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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. In Vivo Characterization of BIRFLU (Figure 1 and Figure 2)
<ol>
	<li>Mouse infection 
	NOTE: Intranasal infection of mice was done as previously described. For a more detailed protocol of IAV infection in vivo using the mouse model of infection, we recommend viewing the video associated with the previous publication. This section only summarizes the steps required for mouse infection with BIRFLU.
	<ol>
		<li>Inspect the mice to evaluate their health and overall physical appearance. Prepare the dilution of BIRFLU in 1x PBS to inoculate mice with 1 x 106 PFU of BIRFLU in a total volume of 30 &#956;L/mouse. Maintain the virus on ice until mouse inoculation. 
		NOTE: Mock-infected (1x PBS) mice will be needed as an internal control for imaging. Place mock-infected mice in a different cage than BIRFLU-infected animals.</li>
		<li>Anesthetize five-to-seven-week-old female BALB/c mice intraperitoneally with 240&#8211;250 mg/kg of tribromoethanol (TBE) by inserting the needle into the caudal 2/3 of the right side of the abdomen. Then, return the mouse to the cage and wait for around 5 min. Check that the mouse is anesthetized before virus inoculation by the absence of the toe-pinch reflex. 
		NOTE: Injectable sedatives are recommended over inhaled sedatives for influenza infections in vivo, as the latter can modify the behavior and uptake of the virus by the airway epithelium. Moreover, they can also affect local immune responses of the lung and interfere with the in vivo imaging of infected mice. Finally, inhaled sedatives have a reduced duration of effect, which would be a problem for in vivo imaging of the infected animals.</li>
		<li>Inoculate the mice intranasally with the 30 &#956;L of the prepared BIRFLU dilution. Check that the mice are breathing properly before returning them to the cage.</li>
	</ol>
	</li>
	<li>Bioluminescence monitoring of mice infected with BIRFLU (Figure 2A) 
	&#8203;NOTE: In this manuscript reporter expression of Nluc or Venus and viral replication in the lungs of mice infected with BIRFLU are determined at 3 days post-infection. However, the nature of bioluminescence imaging allows repeated monitoring of Nluc expression in individual BIRFLU-infected animals without the need to euthanize them for each experimental time point. An in vivo imaging system with an isoflurane anesthesia manifold is required to perform the described experimental procedures. See the Table of Materials for details regarding the imaging instrument and image software used for the image acquisition and data analysis.
	<ol>
		<li>Shave the mice&#39;s chest to improve the bioluminescence signal. Open the imaging software and press Initialize. Next, set the parameters that will be used, including setting the imaging mode to bioluminescence, auto-saving, exposure time to auto, open filter, etc.</li>
		<li>Once the machine is fully initialized, turn on the isoflurane anesthesia system. Place animals in the anesthesia chamber. Mice are simultaneously and lightly anesthetized with a mixture of oxygen gas and vaporized 1&#8211;2% isoflurane.</li>
		<li>Once mice are anesthetized, administer the Nluc substrate (see Table of Materials) diluted 1:10 in 1x PBS (final volume 100 &#181;L/mouse) via retro-orbital route using a syringe with a 22 G needle.</li>
		<li>Immediately after Nluc reagent administration, place the animals in the imaging instrument with their chests facing up and snouts inside the manifold cone to keep the animal anesthetized during imaging. Immediately after closing the imager door, click Acquire in the software program (Figure 2A, top).</li>
		<li>After imaging, return the mice to their cages monitoring them until they have fully recovered, and turn off the isoflurane vaporizer. Then, proceed with the ex vivo imaging of mice lungs to evaluate Venus reporter gene expression (section 1.3).</li>
		<li>Use the imaging software tools to analyze the acquired bioluminescence data. Utilize the tool ROI (region of interest) to designate the specific signal and perform flux measurements in the region of interest (usually around the chest) (Figure 2A, bottom). Although the ROI shape is irrelevant, larger ROIs are generally preferred to capture the entire signal diffusion area.</li>
		<li>Click Measure. Assess the bioluminescence in photons since it provides an absolute photon emission measurement that is comparable to output measurements provided by different parameters or imaging instruments.</li>
	</ol>
	</li>
	<li>Fluorescence analysis in mice infected with BIRFLU (Figure 1 and Figure 2B)
	<ol>
		<li>Collect the mouse lungs after in vivo imaging, as previously described.
		<ol>
			<li>Briefly, euthanize mice with a lethal dose of TBE (500 mg/kg). Disinfect the incision site with 70% ethanol. With a scalpel, make an incision from the sternum to the base of the abdomen and then cut from the bottom of the incision to the sides with scissors. Next, cut the hepatic vein (second physical euthanasia method) to bleed the animal. 
			NOTE: To avoid high background signals during imaging, it is important to minimize the amount of blood in the lungs (see below).</li>
		</ol>
		</li>
		<li>Place the mouse in dorsal recumbency, and use the scissors to cut the pleura and open the rib cage. Subsequently, remove the lungs by snipping the end of the trachea with scissors while gently holding the lungs with forceps.
		<ol>
			<li>Place the lungs in a six-well plate with 2 mL of 1x PBS and wash the lungs three times with 1x PBS. 
			NOTE: To avoid contamination between samples, clean and disinfect the dissecting tools between each animal.</li>
		</ol>
		</li>
		<li>Start the image acquisition software by clicking initialize and setting the parameters for imaging, including setting imaging mode to fluorescence, auto-saving, exposure time to auto, excitation (500 nm), and emission (540 nm) filters.</li>
		<li>When the machine is initialized, place the lungs in a black background tray, ensuring that the tissues are separated from one another, introduce the tray into the imager, and click Acquire on the imaging system program after closing the imager door (Figure 2B, top).</li>
		<li>After imaging, remove the tissues immediately and store them on ice (4 &#176;C) if the samples are processed the same day; or on a tube and dry ice to freeze them quickly, before storing them at -80 &#176;C, if the samples will be processed later on a different day.</li>
		<li>For image processing, select the ROI tool and draw ROIs around each of the individual lungs. Click Measure. Then, using the resulting average radiant efficiency measurements, subtract the values from the mock-infected mice (Figure 2B, bottom). 
		NOTE: With BIRFLU, there is a good correlation of the levels of Nluc and Venus expression, and signal distribution. Therefore, it is important to analyze Nluc (whole mouse) and Venus (excised lungs) expression from the same animal and maintain the same orientation.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Five- to seven-week-old female BALB/c mice</td>
			<td>National Cancer Institute (NCI)</td>
			<td>555</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>1001936040</td>
			<td>Store at Room temperature</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>PerkinElmer</td>
			<td>124262</td>
			<td>This instrument is used for in vivo imaging&#160;</td>
		</tr>
		<tr>
			<td>IX81 Motorized Inverted Microscope</td>
			<td>Olympus</td>
			<td>Olympus IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image 4.7.2 software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>This instrument is used for in vivo imaging&#160;</td>
		</tr>
		<tr>
			<td>Lumicount</td>
			<td>Packard</td>
			<td></td>
			<td>This instrument is used for quantifying luciferase activity&#160;</td>
		</tr>
		<tr>
			<td>Nano-Glo Luciferase Assay Reagent</td>
			<td>Promega</td>
			<td>N1110</td>
			<td>This reagent is used to measure Nluc activity. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retiga 20000R Fast1394 Camera</td>
			<td>Qimaging</td>
			<td>Retiga 2000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>HP</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a luciferase-fluorescent reporter influenza virus to track viral infections

Source: Chiem, K., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59890">A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection.</a> J. Vis. Exp. (150), (2019).
This video demonstrates a method to track viral infections in mouse models using a recombinant luciferase and fluorescence protein-expressing bi-reporter Influenza A virus. This method enables both in vivo and ex vivo studies, enabling researchers to gain insights into viral dynamics and the effects of interventions.

<img alt="Figure 1" src="/files/ftp_upload/21896/21896_Figure1.jpg" /> 
Figure 1: Schematic representation for the study of BIRFLU in mice. Expression of Nluc and Venus reporter genes was evaluated in mice infected with 1 x 106 PFU of BIRFLU using in vivo or ex vivo imaging. Briefly, on day 1, 5 to 7-week-old female BALB/c mice were mock-infected (1x PBS) or inoculated with 1 x 106 PFU of BIRFLU intranasally. At day 3 post-infection, mice were mildly anesthetized using isoflurane, and Nluc substrate was injected retro-orbitally. Nluc signal was directly assessed using in vivo imaging. Immediately after imaging, mice were euthanized and expression of Venus in whole excised lungs was analyzed using ex vivo imaging. Recovered mice lungs were homogenized to evaluate viral replication and stability by plaque assay. Arrows indicate the correlation between fluorescence (Venus), immunostaining (Nluc), and crystal violet staining.
<img alt="Figure 2" src="/files/ftp_upload/21896/21896_Figure2.jpg" /> 
Figure 2: In vivo bioluminescence and fluorescence expression. Female five-to-seven-week-old BALB/c mice were mock-infected (1x PBS) or inoculated with 1 x 106 PFU of BIRFLU intranasally. At day 3 post-infection, Nluc activity (A) in the whole mouse was determined. Representative images of a single mouse showing radiance scale (p/sec/cm2/sr). Bioluminescence radiance values were quantitated and the average total flux is shown (Flux (Log10 p/s). After Nluc imaging, lungs were harvested for ex vivo imaging (B). Representative pictures from whole lungs are shown. To quantify Venus expression, mean values of regions of interest (ROIs) were normalized to lung auto-fluorescence from mock-infected mice, and fold changes were calculated. To analyze the genetic stability of BIRFLU in vivo, viruses recovered from mice lungs were analyzed by plaque assay using fluorescent microscopy (Venus, top), immunostaining (Nluc, middle), and crystal violet staining (bottom) (C). Representative images from one mouse are shown. To evaluate virus replication, whole lungs were homogenized after imaging and used to infect MDCK cells and determine viral titers by plaque assay (PFU/mL) (D). Arrows indicate the correlation between fluorescence (Venus), immunostaining (Nluc), and crystal violet staining. Bars represent the mean &#177; SD of lung virus titers. This figure has been adapted from Logales et al.

A Luciferase-Fluorescent Reporter Influenza Virus to Track Viral Infections

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. In ...

Start with a pathogenic recombinant influenza virus with a genome modified to incorporate compact reporter genes.
These reporter genes encode a small luciferase bioluminescent enzyme and a monomeric fluorescent protein.
Inoculate the virus intranasally into an anesthetized mouse, appropriately shaved for subsequent imaging.
The virus enters the host cell and releases its genetic contents.
Inside the nucleus, the viral genome transcribes into mRNA molecules and subsequently translates to produce luciferase and fluorescent protein in the host cell.
Inject a luciferin substrate retro-orbitally into the mouse.
The luciferase enzyme oxidizes luciferin to oxyluciferin, which on relaxation, emits a bioluminescent signal.
Measure the signal to estimate luciferase levels, enabling viral infection monitoring in a live animal.
For ex vivo analysis, excise the lungs and capture the fluorescence signal through a suitable imaging technique.
Unlike regular variants, monomeric fluorescent proteins do not form aggregates, yielding a clear image to track viral infection accurately.

a-luciferase-fluorescent-reporter-influenza-virus-to-track-viral

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

98 ビュー. 出典:Chiem, K., et al.ウイルス感染のライブイメージングと定量化のためのルシフェラーゼ蛍光レポーターインフルエンザウイルス。J. Vis. Exp.(150)、(2019)。 このビデオでは、組換えルシフェラーゼと蛍光タンパク質発現バイレポーターのインフルエンザAウイルスを使用して、マウスモデルでウイルス感染を追跡する方法を示します。この方法により、in vivo と ex vivo の両方の?...

ビデオ: ウイルス感染を追跡するためのルシフェラーゼ蛍光レポーターインフルエンザウイルス - 概念

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

二重ルシファーゼレポーターシステムによるCRISPRプラスミドの構築と哺乳動物細胞におけるノックアウト効率の検出

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

JoVE Journal

生物工学

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

レポーター発現組換えSARS-CoV-2を用いたK18 hACE2トランスジェニックマウスにおけるウイルス感染のライブイメージングと定量

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

免疫・感染学

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

スプリットルシフェラーゼアッセイ系を用いたHBx-DDB1相互作用の同定阻害剤

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

A Luciferase-Fluorescent Reporter Influenza Virus to Track Viral Infections

記録

A Luciferase-Fluorescent Reporter Influenza Virus to Track Viral Infections