The author acknowledges Jennifer Tipper, Bilan Li, Jesse vanWestrienen, Kevin Harrod, Da-Yuan Chen, Farjana Ahmed, Sonya Mros, Kenneth Yamada, Richard Webby, the BEI Resources (NIAID), the Health Research Council of New Zealand, the Maurice and Phyllis Paykel Trust (New Zealand), the H.S. and J.C. Anderson Trust (Dunedin), and the Department of Microbiology and Immunology and School of Biomedical Sciences (University of Otago).

Regulatory approvals were obtained from the University of Otago Institutional Biological Safety Committee to work with the IAV and mammalian cells. Madin-Darby Canine Kidney (MDCK) or human lung alveolar epithelial A549 cells and IAV H1N1 subtypes were used for the present study. IAV was grown in chicken eggs, as described elsewhere17. Sterile and aseptic conditions were used to work with mammalian cells, and a Biosafety Level 2 (or Physical Containment 2) facility and Class II biosafety cabinet were used to work with IAV subtypes.
1. Assessing the cleavage or degradation of proteins in IAV-infected cells by caspases
<ol>
	<li>Seed 3 x 105 MDCK or A549 cells per well in a 12-well cell culture plate. 
	NOTE: If using MDCK cells, ensure that the antibody against the protein of interest recognizes its canine species.</li>
	<li>Seed the wells in pairs, i.e., a control pair-one well for the uninfected-mock sample and one for the infected-mock sample-and a test pair-one well for uninfected-inhibitor A sample and one for the infected-inhibitor A sample. Increase the number of pairs with each additional inhibitor (see references7,8).</li>
	<li>Incubate the cells at 37 &#176;C under a 5% CO2 atmosphere overnight.</li>
	<li>The next day, infect the cells with IAV (an H1N1 subtype or another subtype).
	<ol>
		<li>For this, prepare the virus inoculum by diluting virus stock in 400 &#181;L of serum-free minimum essential medium (MEM) (see Table of Materials) at a multiplicity of infection (MOI) of 0.5-3.0 plaque-forming units (pfu)/cell7,11 (a factor of the doubling of cell number when calculating the MOI). 
		NOTE: For infecting MDCK cells, supplement the virus inoculum with tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin at a final concentration of 1 &#181;g/mL.</li>
	</ol>
	</li>
	<li>Remove the old culture medium from the cells (step 1.3) and wash the cells with 1 mL/well of serum-free MEM 2x.</li>
	<li>Add 400 &#181;L of virus inoculum (step 1.4.1) to the cells and incubate them for 1 h at 35 &#176;C under a 5% CO2 atmosphere.</li>
	<li>In the meantime, dilute a caspase inhibitor (e.g., Z-DEVD-FMK), a lysosome Inhibitor (e.g., NH4Cl), or a proteasome inhibitor (e.g., MG132) (see Table of Materials) at a final concentration of 40 &#181;M, 20 mM, or 10 &#181;M, respectively, in serum-free MEM7 (see Table of Materials). The latter two inhibitors serve as a control. Dilute an equal volume of the solvent (if other than water) used to reconstitute one of the inhibitors in serum-free medium as a mock.</li>
	<li>Remove the virus inoculum and wash the cells as in step 1.5 (1x).</li>
	<li>Add the serum-free MEM, 1 mL/well, mock or supplemented with an inhibitor, to the cells and incubate the cells as in step 1.6. This time point is considered as 0 h infection.</li>
	<li>After 24 h, harvest the cells by scraping them with a 1 mL syringe plunger (rubber side) and transfer them into a 1.5 mL polypropylene tube.</li>
	<li>Centrifuge the tube at 12,000 x g for 1-2 min at room temperature. Collect the supernatant using a pipette. 
	NOTE: This supernatant could be discarded or used for a plaque assay8 to measure the titer of the released virus progeny.</li>
	<li>Wash the cell pellet with 250 &#181;L of phosphate-buffered saline (PBS) by re-centrifugation as in step 1.11.</li>
	<li>Remove the supernatant and lyse the cells by adding 80-100 &#181;L of cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, and 1x protease inhibitor cocktail, see Table of Materials) and vortexing.</li>
	<li>Heat the tube at 98 &#176;C for 10 min to completely lyse and prepare the total cell lysate. Store the lysates at 4 &#176;C for performing steps 1.15-1.17 the next day, but, for best results, finish these steps on the same day.</li>
	<li>Estimate the protein amount in each sample using a BCA assay kit (see Table of Materials).</li>
	<li>Resolve equal amounts of protein from uninfected and infected samples by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE)11 along with molecular weight markers11.</li>
	<li>Transfer the protein to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane (see Table of Materials). 
	NOTE: PVDF membrane may give a high background in some western blot imagers; check for compatibility.</li>
	<li>Perform western blotting to detect the protein of interest using the method described elsewhere11.</li>
	<li>Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes. 
	NOTE: If the protein level has recovered in the caspase inhibitor-treated infected sample lane, then the protein is cleaved or degraded by caspases. Otherwise, it is degraded by either lysosome or proteasome.</li>
	<li>Quantify the protein recovery by measuring its band intensity in each lane from at least three replicates of the same experiment and normalizing it with the corresponding loading control band.</li>
	<li>Use any contemporary imager and associated software (see Table of Materials) to image the western blots and quantify the protein recovery.</li>
</ol>
2. Confirmation of caspase-mediated cleavage or degradation of proteins in IAV-infected cells by RNA interference
<ol>
	<li>Design or obtain a pre-design small-interfering RNA (siRNA) targeting either canine or human caspase 3, caspase 6, and caspase 7 (executioner caspases1) and a non-targeting control siRNA (see Table of Materials).</li>
	<li>Deliver each siRNA to MDCK or A549 cells by reverse transfection8,11.</li>
	<li>For this, dilute in separate tubes 100 nM of control siRNA or caspase siRNA or a recommended volume of transfection reagent (see Table of Materials) in 100 &#181;L of appropriate medium (like OptiMEM, see Table of Materials), and incubate for 5 min at room temperature.</li>
	<li>Prepare each dilution in duplicate. 
	NOTE: This duplicate includes one replicate for analyzing the knockdown of caspase expression by RT-qPCR or western blotting (if antibodies to caspases are available) and another for assessing the recovery of the protein of interest by western blotting.</li>
	<li>Mix 100 &#181;L of each siRNA solution with 100 &#181;L of transfection reagent solution (step 2.3) and incubate for 20-45 min at room temperature to form the siRNA-transfection reagent complex.</li>
	<li>In the meantime, split and add 1 x 105 MDCK or A549 cells in 800 &#181;L of the complete growth medium, mix with 200 &#181;L of siRNA-transfection reagent complex (step 2.5), and add 1 mL of the suspension to a well of a 12-well culture plate. This dilution brings the final concentration of control siRNA or caspase siRNA to 10 nM.</li>
	<li>Incubate the cells at 37 &#176;C under a 5% CO2 atmosphere for 72 h.</li>
	<li>Harvest the cells from one replicate as in step 1.10, and process them to validate or confirm the knockdown of the expression of caspase 3, caspase 6, and caspase 7 either by RT-qPCR or western blotting, respectively, using standard methods and protocols8,11.</li>
	<li>Infect the cells in the other replicate with IAV as described in steps 1.4-1.9 (without inhibitors).</li>
	<li>After 24 h, harvest, process, and analyze the cells by western blotting and measure and quantify the protein recovery as described in steps 1.10-1.20.</li>
</ol>
3. Site-directed mutagenesis for locating the caspase cleavage site(s) in the polypeptide
<ol>
	<li>Clone the gene-encoding protein of interest in a mammalian expression plasmid11 or obtain it from a research lab or commercial source (see Table of Materials). 
	NOTE: It is preferred if the gene is cloned with an epitope tag or as GFP fusion to distinguish the ectopically expressed polypeptide from the endogenously expressed one during western blotting.</li>
	<li>Using caspase substrate databases4 or protein alignment tools, locate the consensus caspase cleavage motifs, XXXD and DXXD, on the polypeptide. Almost all caspase cleavage motifs possess the aspartic acid at the cleavage site (P1)3,4.</li>
	<li>Mutate the putative P1 aspartic acid to glutamic acid or a non-polar amino acid (alanine, valine) by site-directed mutagenesis followed by DNA sequencing11 using standard methods and protocols.</li>
	<li>Deliver the wild-type (WT) and mutant plasmid DNA to MDCK or A549 cells by reverse transfection11.
	<ol>
		<li>For this, dilute 2 &#181;g of plasmid DNA or a recommended volume of the transfection reagent in 100 &#181;L of an appropriate medium (see step 2.3 and Table of Materials) in separate tubes, and incubate for 5 min at room temperature.</li>
		<li>Mix 100 &#181;L of each plasmid DNA solution with 100 &#181;L of the transfection reagent solution and incubate for 20-45 min at room temperature to form the DNA-transfection reagent complex.</li>
	</ol>
	</li>
	<li>In the meantime, split and add 3 x 105 MDCK or A549 cells to 800 &#181;L of the complete growth medium, mix with 200 &#181;L of DNA-transfection reagent complex, and add the 1 mL suspension to a well of a 12-well culture plate.</li>
	<li>Incubate the cells at 37 &#176;C under a 5% CO2 atmosphere.</li>
	<li>After 48 h, infect the cells with IAV as described in steps 1.4-1.9 (without adding the inhibitors).</li>
	<li>After 24 h, harvest, process, and analyze the cells by western blotting (if applicable, using the antibody to an epitope tag or GFP). Then, measure and quantify the protein recovery as described in steps 1.10-1.20.</li>
</ol>

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Y., Husain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-mediated+degradation+of+host+cortactin+that+promotes+influenza+A+virus+infection+in+epithelial+cells.">Caspase-mediated degradation of host cortactin that promotes influenza A virus infection in epithelial cells.</a> Virology. 497, 146-156 (2016).</li><li>Galvin, H. D., Husain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+virus-induced+host+caspase+and+viral+PA-X+antagonize+the+antiviral+host+factor,+histone+deacetylase+4.">Influenza A virus-induced host caspase and viral PA-X antagonize the antiviral host factor, histone deacetylase 4.</a> Journal of Biological Chemistry. 294 (52), 20207-20221 (2019).</li><li>Husain, M., Harrod, K. 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(97), e52421 (2015).</li><li>L&#252;thi, A. U., Martin, 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=The+CASBAH:+A+searchable+database+of+caspase+substrates.">The CASBAH: A searchable database of caspase substrates.</a> Cell Death &amp; Differentiation. 14 (4), 641-650 (2007).</li><li>Kumar, S., van Raam, B. J., Salvesen, G. S., Cieplak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+cleavage+sites+in+the+human+proteome:+CaspDB,+a+database+of+predicted+substrates.">Caspase cleavage sites in the human proteome: CaspDB, a database of predicted substrates.</a> PLoS One. 9 (10), 110539 (2014).</li><li>Igarashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CutDB:+A+proteolytic+event+database.">CutDB: A proteolytic event database.</a> Nucleic Acids Research. 35 (Database issue). 35, 546-549 (2007).</li><li>Crawford, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DegraBase:+A+database+of+proteolysis+in+healthy+and+apoptotic+human+cells.">The DegraBase: A database of proteolysis in healthy and apoptotic human cells.</a> Molecular &amp; Cellular Proteomics. 12 (3), 813-824 (2013).</li><li>Rawlings, N. D., Tolle, D. P., Barrett, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEROPS:+The+peptidase+database.">MEROPS: The peptidase database.</a> Nucleic Acids Research. 32, 160-164 (2004).</li><li>Lange, P. F., Overall, C. 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The author has no conflicts of interest to disclose.

It is established that viruses tailor the host factors and pathways to their benefit. In turn, the host cells resist that by employing various strategies. One of those strategies is PANoptosis, which host cells use as an antiviral strategy against virus infections. However, viruses like IAV have evolved their own strategies to counter PANoptosis and exploit it to their advantage1,3,6. This interplay involves the cleavage of vari...

Influenza A virus (IAV) is the prototypic member of the Orthomyxoviridae family and is known to cause global epidemics and unpredictable pandemics. IAV causes human respiratory disease, influenza, commonly known as &#34;flu&#34;. The flu is an acute disease that results in the induction of host pro- and anti-inflammatory innate immune responses and the death of epithelial cells in the human respiratory tract. Both processes are governed by a phenomenon called programmed cell death1. The signaling for programmed cell death is induced as soon as various pathogen recognition receptors sense the incoming virus particles in host cells. This leads to the programming of the death of infected cells and signaling to the neighboring healthy cells by three interconnected pathways called pyroptosis, apoptosis, and necroptosis-recently coined as one process, PANoptosis1.
PANoptosis involves the proteolytic processing of many host and viral proteins from induction to execution. Such processing of proteins is primarily spearheaded by a family of cysteine proteases called caspases1,2. Up to 18 caspases (from caspase 1 to caspase 18) are known3. Most caspases are expressed as pro-caspases and activated by undergoing their own proteolytic processing either by autocatalysis or other caspases4 in response to a stimulus like a virus infection. The PANoptosis of IAV-infected cells was thought to be a host defense mechanism, but IAV has evolved ways to evade and exploit it to facilitate its replication1,2,5,6. One of them is to antagonize the host factors via caspase-mediated cleavage or degradation that are either inherently antiviral or interfere with one of the steps of the IAV life cycle. To this end, host factors, cortactin, HDAC4, and HDAC6 have been discovered to undergo caspase-mediated cleavage or degradation in IAV-infected epithelial cells7,8,9. The HDAC4 and HDAC6 are anti-IAV factors8,10, and cortactin interferes with IAV replication at a later stage of infection, potentially during viral assembly and budding11.
In addition, various caspases are also activated, which, in turn, cleave multiple proteins to activate the host inflammatory response during IAV infection1,2. Furthermore, nucleoprotein (NP), ion-channel M2 protein of IAV12,13,14, and various proteins of other viruses3,15,16 also undergo caspase-mediated cleavage during infection, which influences viral pathogenesis. Therefore, there is a continuous need to study caspase-mediated cleavage or degradation of host and viral proteins during IAV and other virus infections to understand the molecular basis of viral pathogenesis. Herein, the methods are presented to (1) assess the cleavage or degradation of such proteins by caspases, (2) identify those caspases, and (3) locate the cleavage sites.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CRM-CCL-185</td>
			<td>Human, epithelial, lung</td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase 3 Inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>264156-M</td>
			<td>Also known as &#39;InSolution Caspase-3 Inhibitor II - Calbiochem&#39;</td>
		</tr>
		<tr>
			<td>cOmplete, Mini Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-NP antibody</td>
			<td></td>
			<td></td>
			<td>Gift from Richard Webby (St Jude Children&#8217;s Research Hospital, Memphis, USA) to MH</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>13778150</td>
			<td></td>
		</tr>
		<tr>
			<td>MDCK cells</td>
			<td>ATCC</td>
			<td>CCL-34</td>
			<td>Dog, epithelial, kidney</td>
		</tr>
		<tr>
			<td>MG132</td>
			<td>Sigma-Aldrich</td>
			<td>M7449</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11095080</td>
			<td>Add L-glutamine, antibiotics or other supplements as required</td>
		</tr>
		<tr>
			<td>MISSION siRNA Universal Negative Control #1</td>
			<td>Sigma-Aldrich</td>
			<td>SIC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Fc imager with Image Studio Lite software 5.2&#160;</td>
			<td>LI-COR</td>
			<td></td>
			<td>Odyssey Fc has been replaced with Odyssey XF and Image Studio Lite software has been replaced with Empiria Studio software.</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid expressing human cortactin-GFP fusion&#160;</td>
			<td>Addgene</td>
			<td>50728</td>
			<td>Gift from Kenneth Yamada to Addgene</td>
		</tr>
		<tr>
			<td>Pre-designed small interferring RNA (siRNA) to caspase 3</td>
			<td>Sigma-Aldrich</td>
			<td>NM_004346</td>
			<td>siRNA ID: SASI_Hs01_00139105</td>
		</tr>
		<tr>
			<td>Pre-designed small interferring RNA to caspase 6</td>
			<td>Sigma-Aldrich</td>
			<td>NM_001226</td>
			<td>siRNA ID: SASI_Hs01_00019062</td>
		</tr>
		<tr>
			<td>Pre-designed small interferring RNA to caspase 7</td>
			<td>Sigma-Aldrich</td>
			<td>NM_001227</td>
			<td>siRNA ID: SASI_Hs01_00128361</td>
		</tr>
		<tr>
			<td>Pre-designed SYBR Green RT-qPCR Primer pairs</td>
			<td>Sigma-Aldrich</td>
			<td>KSPQ12012</td>
			<td>Primer Pair IDs: H_CASP3_1; H_CASP6_1; H_CASP7_1</td>
		</tr>
		<tr>
			<td>Protran Premium nitrocellulose membrane</td>
			<td>Cytiva (Fomerly GE Healthcare)</td>
			<td>10600003</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-actin antibody</td>
			<td>Abcam</td>
			<td>ab8227</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-cortactin antibody</td>
			<td>Cell Signaling</td>
			<td>3502</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFP antibody</td>
			<td>Takara</td>
			<td>632592</td>
			<td></td>
		</tr>
		<tr>
			<td>SeeBlue Pre-stained Protein Standard</td>
			<td>ThermoFisher Scientific</td>
			<td>LC5625</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection medium, Opti-MEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Triton X-100</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, TPCK-Treated</td>
			<td>Sigma-Aldrich</td>
			<td>4370285</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying caspases and their motifs that cleave proteins during influenza a virus infection

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Treatment with caspase 3 inhibitor 
It has been discovered that host cortactin, HDAC4, and HDAC6 polypeptides undergo degradation in response to IAV infection in both canine (MDCK) and human (A549, NHBE) cells7,8,9. By using the above approaches, it was uncovered that IAV-induced host caspases, particularly caspase 3, cause their degradation7,8...

Watch this Scientific Journal Video about Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection at JoVE.com

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially ...

identifying-caspases-their-motifs-that-cleave-proteins-during

Research

JoVE Journal

Immunology and Infection

1.4K Views.  University of Otago. Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Video: Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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, ...

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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

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

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

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

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.