Research on influenza virus in LM-S laboratory is partially funded by The New York Influenza Center of Excellence (NYICE) (NIH 272201400005C), a member of the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract No. HHSN272201400005C (NYICE) and by the Department of Defense (DoD) Peer Reviewed Medical Research Program (PRMRP) grant W81XWH-18-1-0460.

The authors have nothing to disclose.

Researchers have relied on recombinant reporter-expressing viruses as vital molecular tools to understand and expand upon the current understanding of viral replication and pathogenesis26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,54. The most commonly favored reporter genes are luciferases and fluorescent proteins, mainly due to the technological advancements in their identification, development of improved variants, and detection by imaging technologies43,44,45,46,47,48. Recombinant reporter viruses are often used to accelerate virological assays, study the dynamics of viruses in vitro and in vivo, and to test the effectiveness of currently approved or new vaccine and therapeutic approaches26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,54. Unfortunately, in the case of IAV, past studies were limited to the expression of a single reporter gene, which hinders the type of study that could be conducted 26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,54. To avoid this limitation, we have generated a replication-competent bi-reporter IAV that expresses a Nluc luciferase and a Venus fluorescent protein (BIRFLU).
In this report, we describe the in vitro characterization of BIRFLU and the experimental approaches to use BIRFLU to track viral infection in vivo using a mouse model of IAV infection. BIRFLU Nluc and Venus expression correlated with viral titers. In addition, BIRFLU remained stable and continued to express both reporter genes after being recovered from the lungs of infected mice. This approach provides researchers with an excellent opportunity to study IAV in cultured cells and in animal models, including the identification and develop of new therapeutic alternatives for the treatment of IAV infections.
Although BIRFLU has been generated using the backbone of PR8, other recombinant IAV using different type, subtype or viral strain backbones could be generated using the same experimental approach. Likewise, in this report we described the experimental procedures for the use of BIRFLU in a mouse model of IAV. However, BIRFLU could be a valuable technology to evaluate IAV infection in other animal models.

Influenza A virus (IAV) is an enveloped single-stranded negative-sense segmented RNA virus of the Orthomyxoviridae family1,2,3. The World Health Organization (WHO) estimates 3-5 million annual influenza cases and over 250,000 deaths from influenza worldwide4,5,6. Groups that are particularly vulnerable to influenza include the elderly, immunocompromised individuals, and children7,8,9,10,11. Although vaccines are available and represent the most common and effective intervention against viral infection, IAV is able to rapidly evolve and escape preexisting immunity3,12,13,14,15. The re-emergence of a pandemic H1N1 strain in 2009 and the emergence of pathogenic IAV reiterates the constant threat to human public health worldwide4,16.
During an epidemic or pandemic, it is crucial to rapidly determine the pathogenicity and transmissibility of newly isolated viruses. Currently available techniques to detect the virus are time-consuming and sometimes require the use of laborious approaches, which can delay the completion of these analyses17,18,19,20. Moreover, present viral assays are difficult to scale up, which could be necessary during the event of an outbreak. Finally, the use of validated animal models of infection, such as mice, guinea pigs and ferrets are routinely used and are vital in studying influenza infections, immune responses, and the efficacy of new vaccines and/or antivirals. However, these models are restrictive due to the inability to observe viral dynamics in real time; this limits the studies to static imaging of viral infections21,22,23,24,25. Animals used in these assays are also euthanized in order to determine viral load, thus increasing the number of animals required to complete these studies26. To circumvent all these limitations, many researchers rely on the use of recombinant replication-competent, reporter-expressing IAVs, which are capable of accelerating virological assays and detecting viral load and dissemination in vivo in real-time26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41. Importantly, these reporter-expressing IAVs are able to replicate similarly to wild-type (WT) IAVs in cell culture and in animal models of infection33,42.
Fluorescent and bioluminescent proteins are two reporter systems commonly used by researchers due to their sensitivity, stability and ease of use. In addition, there is tremendous support and advancement in fluorescent and bioluminescent protein detection technologies43,44,45,46,47,48. Fluorescent proteins and luciferase have different properties that allow them to glow, specifically differing in how excited states are generated and how emittance is detected43,44,45,46,47,48. Fluorescent proteins are first excited by absorbing energy, which is subsequently released as light at a different wavelength as the molecules decrease to a lower energy state43. On the other hand, bioluminescence is derived from a chemical exothermic reaction that involves a substrate, oxygen, and sometimes ATP in order to produce light45. Due to the varying properties of these two types of reporter proteins, one maybe more advantageous than the other depending on the study of interest. While fluorescent proteins are widely used to observe cellular localization28,41, their in vivo signals have inadequate intensity and are often obscured by autofluorescence in live tissues49. Therefore, researchers rely on luciferases to evaluate viral dynamics in live organisms, although fluorescent proteins can be preferred for ex vivo studies50,51,52,53. Unlike fluorescent proteins, luciferases are more convenient for in vivo studies and more applicable in non-invasive approaches26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,54. Ultimately, based on the type of study, researchers must choose between the use of either a fluorescent or a luciferase reporter protein as their readout, which subjects their study to a trade-off of functionalities and sensitivities, and severely restricts the usefulness of the recombinant reporter viruses. Moreover, there are concerns regarding the correlation among the expression of different reporter genes using fluorescence or luciferase systems and viral replication or dissemination, which might jeopardize the interpretation of the data obtained with reporter-expressing IAVs.
We have overcome this limitation by generating a recombinant replication-competent bi-reporter IAV (BIRFLU) that encodes for both a fluorescent and a luciferase protein in the same viral genome55 (Figure 1). Here, NanoLuc luciferase (Nluc), a small and bright bioluminescent protein48, was inserted upstream of the hemagglutinin (HA) sequence in the viral HA segment of influenza A/Puerto Rico/08/1934 H1N1 (PR8)24,33,40,55,56,57. In addition, Venus, a frequently used monomeric fluorescent protein, was inserted into the non-structural (NS) viral segment32,33,36,41,55. Since BIRFLU encodes for both fluorescent and luciferase reporter genes, either reporter protein signal can be used as readout to determine viral replication and dissemination in vitro or in vivo55. Additional information regarding the generation and in vitro or in vivo characterization of BIRFLU can be found in our recent publication55. BIRFLU can be used to test the effectiveness of antiviral drugs or neutralizing antibodies via a novel fluorescent- and bioluminescent-based microneutralization assay55. Moreover, BIRFLU can also be used to evaluate viral dynamics in a mouse model of infection55. In this manuscript, we describe the procedures to characterize BIRFLU55 in vitro and how to study BIRFLU infection in mice using in vivo luminescence imaging systems for the detection of Nluc in vivo or of Venus ex vivo.
The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, brings researchers the unique opportunity to use BIRFLU for IAV research, including the study of virus-host interactions, dynamics of viral infection; the development of novel vaccine approaches for the therapeutic treatment of IAV infections or the potential use of IAV as a vaccine vector for the treatment of other pathogen infections.

<table>
	<tbody>
		<tr>
			<td>12-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>665102</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Photoshop CS4</td>
			<td>Adobe</td>
			<td></td>
			<td>This software is used in 3.1.10 and 4.4.7</td>
		</tr>
		<tr>
			<td>Bovin Albumin solution (BA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7409</td>
			<td>Store at 4&#176; C</td>
		</tr>
		<tr>
			<td>Bovin Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Cell Culture dishes 100mm</td>
			<td>Greiner Bio-one</td>
			<td>664-160</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc MP Imaging System</td>
			<td>BioRad</td>
			<td></td>
			<td>This instrument is used in 4.4.7</td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Thermo Fisher Scientific</td>
			<td>C581-100</td>
			<td>Store at Room temperature</td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinders</td>
			<td>Thomas Scientific</td>
			<td>7722-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Corning Cellgro</td>
			<td>15-013-CV</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Seradigm</td>
			<td>1500-050</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<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 (4.2 and 4.3)</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 (4.2 and 4.3)</td>
		</tr>
		<tr>
			<td>Lumicount</td>
			<td>Packard</td>
			<td></td>
			<td>This instrument is used for quantifying luciferase activity (3.2.6)</td>
		</tr>
		<tr>
			<td>Madin-Darby Canine Kidney (MDCK) epithelial cells</td>
			<td>ATCC</td>
			<td>CCL-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Antibody anti-NP Influenza A Virus HB-65</td>
			<td>ATCC</td>
			<td>H16-L10-4R5</td>
			<td>Store at -20 &#176;C</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>Neutral Buffered Formalin 10%</td>
			<td>EMD</td>
			<td>65346-85</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well Microplates</td>
			<td>Thermo Fisher Scientific</td>
			<td>269620</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS) 100x</td>
			<td>Corning</td>
			<td>30-00-CI</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/L-Glutamine (PSG) 100x</td>
			<td>Corning</td>
			<td>30-009-CI</td>
			<td>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>
		<tr>
			<td>Texas Red-conjugated anti-mouse -rabbit secondary antibodies</td>
			<td>Jackson</td>
			<td>715-075-150</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T8802</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>J.T.Baker</td>
			<td>X198-07</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Vmax Kinetic plate reader</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Generation and characterization of BIRFLU in vitro (Figure 1 and Figure 2)
A recombinant replication-competent IAV expressing two different reporter genes (BIRFLU) was constructed using state of the art molecular biology and plasmid-based reverse genetics techniques (Figure 1). Here, we chose to use Nluc due to several advantages over other luciferases, including its small size, ATP-independence, greater intensity, and optimized substrate48,60. Nluc was cloned into the HA segment of IAV PR8 followed by the porcine teschovirus (PTV) 2A cleavage site (2A) in front of the open reading frame (ORF) of HA (Figure 1). The ORF of HA included silent mutations to remove the original packing signals and avoid any possible recombination. The complete HA packaging signal was added in front of Nluc to allow proper incorporation of the modified HA segment into the virion and Nluc and HA expression from the same viral RNA segment (Figure 1). In addition, the fluorescent protein Venus was cloned into a modified IAV PR8 NS segment which encodes the two viral proteins NS1 and NEP from a single transcript32,36,41,54,57. To that end, Venus was fused to the C-terminal of NS1 and the entire NEP ORF was cloned downstream of the PTV 2A cleavage site that was placed between the NS1-Venus and NEP sequences (Figure 1). Ultimately, these two modified HA and NS viral plasmid constructs were used in combination with the rest of the IAV PR8 reverse genetics plasmids to generate BIRFLU (Figure 1). The in vitro and in vivo characterization of BIRFLU has been described previously55.
In Figure 2, we characterized in vitro BIRFLU by determining Venus, Nluc, and NP expression levels using fluorescence and indirect immunofluorescence approaches (Figure 2A,B). Confluent monolayers of MDCK cells were either mock-infected or infected (MOI 0.1) with WT or BIRFLU PR8 viruses and, at 18 h post-infection, Venus expression was directly evaluated using fluorescence microscopy (Figure 2A,B). Nluc (Figure 2A) and NP (Figure 2B) expression were visualized by indirect immunofluorescence using antibodies specific for each protein. As anticipated, Venus and Nluc expression were detected only in cells infected by BIRFLU and not in cells infected by WT PR8 virus. In addition, indirect immunofluorescence microscopy revealed NP expression in both WT and BIRFLU PR8-infected cells. No expression of Venus, Nluc or NP were detected, as expected, in mock-infected cells (Figure A,B).
To evaluate Nluc expression levels in vitro, MDCK cells were infected (MOI 0.001) with WT or BIRFLU PR8 viruses and Nluc activity in tissue culture supernatants was assessed at 24, 48, 72 and 96 h post-infection (Figure 2C). Only Nluc activity was detected in tissue culture supernatants of MDCK cells infected with BIRFLU (Figure 2C). Nluc activity in the tissue culture supernatants was detected as early as 24 h post-infection with higher expression levels at 96 h post-infection, most probably because the cytopathic effect (CPE) induced during viral infection release the Nluc protein retained into the cell. To evaluate the fitness of BIRFLU in cultured cells, growth kinetics of WT and BIRFLU PR8 viruses were also evaluated (Figure 2D) and the presence of infectious virus in tissue culture supernatants was determined by immune-focus assay (Figure 2D). Notably, BIRFLU replication kinetics were comparable to those of WT PR8 virus, although BIRFLU replication was slightly delayed and did not reach same viral titers as WT PR8. However, BIRFLU was able to reach titers of 5 x 107 PFU/mL (Figure 2D), indicating that expression of two reporter genes in the viral genome does not significantly interfere with BIRFLU replication in MDCK cells.
Tracking BIRFLU infection in mice (Figure 3 and Figure 4)
Figure 3 is a schematic flow chart for the assessment of BIRFLU dynamics in a mouse model of IAV infection. Five-to-seven-week-old female BALB/C mice were either mock-infected with 1x PBS or infected with 1 x 106 PFU of BIRFLU intranasally. At 3 days post-infection, mice were anesthetized with isoflurane and then injected with Nluc substrate retro-orbitally. All mice were immediately placed in the IVIS instrument and Nluc signal was assessed in vivo using the IVIS. Next, mice were euthanized and lungs were harvested. Excised lungs were then analyzed ex vivo using the in vivo imager to determine fluorescence intensity via Venus expression. Lastly, mice lungs were homogenized, and viral titers and stability were determined by plaque assay. Plaques were assessed by the direct fluorescence of Venus, by immunostaining using antibodies specific for Nluc and by crystal violet staining.
Previously described replication-competent reporter-expressing IAVs express a single reporter gene, most frequently either a fluorescent or a bioluminescent protein, as surrogate for viral infection and replication. However, BIRFLU, is able to express both types of reporter genes upon viral infection. To assess the correlation between bioluminescence (in vivo imaging) and fluorescence (ex vivo imaging) after BIRFLU infection, five-to-seven-week-old female BALB/c mice were mock-infected with 1x PBS or inoculated with BIRFLU (106 PFU) intranasally. Nluc activity (Figure 4A) was evaluated by administration of Nluc substrate injected retro-orbitally at 3 days post-infection using an in vivo imaging instrument. We chose to evaluate bioluminescence at day 3 because previous studies indicated that IAV replication, including PR8, peaks between days 2 and 4 post-infection24,54. Bioluminescence was monitored (Figure 4A, top) and used to calculate the average total flux (Flux (log10 p/s) (Figure 4A, bottom). As predicted, mice inoculated with BIRFLU displayed high bioluminescence activity but no signal was detected in mock-infected mice. Thereafter, the lungs of infected mice were harvested and Venus expression was assessed using ex vivo imaging (Figure 4B, top). Moreover, the fluorescence average radiant efficiency was calculated (Figure 4B, bottom). The excised mice lungs were also homogenized to determine the viral titers and the genetic stability of BIRFLU in vivo (Figure 4C,D). Genetic stability of BIRFLU was analyzed through plaque assay using the viruses isolated from mice lungs and fluorescent microscopy (Venus, top), immunostaining (Nluc, middle) and crystal violet staining (bottom). BIRFLU recovered from mice lungs were able to form plaques and stably expresses both reporter genes (Figure 4C). Notably, we observed a good correlation between bioluminescence and fluorescence signals with viral replication.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59890/59890fig1.jpg" /> 
Figure 1: Schematic representation of IAV PR8 WT and BIRFLU virion structure and genome segments. IAV are surrounded by a lipid bilayer containing the two major viral glycoproteins hemagglutinin (HA; black) and neuraminidase (NA; blue). IAV contain eight single-stranded, negative-sense, RNA segments (PB2, PB1, PA, HA, NP, NA, M, and NS). Each viral segment contains non-coding regions (NCR) at the 3&#8217; and 5&#8217; ends (black boxes). Also, at the 3&#8217; and 5&#8217; end of the viral (v)RNAs are the packaging signals, responsible for the efficient encapsidation of vRNAs into nascent virions (white boxes). IAV PR8 HA and NS viral segments and products are indicated in black. Sequences of Nluc, Venus, and PTV 2A are indicated in red, green and striped boxes, respectively. The schematic representation of the modified HA and NS segments expressing Nluc and Venus, respectively, in BIRFLU are also indicated. This figure has been adapted from Nogales et al.55. <a href="https://www.jove.com/files/ftp_upload/59890/59890fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59890/59890fig2.jpg" /> 
Figure 2: In vitro characterization of BIRFLU. (A, B) Analysis of protein expression by direct fluorescence and immunofluorescence. MDCK cells were mock-infected or infected (MOI 0.1) with PR8 WT or BIRFLU viruses. Infected cells were fixed at 18 h post-infection to directly visualize Venus expression by direct fluorescent microscopy and to visualize Nluc (A) and viral NP (B) expression using specific antibodies and indirect immunofluorescence. Nuclei were stained with DAPI. Representative images (20x magnification) are shown. Scale bars = 100 &#956;m. (C, D) Growth kinetics of PR8 WT and BIRFLU. Nluc activity (C) and viral titers (D) in tissue culture supernatants from MDCK cells infected (MOI 0.001) with WT and BIRFLU PR8 viruses were assessed at the indicated times post-infection. Data represent the means &#177; SD of triplicates. Viral titers were determined by immune-focus assay (FFU/mL). Dotted line denotes the limit of detection (200 FFU/ml). This figure has been adapted from Nogales et al.55. <a href="https://www.jove.com/files/ftp_upload/59890/59890fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59890/59890fig3.jpg" /> 
Figure 3: 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 correlation between fluorescence (Venus), immunostaining (Nluc) and crystal violet staining. <a href="https://www.jove.com/files/ftp_upload/59890/59890fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59890/59890fig4.jpg" /> 
Figure 4: 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 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.55. <a href="https://www.jove.com/files/ftp_upload/59890/59890fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tissue culture media and solutions</td>
			<td>Composition</td>
			<td>Storage</td>
			<td>Use</td>
		</tr>
		<tr>
			<td>Tissue culture media: Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM), 10 % Fetal Bovine Serum (FBS), 1% Penicillin-Streptomycin-L-glutamine (PSG) (DMEM 10 % FBS 1% PSG).</td>
			<td>445 ml DMEM, 50 mL of FBS and 5 mL of 100x PSG.</td>
			<td>4 &#176;C</td>
			<td>Maintenance of MDCK cells</td>
		</tr>
		<tr>
			<td>Post-infection media: DMEM 0.3% Bovine Albumin (BA), 1% PSG (DMEM 0.3 % BA 1% PSG).</td>
			<td>491 ml DMEM, 4.2 ml of 35 % BA and 5 ml of 100x PSG</td>
			<td>4 &#176;C</td>
			<td>Maintenance of MDCK cells after viral infection</td>
		</tr>
		<tr>
			<td>10x Phosphate buffered saline (PBS)</td>
			<td>80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3</td>
			<td>Room temperature</td>
			<td>To prepare 1x PBS</td>
		</tr>
		<tr>
			<td>1x PBS </td>
			<td>Dilute 10x PBS with ddH2O</td>
			<td>Room temperature</td>
			<td>Wash cells</td>
		</tr>
		<tr>
			<td>Infection media: 1x PBS, 0.3% BA, 1% Penicillin-Streptomycin (PS) (PBS/BA/PS).</td>
			<td>487 mL 1x PBS sterile, 4.2 mL of 35% BA and 5 ml of 100x 1% PS(100 U/mL)</td>
			<td>4 &#176;C</td>
			<td>Viral infections</td>
		</tr>
		<tr>
			<td>Fixation/permeabilization solution: 4% formaldehyde, 0.5% triton X-100 diluted in 1x PBS.</td>
			<td>400 mL neutral buffered formalin 10%, 5 ml of Triton X-100 and 595 mL of 1x PBS</td>
			<td>Room temperature</td>
			<td>Fix and permeabilization of MDCK cells.</td>
		</tr>
		<tr>
			<td>Blocking solution: 2.5% Bovine Serum Albumin (BSA) in 1x PBS.</td>
			<td>2.5 g of BSA in 97.5 mL of 1x PBS</td>
			<td>4 &#176;C</td>
			<td>Blocking solution for immunofluorescence and plaque assays.</td>
		</tr>
		<tr>
			<td>Antibody dilution solution (1% BSA in 1x PBS)</td>
			<td>1 g of BSA in 99 mL of 1x PBS</td>
			<td>4 &#176;C</td>
			<td>Dilution of primary and secondary antibodies.</td>
		</tr>
		<tr>
			<td>0.1% crystal violet solution</td>
			<td>1 g of crystal violet in 400 mL of methanol. Add 600 ml of ddH2O</td>
			<td>Room temperature</td>
			<td>Staining of MDCK cells in plaque assays.</td>
		</tr>
		<tr>
			<td>Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin</td>
			<td>Prepare a 1,000x stock solution at 1 mg/mL in ddH2O</td>
			<td>-20 &#176;C</td>
			<td>For viral infections.</td>
		</tr>
	</tbody>
</table>
Table 1: Tissue culture media and solutions.

Watch this Scientific Journal Video about A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection at JoVE.com

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

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.

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

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JoVE Journal

Immunology and Infection

24.9K Views.  University of Rochester School of Medicine and Dentistry. 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.

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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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.

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

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

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.

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 ...