This work was supported by FWO-Vlaanderen, the IOF project IOF10/StarTT/027 and Ghent University Special Research Grant BOF12/GOA/014.

Note: The following transfection and co-immunoprecipitation protocol is established for a 9 cm Petri dish format. Other formats are also possible after scaling the protocol.
1. Seeding the Human Embryonic Kidney (HEK) 293T Cells
<ol>
	<li>Seed the HEK293T cells one day before transfection at 1.2 x 106 cells per 9 cm Petri dish in 12 ml of Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.4 mM Na-pyruvate, 0.1 mM non-e...

<ol>
	<li>Verhelst, J., Hulpiau, P., Saelens, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mx+proteins:+antiviral+gatekeepers+that+restrain+the+uninvited.">Mx proteins: antiviral gatekeepers that restrain the uninvited.</a> Microbiol Mol Biol Rev. 77 (4), 551-566 (2013).</li><li>Goujon, C., 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=Human+MX2+is+an+interferon-induced+post-entry+inhibitor+of+HIV-1+infection.">Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection.</a> Nature. 502 (7472), 559-562 (2013).</li><li>Kane, M., 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=MX2+is+an+interferon-induced+inhibitor+of+HIV-1+infection.">MX2 is an interferon-induced inhibitor of HIV-1 infection.</a> Nature. 502 (7472), 563-566 (2013).</li><li>Liu, Z., 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+interferon-inducible+MxB+protein+inhibits+HIV-1+infection.">The interferon-inducible MxB protein inhibits HIV-1 infection.</a> Cell Host Microbe. 14 (4), 398-410 (2013).</li><li>Kochs, G., Haller, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-induced+human+MxA+GTPase+blocks+nuclear+import+of+Thogoto+virus+nucleocapsids.">Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids.</a> Proc Natl Acad Sci U S A. 96 (5), 2082-2086 (1999).</li><li>Flohr, F., Schneider-Schaulies, S., Haller, O., Kochs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+central+interactive+region+of+human+MxA+GTPase+is+involved+in+GTPase+activation+and+interaction+with+viral+target+structures.">The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures.</a> FEBS Lett. 463 (1-2), 24-28 (1999).</li><li>Kochs, G., Haller, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTP-bound+human+MxA+protein+interacts+with+the+nucleocapsids+of+Thogoto+virus+(Orthomyxoviridae).">GTP-bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae).</a> J Biol Chem. 274 (7), 4370-4376 (1999).</li><li>Mitchell, P. S., 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=Evolution-guided+identification+of+antiviral+specificity+determinants+in+the+broadly+acting+interferon-induced+innate+immunity+factor+MxA.">Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA.</a> Cell Host Microbe. 12 (4), 598-604 (2012).</li><li>Patzina, C., Haller, O., Kochs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+requirements+for+the+antiviral+activity+of+the+human+MxA+protein+against+Thogoto+and+influenza+A+virus.">Structural requirements for the antiviral activity of the human MxA protein against Thogoto and influenza A virus.</a> J Biol Chem. 289 (9), 6020-6027 (2014).</li><li>Turan, K., 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=Nuclear+MxA+proteins+form+a+complex+with+influenza+virus+NP+and+inhibit+the+transcription+of+the+engineered+influenza+virus+genome.">Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome.</a> Nucleic Acids Res. 32 (2), 643-652 (2004).</li><li>Dittmann, J., 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=Influenza+A+virus+strains+differ+in+sensitivity+to+the+antiviral+action+of+Mx-GTPase.">Influenza A virus strains differ in sensitivity to the antiviral action of Mx-GTPase.</a> J Virol. 82 (7), 3624-3631 (2008).</li><li>Zimmermann, P., Manz, B., Haller, O., Schwemmle, M., Kochs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viral+nucleoprotein+determines+Mx+sensitivity+of+influenza+A+viruses.">The viral nucleoprotein determines Mx sensitivity of influenza A viruses.</a> J Virol. 85 (16), 8133-8140 (2011).</li><li>Manz, B., 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=Pandemic+influenza+A+viruses+escape+from+restriction+by+human+MxA+through+adaptive+mutations+in+the+nucleoprotein.">Pandemic influenza A viruses escape from restriction by human MxA through adaptive mutations in the nucleoprotein.</a> PLoS Pathog. 9 (3), e1003279 (2013).</li><li>Verhelst, J., Parthoens, E., Schepens, B., Fiers, W., Saelens, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-inducible+protein+Mx1+inhibits+influenza+virus+by+interfering+with+functional+viral+ribonucleoprotein+complex+assembly.">Interferon-inducible protein Mx1 inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly.</a> J Virol. 86 (24), 13445-13455 (2012).</li><li>Brewer, C. F., Riehm, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+possible+nonspecific+reactions+between+N-ethylmaleimide+and+proteins.">Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins.</a> Anal Biochem. 18 (2), 248-255 (1967).</li><li>Wu, C. Y., Jeng, K. S., Lai, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SUMOylation+of+matrix+protein+M1+modulates+the+assembly+and+morphogenesis+of+influenza+A+virus.">The SUMOylation of matrix protein M1 modulates the assembly and morphogenesis of influenza A virus.</a> J Virol. 85 (13), 6618-6628 (2011).</li><li>Chen, Z., Zhou, J., Zhang, Y., Bepler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+ribonucleotide+reductase+M1-gemcitabine+interaction+in+vivo+by+N-ethylmaleimide.">Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide.</a> Biochem Biophys Res Commun. 413 (2), 383-388 (2011).</li><li>Rennie, M. L., McKelvie, S. A., Bulloch, E. M., Kingston, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+dimerization+of+human+MxA+promotes+GTP+hydrolysis,+resulting+in+a+mechanical+power+stroke.">Transient dimerization of human MxA promotes GTP hydrolysis, resulting in a mechanical power stroke.</a> Structure. 22 (10), 1433-1445 (2014).</li><li>Gifford, J. L., Walsh, M. P., Vogel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+and+metal-ion-binding+properties+of+the+Ca2+-binding+helix-loop-helix+EF-hand+motifs.">Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs.</a> Biochem J. 405 (2), 199-221 (2007).</li><li>Gallagher, S., Chakavarti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblot+analysis.">Immunoblot analysis.</a> J Vis Exp. (16), (2008).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Pavlovic, J., Haller, O., Staeheli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+mouse+Mx+proteins+inhibit+different+steps+of+the+influenza+virus+multiplication+cycle.">Human and mouse Mx proteins inhibit different steps of the influenza virus multiplication cycle.</a> J Virol. 66 (4), 2564-2569 (1992).</li></ol>

Studying the interaction between antiviral proteins and their viral targets is very important to understand the details of the antiviral mechanism of these proteins. This can give new insights into how viruses and their hosts co-evolved and be the basis for the development of new antiviral strategies. The optimized co-immunoprecipitation protocol described here allows to study the interaction between the mouse Mx1 protein and its viral target, the influenza NP protein. The most important aspect of this protocol is the ad...

Myxovirus resistance (Mx) proteins are an important part of the innate immune defense against viral pathogens. These proteins are large dynamin-like GTPases that are induced by type I and type III interferons. The corresponding Mx genes are present in nearly all vertebrates in one or multiple copies and their gene products inhibit a wide range of viruses, including Orthomyxoviridae (e.g., influenza virus), Rhabdoviridae (e.g., vesicular stomatitis virus), Bunyaviridae (e.g., la crosse virus) and Retroviridae (e.g., human immunodeficiency virus-1)1-4. It is unclear how these proteins recognize such a broad array of viruses, without any apparent shared primary sequence motifs in these viruses. Analyzing the interaction of Mx proteins with their viral targets, potentially involving higher order complexes with other host cell factors, will help to understand the molecular mechanisms that have evolved in the arms race between viruses and their hosts.
The interaction between mammalian Mx proteins and viral targets has been studied most extensively for human MxA. Human MxA can inhibit the replication of many viruses, including the orthomyxoviruses influenza A and Thogoto virus. MxA binds the Thogoto virus ribonucleoprotein complexes (vRNPs), thereby preventing their nuclear entry, which results in block of infection5. This interaction between MxA and Thogoto virus vRNPs has been demonstrated with co-sedimentation and co-immunoprecipitation experiments6-9. How Mx proteins hinder influenza A viruses is less clear. One major problem is that it is not straightforward to demonstrate an interaction between an Mx protein and an influenza gene product. One report demonstrated an interaction between human MxA and the NP protein in influenza A virus infected cells10. This interaction could only be shown by co-immunoprecipitation if the cells had been treated with the cross-linking reagent dithiobis (succinimidyl propionate) before lysis, suggesting that the interaction is transient and/or weak. More recent studies have shown that the differential Mx sensitivity of different influenza A strains is determined by the origin of the NP protein11,12. In line with this, influenza A viruses can partly escape from Mx control by mutating specific residues in the NP protein13. This suggests that the main target of influenza A viruses for host Mx is the NP protein, most probably NP assembled in vRNP complexes. However, none of these more recent studies demonstrated an interaction between influenza NP or vRNPs and either human MxA or mouse Mx1.
Recently we showed, for the first time, an interaction between the influenza NP and the mouse Mx1 protein with an optimized co-immunoprecipitation protocol14, which is described here in detail. In general, co-immunoprecipitation is one of the most frequently used biochemical approaches to investigate protein-protein interactions. This technique is often preferred over alternative techniques, e.g., yeast two hybrid, since it allows to investigate protein-protein interactions in their natural environment. Co-immunoprecipitation can be carried out on endogenously expressed proteins if antibodies against the proteins of interest are available. Alternatively, the proteins of interest can be expressed in the cell through transfection or infection and an affinity tag may be used. In addition to the above-mentioned advantages, the described co-immunoprecipitation protocol allows the detection of weak and/or transient protein interactions. The main component in this optimized protocol is the addition of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM is an alkylating reagent that reacts with free thiol groups such as present in cysteines, at a pH of 6.5-7.5, to form a stable thio-ester (Figure 1). At higher pH, NEM can also react with amino groups or undergo hydrolysis15. NEM is typically used to block free thiol groups, in order to prevent disulfide bond formation or inhibit enzymatic activity. For example, NEM is often used to block desumoylating enzymes, which are cysteine proteases. In the described co-immunoprecipitation protocol, NEM was initially included in the lysis buffer because it had been reported that the sumoylation of influenza proteins can influence the interaction between viral proteins16. Unexpectedly, the addition of NEM proved to be key to document the interaction between influenza NP and mouse Mx1 by co-immunoprecipitation. It is unclear why the addition of NEM is crucial to detect the NP&#8211;Mx1 interaction. Possibly the interaction is too transient and/or weak. NEM could stabilize the interaction, e.g., by preserving a specific conformation of Mx1, a viral protein or even an unknown third component. Such a stabilizing effect of NEM has been observed before, e.g., for the interaction between the ribonucleotide reductase M1 and its inhibitor gemcitabine (F2dC)17. Mx1 and NP both contain multiple cysteine residues which could be modified by NEM. For example, a recent study by Rennie et al. demonstrated that a stalkless MxA-variant contains three solvent exposed cysteine residues which can be modified by iodoacetamide. Mutating these residues to serines did not influence the enzymatic activity of MxA, but prevented disulfide-mediated aggregation18. As these cysteines are conserved in Mx1, this suggests that the analogous cysteines in Mx1 can be modified by NEM and as such influence its conformation or solubility. In addition, NEM might also affect the GTPase activity of Mx1, which is essential for the anti-influenza activity of Mx1, and thereby stabilize the interaction between Mx1 and NP. However, a direct effect of NEM on the GTPase activity of Mx1 is unlikely, as NEM is also required to detect the interaction between influenza NP and GTPase inactive mutants of the Mx1 protein14. Clearly, more research is needed to unravel the effect of NEM on the NP&#8211;Mx1 interaction.
In summary, the described co-immunoprecipitation protocol allows to study the interaction between the antiviral Mx1 protein and its viral target, the influenza NP protein. This protocol could also be used to study other weak or transient interactions that depend on the stabilization of specific protein conformations. Protein-protein interaction that depend on specific conformations have been described before, e.g., for calcium-binding proteins such as calmodulin19. Finally, the beneficial role of NEM could also be used in other methods that detect protein-protein interactions, such as co-sedimentation assays.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM high glucose</td>
			<td>Gibco</td>
			<td>52100-047</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Ethylmaleimide</td>
			<td>Sigma</td>
			<td>E-3876</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma</td>
			<td>I-30212</td>
			<td>also known as NP40</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11 873 580 001</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-NP monoclonal antibody</td>
			<td>NIH Biodefense and Emerging Infections Research Resources Repository</td>
			<td>NR-4282</td>
			<td>ascites blend of clones A1 and A3</td>
		</tr>
		<tr>
			<td>anti-RNP polyclonal serum</td>
			<td>NIH Biodefense and Emerging Infections Research Resources Repository</td>
			<td>NR-3133</td>
			<td>directed against A/Scotland/840/74 (H3N2)</td>
		</tr>
		<tr>
			<td>Protein G Sepharose 4FF</td>
			<td>GE Healthcare</td>
			<td>17-0618-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyperfilm ECL 18 x 24 cm</td>
			<td>GE Healthcare</td>
			<td>28-9068-36</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Western Blotting Substrate</td>
			<td>Pierce</td>
			<td>32106</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

N-ethylmaleimide is an organic compound that can be used to irreversibly modify free thiol groups, e.g. to inhibit cysteine proteases (Figure 1).
The antiviral Mx1 protein inhibits influenza A virus replication by interacting with the viral nucleoprotein. The optimized co-immunoprecipitation protocol described here allows to study this NP&#8211;Mx1 interaction. HEK293T cells were transfected with expression vectors for the antiviral Mx1 protein in the absence or prese...

Watch this Scientific Journal Video about Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein at JoVE.com

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

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 ...

co-immunoprecipitation-mouse-mx1-protein-with-influenza-virus

Research

JoVE Journal

Immunology and Infection

17.6K vues.  VIB. 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.

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

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, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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 ...

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 ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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 ...

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.