This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

All animal experiments were approved by the Animal Care and Use Committee at the University of Texas Medical Branch.
1. Isolation of DCs from Non-infected and WNV-infected Mice
<ol>
	<li>Age- and sex- match 6-10 week-old, wild-type C57BL/6 (B6) and MyD88&#8722;/&#8722;mice. Inoculate intraperitoneally (i.p.) with 500 plaque forming unit (PFU) of WNV NS4B- P38G mutant. At day 3 post-infection, euthanize B6 and MyD88&#8722;/&#8722; mice with CO2. Use non-infected mice of both groups as controls. Use three mice from each group.</li>
	<li>Wet fur on left side of sacrificed mouse using 70% ethanol and cut away the fur along the left side of the mouse, about half-way between the front and back legs. Cut open the body cavity. Remove the spleen by using the forceps. Place the spleen in a Petri dish with RPMI.</li>
	<li>Isolate splenic DCs by using anti-CD11c magnetic beads according to the manufacturer&#8217;s instructions.</li>
</ol>
2. Purification and Labeling of T Cells of OTII Transgenic Mice
<ol>
	<li>Harvest one spleen from a na&#239;ve OTII mouse as described in step 1.2 above and homogenize the spleen between the frosted ends of two slides. Transfer the cell suspension to a 15 ml conical tube, add RPMI medium up to 14 ml. Let the suspension sit for 5 min and transfer 13 ml of it to a new 15 ml conical tube.</li>
	<li>Isolate splenic CD4+ T cells by using a CD4+ T cell isolation kit according to the manufacturer&#8217;s instructions.</li>
	<li>Transfer cells into a 50 ml conical tube, wash twice with PBS. Resuspend cells in PBS with 0.5% bovine serum albumin (BSA) at 1 x 106 cells per ml, and add 0.5 &#181;mol/ml of CFSE. Incubate at 37 &#176;C for 10 min, protected from light.</li>
	<li>Add 5 ml cold complete medium (RPMI-1640 with 10% heat-inactivated fetal bovine serum, 1/100 vol antibiotics/antimycotics, 1/100 vol L-glutamine and 1/1,000 vol of 1,000 x 2-mercaptoethanol).</li>
	<li>Incubate on ice for 5 min. Wash cells once with PBS, fix 0.2 x 106 labeled CD4+ T cells in 2% PFA and acquire on a flow cytometer. Use this sample to determine the basal level of CFSE. Re-suspend the rest of labeled cells in complete medium.</li>
</ol>
3. Co-culture OTII T cells with DCs of WNV-infected Mice
<ol>
	<li>Culture 2 x 105 purified CD4+ T cells of OTII mice alone or with purified DCs of wild-type or MyD88&#8722;/&#8722; mice (2 x 104) in a 24 well plate with or without OVA residue 323-339 (1 g/ml) in 1 ml complete medium per well.</li>
	<li>Incubate cells at 37 &#176;C for 5 days. Harvest cells, wash twice in FACS buffer (PBS with 1% FBS) and fix in 0.25 ml 2% PFA. Vortex immediately.</li>
</ol>
4. Flow Cytometry Analysis of In Vitro T Cell Priming
<ol>
	<li>For acquisition, double click the flow cytometry software icon. For a density plot of linear FSC-A vs. linear SSC-A, select channels 0 to 200,000 on FSC-A and 0 to 200,000 on SSC-A. Then create a gate on live cells (P2; see Figure 1A).</li>
	<li>To analyze the fluorescence intensity of CFSE, open the histogram for the FL1 channel and acquire 20,000 events on the gated population. Set up a marker on samples for OTII cells cultured alone (Figure 1A).</li>
	<li>Acquire samples for OTII cells co-cultured with wild-type DCs (Figure 1B) or MyD88&#8722;/&#8722; DCs (Figure 1C) in a similar way.</li>
</ol>
5. Isolation and Stimulation of Splenocytes for Cytokine Assays
<ol>
	<li>Infect B6 and MyD88&#8722;/&#8722;mice with WNV NS4B-P38G mutant using the same procedure as described in step 1.1. At day 30 post-infection, re-challenge surviving mice with 2000 PFU of the wild-type WNV strain i.p. At days 8 and 21 post-primary WNV infection or at day 4 following secondary infection, euthanize mice with CO2 for collection of spleens. 
	NOTE: We used 2-3 mice per group for each time point.</li>
	<li>Make a single cell suspension of splenocytes as described above in step 1.2 &#38; step 2.2. Count cells using a hemocytometer and re-suspend them in 10 ml complete medium.</li>
	<li>Dilute cells with complete medium to 2.5 x 106 cells/ml. Add 1 ml of splenocytes in a 1.5 ml micro-centrifuge tube.</li>
	<li>To simulate CD8+ T cells, dilute WNV-specific NS4B and E peptides (SSVWNATTA and IALTFLAV) to 1 mg/ml in DMSO. For stimulation of CD4+ T cells, dilute WNV-specific NS3 and E peptides (RRWCFDGPRTNTILE and PVGRLVTVNPFVSVA) to 1 mg/ml in DMSO. Add 10 &#181;l of peptides to the cells followed by 1 &#181;l of Brefeldin A solution. Use cells without peptides treatment as controls. Mix the cells.</li>
	<li>Punch two holes with an 18 G needle in the cap of the tube. Incubate cells at 37 &#176;C for 5 hr.</li>
</ol>
6. Intracellular Cytokine Staining
<ol>
	<li>Transfer cells to a new micro-centrifuge tube. Spin cells for 5 min at 300-400 x g and pour off supernatant. Add 1 ml FACS buffer, spin 5 min at 300-400 x g and re-suspend cells by pipetting with 120 &#181;l FACS buffer.</li>
	<li>Add 2 &#181;l Fc blocker and incubate cells at room temperature for 10 min. Add 1 ml FACS buffer to each tube. Spin 5 min at 300 - 400 x g.</li>
	<li>Re-suspend cells in 300 &#181;l FACS buffer and split them into three tubes (about 0.8 x 106 cells per tube). As shown in Table 1, reserve the first tube of each culture condition for rat IgG-PE staining. For the second tube, add 3 &#181;l of anti-CD4 APC to CD4 peptides-treated cells, 3 &#181;l of anti-CD8 FITC to CD8 peptides-treated cells or both antibodies to cells without peptides treatment. Use the third tube for compensation staining. For each culture condition, use three tubes of cells stained with APC-conjugated CD4, FITC-conjugated CD8 or PE- labeled CD3 antibodies alone (3 &#181;l per tube) as compensation controls for FL1, FL2 and FL4 channels.</li>
	<li>Vortex cells briefly. Leave on ice for 20 min and protect from light. Add 1.4 ml FACS buffer. Spin 5 min at 300-400 x g and pour off supernatant.</li>
	<li>Agitate to disrupt cell pellet (or briefly vortex). Re-suspend cell pellet in 250 &#181;l fixation/permeabilization solutionby pipetting. Incubate at room temperature for 20 min and protect from light.</li>
	<li>Add 1.2 ml FACS buffer. Spin 5 min at 300-400 x g and resuspend cells in 300 &#181;l FACS buffer. 
	NOTE: At this point, cells can be transferred to a BL2 laboratory for further processing or stored in a refrigerator and protected from light for up to three days.</li>
	<li>Add 500 &#181;l FACS buffer. Spin 5 min at 300-400 x g and resuspend cells in 500 &#181;l of 1x permeabilization/wash buffer. Spin 5 min at 300-400 x g.</li>
	<li>Re-suspend cells in 100 &#181;l 1x Perm/Wash, add 3.5 &#181;l anti-IFN&#947;-PE to the tube previously added with antibodies for CD4 and/or CD8 T cell markers in step 6.3 and 3.5 &#181;l rat IgG-PE in the tube reserved for IgG control for 25 min on ice and protect from light. Wash cells by adding 1.4 ml of 1x permeabilization/wash buffer. Spin 5 min at 300-400 x g. Repeat the washing step by adding 1.4 ml 1x permeabilization/wash buffer.</li>
	<li>Spin 5 min at 300-400 x g and decant supernatant. Wash cells by adding 1.4 ml of FACS buffer and re-suspend in 400 &#181;l of FACS buffer for final acquisition.</li>
</ol>
7. Flow Cytometry Analysis of Intracellular Cytokine Staining
<ol>
	<li>Use the same acquisition settings as in step 4.1. Create a gate on live cells (P2; see Figure 2A). Adjust the voltages using the compensation tubes for each group.</li>
	<li>Open two new dot plots, one to display data collected for FL1 vs. FL2 (CD8 vs. IFN-&#947;), and the other one is to display data collected for FL4 vs. FL2 (CD4 vs. IFN-&#947;). Acquire 50,000 events for the gated population of each sample.</li>
</ol>

<ol>
	<li>Campbell, G. L., Marfin, A. A., Lanciotti, R. S., Gubler, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus.">West Nile virus.</a> Lancet Infect. Dis. 2, 519-529 (2002).</li><li>Welte, T., 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=Immune+responses+to+an+attenuated+West+Nile+virus+NS4B-P38G+mutant+strain.">Immune responses to an attenuated West Nile virus NS4B-P38G mutant strain.</a> Vaccine. 29, 4853-4861 (2011).</li><li>Iwasaki, A., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+control+of+the+adaptive+immune+responses.">Toll-like receptor control of the adaptive immune responses.</a> Nat. Immunol. 5, 987-995 (2004).</li><li>Akira, S., Hemmi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+pathogen-associated+molecular+patterns+by+TLR+family.">Recognition of pathogen-associated molecular patterns by TLR family.</a> Immunol. Lett. 85, 85-95 (2003).</li><li>Xie, G., 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=A+West+Nile+virus+NS4B-P38G+mutant+strain+induces+adaptive+immunity+via+TLR7-MyD88-dependent+and+independent+signaling+pathways.">A West Nile virus NS4B-P38G mutant strain induces adaptive immunity via TLR7-MyD88-dependent and independent signaling pathways.</a> Vaccine. 31 (38), 4143-4151 (2013).</li><li>Fang, H., 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=gammadelta+T+cells+promote+the+maturation+of+dendritic+cells+during+West+Nile+virus+infection.">gammadelta T cells promote the maturation of dendritic cells during West Nile virus infection.</a> FEMS Immunol. Med. Microbiol. 59, 71-80 (2010).</li><li>Silva, M. C., Guerrero-Plata, A., Gilfoy, F. D., Garofalo, R. P., Mason, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+activation+of+human+monocyte-derived+and+plasmacytoid+dendritic+cells+by+West+Nile+virus+generated+in+different+host+cells.">Differential activation of human monocyte-derived and plasmacytoid dendritic cells by West Nile virus generated in different host cells.</a> J. Virol. 81, 13640-13648 (2007).</li><li>Shrestha, B., Samuel, M. A., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+Cells+Require+Perforin+To+Clear+West+Nile+Virus+from+Infected+Neurons.">CD8+ T Cells Require Perforin To Clear West Nile Virus from Infected Neurons.</a> J. Virol. 80, 119-129 (2006).</li><li>Wang, Y., Lobigs, M., Lee, E., Mullbacher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+mediate+recovery+and+immunopathology+in+West+Nile+virus+encephalitis.">CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis.</a> J. Virol. 77, 13323-13334 (2003).</li><li>Plebanski, M., Katsara, M., Sheng, K. C., Xiang, S. D., Apostolopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+measure+T-cell+responses.">Methods to measure T-cell responses.</a> Expert Rev. Vaccines. 9, 595-600 (2010).</li><li>Lyons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+cell+division+in+vivo+and+in+vitro+using+flow+cytometric+measurement+of+CFSE+dye+dilution.">Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution.</a> J. Immunol. Methods. 243, 147-154 (2000).</li><li>Kraus, A. A., Priemer, C., Heider, H., Kruger, D. H., Ulrich, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Hantaan+virus-containing+samples+for+subsequent+investigations+outside+biosafety+level+3+facilities.">Inactivation of Hantaan virus-containing samples for subsequent investigations outside biosafety level 3 facilities.</a> Intervirology. 48, 255-261 (2005).</li><li>Saxena, V., 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=A+hamster-derived+west+nile+virus+isolate+induces+persistent+renal+infection+in+mice.">A hamster-derived west nile virus isolate induces persistent renal infection in mice.</a> PLoS Negl. Trop. Dis. 7, 2275 (2013).</li><li>Welte, T., 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=Vgamma4++T+cells+regulate+host+immune+response+to+West+Nile+virus+infection.">Vgamma4+ T cells regulate host immune response to West Nile virus infection.</a> FEMS Immunol. Med. Microbiol. 63, 183-192 (2011).</li></ol>

The authors have nothing to disclose.

WNV is a BL3 pathogen. Due to safety regulations, immunological assays with WNV-infected samples are often restricted by the availability of equipment at BL3 facilities or more lengthy and tedious to perform. In a recent study, we used two flow cytometry-based methods to study cell mediated immune responses during WNV infection5. In both assays, WNV-infected cells were treated with 1-2% PFA directly or with fixation/permeabilization solution containing 4% PFA. It is known that 1% PFA fixation of virus-infected...

West Nile virus (WNV), a neurotropic, plus-sensed flavivirus, is an emerging public health threat. Currently, no vaccines have been approved for human use1. An attenuated WNV strain, which has a P38G substitution in the nonstructural (NS)4B protein, is known to induce no lethality in mice but higher innate cytokines and T cell responses in mice than wild-type WNV NY99 strain2. Mice immunized with the NS4B-P38G mutant were all protected from a secondary challenge with lethal wild-type WNV. This suggests that the NS4B-P38G mutant has suitable features for an ideal vaccine candidate. The mechanisms by which the NS4B-P38G mutant induces high protective adaptive immunity are not clearly understood yet. Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, play an essential role in the initiation of innate immunity to viral infection. The core TLR signaling pathway utilizes myeloid differentiation primary response gene 88 (MyD88) as the primary adaptor3,4. In a recent study, MyD88 signaling was shown to play an important role in development of cell mediated immunity during WNV NS4B- P38G mutant infection in mice5. Dentritic cells (DCs) are one of the most important antigen-presenting cells exhibiting the unique capacity to initiate primary T cell responses during viral infection6,7. CD4+ and CD8+ T-cells both contribute to long-lasting protective immunity and are important for host survival following wild-type WNV infection8,9. Two immunological assays were used in this study to assess the functions of these cells in the NS4B-P38G mutant infected mice.
First, an in vitro T cell priming assay was utilized to compare the antigen-presenting capability of DCs of WNV-infected wild-type and MyD88&#8722;/&#8722; mice. To increase sensitivity of the assay, na&#239;ve CD4+ T cells were isolated from OTII transgenic mice, which express a V&#945;2/V&#946;5 TCR specific for the chicken ovalbumin (OVA) peptide 323 - 339. DCs of WNV infected mice were purified, and co-cultured with carboxyfluorescein succinimidyl easter (CFSE)-labeled CD4+ T cells in the presence of OVA peptide. After 5 days of co-culture, cells were harvested and fixed with paraformaldehyde (PFA) and analyzed by flow cytometry. Proliferative assays have been traditionally carried out through incorporation of 5-bromo-2&#8217;-deoxyuridine (BrdU) or tritiated thymine deoxyriboside (3HTdr)10. Nevertheless, these assays are either radioactive and/or in need of special equipments at biosafety level (BL) 3 facilities, where WNV studies are conducted. The flow cytometric analysis of lymphocyte proliferation by serial halving of the fluorescence intensity of the vital dye CFSE has become more commonly used in immunological assays as the dye is more stably and evenly incorporated into cells, detected easily by flow cytometry, and is nonradioactive11. The assay also has the ability to assess the number of cell divisions. One major advantage of using this assay in WNV studies is that fixation of the infected cells with 1-2% PFA could inactivate WNV12, which will enable sample acquisition with a flow cytometer in a BL2 laboratory.
Next, a modified intracellular cytokine staining (ICS) procedure was used to study the role of MyD88 signaling in regulation of WNV specific T cell responses in NS4B-P38G mutant-infected mice. In this assay, splenocytes isolated from infected mice were treated in vitro with WNV specific peptides. Brefeldin A was added to retain the cytokines within the cell. After 5 hr incubation, cells were harvested, washed and stained for T cell subsets. Cells were then fixed in PFA, permeabilized and stained for interferon (IFN)-&#947; and analyzed by flow cytometry. As with other flow cytometry-based assay, once the cells are treated with fixation and permeabilization buffer containing PFA, infected samples can be transferred to a BL2 laboratory for further processing and analysis. In several published studies, we have used ICS to measure T cell effector functions in WNV-infected mice13,14. Although it&#8217;s well established, one major drawback of this assay is that the procedure is very lengthy and could be more time consuming when performed inside BL3 facilities. Here, a micro-centrifuge tube-based ICS method was shown to be more feasible, easier to proceed and less time consuming when performed within a BL3 laboratory.

<table><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>11875</td><td>warm up at 37 &amp;deg;C</td></tr><tr><td>anti-CD11c magnetic beads</td><td>Miltenyi Biotec</td><td>130-052-001</td><td>follow the manufacturer&amp;rsquo;s instructions</td></tr><tr><td>anti-CD4 magnetic beads</td><td>Miltenyi Biotec</td><td>130-095-248</td><td>follow the manufacturer&amp;rsquo;s instructions</td></tr><tr><td>CFSE</td><td>Invitrogen</td><td>C34554</td><td></td></tr><tr><td>OVA residue 323-339</td><td>Genscript Corporation</td><td>RP10610</td><td></td></tr><tr><td>Peptides</td><td>Proimmune</td><td>PC0AD-D</td><td></td></tr><tr><td>Brefeldin A solution</td><td>BD Bioscience</td><td>555029</td><td></td></tr><tr><td>Mouse Fc Blocker</td><td>e-Bioscience</td><td>14-0161-85</td><td></td></tr><tr><td>APC-conjugated CD4</td><td>e-Bioscience</td><td>17-0041-81</td><td></td></tr><tr><td>FITC-conjugated CD8</td><td>e-Bioscience</td><td>11-0081-82</td><td></td></tr><tr><td>Fixation/Permeabilization Solution</td><td>BD-Bioscience</td><td>554722</td><td></td></tr><tr><td>Permeabilization/wash buffer</td><td>BD-Bioscience</td><td>554723</td><td></td></tr><tr><td>anti-IFN&amp;gamma;-PE</td><td>e-Bioscience</td><td>12-7311-82</td><td></td></tr><tr><td>Accuri flow cytometer</td><td>BD Bioscience</td><td></td><td></td></tr></tbody></table>

in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In the T cell priming assay, CFSE labeled CD4+ T cells were cultured with purified DCs from the NS4B-P38G mutant-infected wild-type and MyD88&#8722;/&#8722; mice in the presence or absence of OVA peptides. Labeled T cells cultured alone with or without OVA for 5 days were used as negative controls. As shown in Figure 1A, total T cells were gated for analysis of fluorescence intensity on the FL1 channel. The marker was set up based on the freshly labeled CD4<sup...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

in-vitro-analysis-myd88-mediated-cellular-immune-response-to-west

Research

JoVE Journal

Immunology and Infection

8.1K Views.  The University of Texas Medical Branch. Two flow cytometry-based methods – an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Video: In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

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 ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

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

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Four methods were used to detect intracellular Wolbachia, which complemented each other and improved the detection accuracy of Wolbachia infection of Aedes albopictus-derived Aa23 and Aa23-T&#160;cured of native Wolbachia infection using antibiotics.

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Summary

Explore More Videos

Chapters in this video

Measurement of γHV68 Infection in Mice

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Influenza A Virus Studies in a Mouse Model of Infection

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1^-/- Mice

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines