/53053fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus at JoVE.com

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Imunofluorescência para monitorar a Incorporação celular de lactoferrina humana e sua Associated atividade antiviral contra o vírus da hepatite C

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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13.3K visualizações.  Université de Sherbrooke. The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Vídeo: Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C 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, ...

Imunologia e Infecção

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Dois métodos de formação de heterocários Descobrir Fatores de restrição HCV

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Um protocolo para hepatite C Analisando replicação de vírus

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Um método para avaliar funções efetoras mediada por Fc induzidas por anticorpos específicos de hemaglutinina de gripe

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Desenvolvimento e validação de uma única molécula ultra-sensível de matriz Digital ensaio imunossorvente ligado à enzima para humana Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

Um elevado-throughput Cre-Lox ativado ensaio de fusão da membrana Viral para identificar inibidores de fusão da membrana Viral do HIV-1

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Biologia

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Imagem de fluorescência multiplexada de célula única para visualizar ácidos nucleicos virais e proteínas e monitorar HIV, HTLV, HBV, HCV, Zika Virus e Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The antibodies neutralize the virus, rendering it unable to infect the host cells, while non-neutralized viruses infect the cells. After incubation, the extent of neutralization is determined by quantifying the virus-infected cells using immunofluorescence.

Um método de imunofluorescência para medir a neutralização do citomegalovírus humano

1. Preparation of target cells for infection

	Seed target cells the day prior to performing the experiment in a 96-well plate (the &#39;target&#39; ...

JoVE Encyclopedia of Experiments

Imunologia

Imunodiagnóstico

Imaging and Visualization Techniques

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

Um ensaio de fixação viral para triagem de compostos antivirais

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...