Name: immunofluorescence to monitor the cellular uptake of human lactoferrin and its associated antiviral activity against the hepatitis c virus
Uploaded: 2015-10-01T13:30:00.000+00:00
Duration: 6 min 28 s
Description: 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

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. Zellen Vorbereitung und Behandlung <ol><li> In einer 24-Well-Platte, legte ein Glasabdeckung am Boden der Wells. Mit phosphatgepufferter Salzlösung (PBS) Waschen Sie jede Vertiefung geben. </li><li> Samen Huh-7 Zellen Stütz HCV subgenomische Replikon in jeder Vertiefung bis zu einer Dichte von 5 × 10 4 Zellen / Vertiefung. Wenn es keine Behandlung, um zu tun, können die Zellen in einer höheren Dichte ausgesät werden. Das Kulturmedium ist Dulbecco modifiziertem Eagle Medium (DMEM) mit 10% Fetal Bovine Serum (FBS), 2 mM L-Glutamin, 1 mM Natriumpyruvat und 250 mg / ml G-418 (um das Replikon zu erhalten) ergänzt. </li><li> Inkubieren bei 37 ° C und 5% CO 2 am nächsten Tag zu behandeln, die Zellen mit 3 &amp; mgr; M hLF aus menschlicher Milch gereinigt. </li></ol> 2. Paraformaldehyd / Saccharose-Herstellung (12% PAF und 12% Saccharose) <ol><li> Setzen 500 ml PBS in einem Becherglas und Wärme zwischen 20 ° C und 30 ° C. 60 g Saccharose in PBS. 60 g des paraformaldehyd (PAF) in PBS / Saccharose-Lösung. Langsam NaOH 2N (3-7 ml), bis eine klare Lösung erhalten wird. </li><li> PH-Wert auf 7,4 mit HCl. Gesamte Volumen auf 500 ml mit PBS. Filter durch die Schwerkraft auf Whatman-Filter. Vor Gebrauch verdünnte die Lösung mit PBS auf eine Endkonzentration von 4% PAF und 4% Saccharose (Lagerung 4 ° C für 1 Woche Maximum). </li></ol> 3. Immunfluoreszenz <ol><li> Zum gewünschten Zeitpunkt (0 h, 2 h oder 24 h der Behandlung), waschen Sie die Zellen zweimal (1 min) mit PBS und fixieren mit PBS / 4% PAF / 4% Saccharose für 20 min bei RT. Die Zellen werden typischerweise bei 30-40% Konfluenz und 1,5x10 5 Zellen verwendet werden. </li><li> Permeabilisierung der Zellen mit 0,15% Triton X-100 in PBS für 5 min bei RT und Block in 10% normalem Ziegenserum (NGS) in PBS für 20 min. </li><li> Verdünnen Sie die primären Antikörper des Proteins von Interesse in 10% NGS (Beispiel: primäre Maus-Anti-hLF Antikörper verdünnt 1: 1.000). Wenden Sie den primären Antikörper zu den Zellen. </li><li> Nach 4 h bei RT oder O /N bei 4 ° C, waschen Sie die Zellen dreimal 5 min mit 10% NGS. Färben die Zellen mit Sekundärantikörper mit Fluorochrom in 10% NGS verdünnt markierten (Beispiel: Fluorochrom mit einer Anregungswellenlänge von 488 nm auf einen Antikörper, anti-Maus-1 verbunden: 1000) für 1 h bei RT im Dunkeln. </li><li> Die Zellen werden einmal für 5 Minuten mit PBS und färben die Zellen, die mit 4 &#39;, 6-Diamidino-2-phenylindol (DAPI) (1 ug / ml) für 15 min bei RT im Dunkeln. </li><li> Für 5 Minuten mit PBS waschen Sie die Zellen zweimal. Berg decken Gläser auf Slowfade Eindeckmedium vor Visualisierung durch Mikroskopie. Die Zellen sind nun bereit für die Analyse durch Epifluoreszenz oder konfokalen Mikroskopie. </li><li> Verwenden Sie verschiedene Steuerelemente, um die Spezifität des Nachweises zu gewährleisten. Zum Beispiel können alle Antikörper, um die Autofluoreszenz zu detektieren weggelassen. Der primäre Antikörper weggelassen werden, um die nicht-spezifische Färbung mit dem sekundären Antikörper zu bestimmen. Schließlich, wenn möglich, zu steuern Zellen oder Gewebe, die nichtexprimieren das Molekül von Interesse kann auch verwendet werden. </li><li> Visualisieren Sie die Proben unter Verwendung von geeigneten Fluoreszenzmikroskop (Epifluoreszenz oder konfokalen) und Filtersätze für die Fluoreszenzmarkierung verwendet angemessen. Dias können auch in einer Diashow Fach an &lt;-20 ° C für die spätere Prüfung abgelegt werden. </li></ol> 4. Fluoreszenzmikroskopie <ol><li> Bewahren Sie die Proben in der Dunkelheit, um Photobleichung zu verhindern. Einschalten der Lichtquelle der ausgewählten Mikroskop (Epifluoreszenz oder konfokalen). </li><li> Schalten Sie das Mikroskop. Einschalten der Kamera für das Mikroskop. Legen Sie die Folie unter dem Mikroskop auf Visualisierung. In Immersionsöl auf der Folie, um die numerische Apertur der Objektivlinse zu erhöhen. </li><li> Wählen Sie die entsprechenden Filter. Stellen Sie den Fokus und entsprechende Ziel. Passen Sie die Fensterläden, um die entsprechenden Fluorochrom erkennen, sondern verhindern, Photobleichen. Halten Deckenbeleuchtung aus, um Hintergrundlicht zu minimieren, wenn man Probe. </li><li> Th ändernE-Filter, um verschiedene Fluorochrome (zB DAPI und für eine Anregungswellenlänge von 488 nm) zu visualisieren. Nehmen Sie Bilder mit der Kamera. </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.

Die HCV-Epidemie bleibt eine globale Bedrohung, mit 80% der neu infizierten Patienten entwickeln eine chronische Infektion, indem sie mit einem Risiko für Leberzirrhose, Leberversagen oder Leberzellkarzinom. Direkt wirkende antivirale Mittel Targeting HCV-Replikation und Reifung stellen erstklassige Anti-HCV-Mittel, wie kürzlich von der behördlichen Genehmigung von zwei NS3 N-Terminus-Protease-Inhibitoren (Boceprevir und Telaprevir) demonstriert. HLF anti-HCV-Aktivität wird derzeit angenommen, dass als das Eindringe...

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

Immunfluoreszenz ist eine Labortechnik häufig verwendet, um viele Aspekte der Biologie zu studieren. Er wird üblicherweise verwendet, um die Verteilung und / oder die Lokalisierung eines Zielmoleküls in Zellen und Gewebe zu visualisieren. Immunfluoreszenz beruht auf der Spezifität von Fluoreszenz-markierten Antikörpern gegen die entsprechenden Antigene in der Zelle. Direkte und indirekte Immunofluoreszenz Ansätze können verwendet werden, die auf der Verwendung von Antikörpern, die mit einem Fluorochrom gebunden verlassen. Direkte Immunfluoreszenz wird seltener verwendet, da sie geringere Signal, mit höheren Kosten und weniger Flexibilität. Im Gegensatz dazu ist die indirekte Immunfluoreszenz mehr allgemein wegen seiner hohen Empfindlichkeit verwendet und liefert ein verstärktes Signal seit mehr als einem sekundären Antikörper kann an jeden primären Antikörper zu befestigen. In diesem Manuskript wurden sowohl Epifluoreszenzmikroskopie und konfokale Mikroskopie verwendet, um die Internalisierung von menschlichem Lactoferrin, eine wichtige Komponente der Immun überwachenSystem, in Leberzellen. Außerdem überwacht wir die Hemmpotenzial hLF auf die intrazelluläre Replikation des Hepatitis C-Virus mittels Immunfluoreszenz. Sowohl die Vorteile und Nachteile, die mit diesen Ansätzen verbunden sind, diskutiert.

Die Fähigkeit der Leber Huh-7-Zelllinie exogen administrated hLF internalisiert wurde mittels Immunfluoreszenz auf einem konfokalen miscroscope überwacht. hLF wurde zu dem Kulturmedium zugegeben, und die Internalisierung wurde 24 h erlaubt, wonach wurde extrazelluläres hLF mit PBS gewaschen und restliche Membran gebunden hLF wurde mit einer 5 min Trypsinbehandlung (1 ml) abgebaut. Die Zellen wurden ausgesät und man ließ sie wieder halten 18 Stunden vor der Immunfluoreszenzfärbung. Um die cytoplasmatischen Grenzen ...

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.

Immunfluoreszenz, um die zelluläre Aufnahme von menschlichem Lactoferrin und die damit verbundenen antivirale Aktivität gegen das Hepatitis C Virus-Monitor

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

immunofluorescence-to-monitor-cellular-uptake-human-lactoferrin-its

Research

JoVE Journal

Immunology and Infection

13.3K Aufrufe.  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.

Serie: 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.

High-Throughput Nachweisverfahren für 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, ...

Forschung

Immunologie und Infektion

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.

Zwei Methoden der Heterokaryon Formation zu entdecken HCV Restriktionsfaktoren

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.

Ein Protokoll für die Analyse von Hepatitis-C-Virus-Replikation

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.

Eine Methode zur Bewertung von Fc-vermittelten Effektor-Funktionen durch Influenza Hämagglutinin spezifische Antikörper induziert

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

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.

Nachweis viraler RNA durch Fluoreszenz<em&gt; In-situ-</em&gt; Hybridisierung (FISH)

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

Biologie

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Ein luziferasefluoreszierendes Reporter-Influenzavirus zur Live-Bildgebung und Quantifizierung von Virusinfektionen

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

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.

Einzelzell-Multiplex-Fluoreszenzbildgebung zur Visualisierung viraler Nukleinsäuren und Proteine und zur Überwachung von HIV-, HTLV-, HBV-, HCV-, Zika-Virus- und Influenza-Infektionen

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

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Source: Bailey, M. J., et al. <a href="https://app.jove.com/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

Messung der Aktivierung von Fc-vermittelten Effektorfunktionen durch HA-Antikörper

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

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.

Eine Immunfluoreszenzmethode zur Messung der Neutralisation des humanen Cytomegalievirus

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; ...

Immunodiagnostics

Imaging and Visualization Techniques

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app.jove.com/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.

Ein Virus-Attachment-Assay für das Screening von antiviralen Verbindungen

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