We would like to acknowledge the NSF for funding (CBET- 0846259).

Refer to Table 1 for preparation of all media, buffers and solutions referenced in this protocol.
1. Production of Aha-Labeled Adenovirus
<ol>
 <li> Prepare eleven 100-mm tissue culture dishes of human embryonic kidney cells (HEK 293) maintained in HEK 293 cell growth medium (see Table 1) at 37 &deg;C until they have reached 80 - 90% confluency. (Note: Cultures with confluency over 90% may be difficult to infect and may not produce viruses as desired.) </li>
 <li> Loosen the cells in one of the dishes by first removing the growth medium, rinsing once with 5 ml of TD buffer, then incubating the cells for one min in 1 ml of trypsin-EDTA (1&times;). </li>
 <li> Add 9 ml of HEK 293 cell growth medium, and use a hemocytometer to count the number of harvested HEK 293 cells from this dish. Use this number to calculate the amount of adenovirus type 5 (Ad5) required for infection using a multiplicity of infection (MOI) value of 10 PFU / cell. Account for the fact that for normal Ad5, the infectivity index (ratio of infectious particles to total number of particles) is typically 1:20. </li>
 <li> Prepare 10 ml of infection buffer. </li>
 <li> Slowly remove the HEK 293 cell growth medium from the ten remaining culture dishes, then wash with 5 ml of TD buffer. Add 1 ml of infection buffer to each dish and incubate at 37 &deg;C for 1 hr. During this infection period, gently rock each culture dish side to side at 15-min intervals. </li>
 <li> After infection, add 9 ml of fresh HEK 293 cell growth medium and incubate at 37 &deg;C for 18 hr. </li>
 <li> Prepare 100 ml of Aha labeling medium (or Met control medium for the control). Be sure to filter the concentrated stock solutions with a 0.2-&mu;m sterile syringe filter before preparing the labeling media. </li>
 <li> Carefully (so as not to wash away the infected cells) remove the HEK 293 cell growth medium after 18 hr and wash each culture dish with 5 ml of TD buffer. </li>
 <li> Remove the TD buffer wash solution and add 10 ml of Aha labeling medium to each culture dish. For a control, methionine control medium should be used instead of the Aha labeling medium at this step. </li>
 <li> Incubate the infected cells in labeling medium at 37 &deg;C for 6 hr. Labeling can be carried out anytime between 12 - 24 hr post-infection but should not be longer than 6 hr in duration. This interval has been determined to maximize overlap with viral structural protein translation while ensuring infectivity is not significantly diminished. 7 </li>
 <li> Carefully remove the labeling medium so as not to disrupt the cells. Add 10 ml of fresh HEK 293 cell growth medium to each dish and incubate the cells at 37 &deg;C for another 18 hr. To minimize loss of infected cells during this step, it is not necessary to remove the labeling solution entirely. The HEK 293 cell growth medium contains an excess of Met which will outcompete any residual Aha. </li>
 <li> Proceed to step 3 for purification. </li>
</ol>
2. Production of Azido-Sugar-Labeled Adenovirus
<ol>
 <li> Follow the procedure titled Production of Aha-Labeled Adenovirus up to and including step 1.5. </li>
 <li> Prepare 100 ml of Azido-sugar labeling medium. </li>
 <li> Cover the cells with 10 ml of fresh Azido-sugar labeling medium and incubate at 37 &deg;C for 44 hr. </li>
</ol>
3. Purification of Azide-Labeled Adenoviral Particles
<ol>
 <li>Remove the media along with the infected cells from each culture dish and transfer to conical tubes. Centrifuge at 2,000 g for 10 min at 4 &deg;C. Remove the supernatant and resuspend the pellet in TD buffer (using 8 ml of TD buffer per 10 dishes). </li>
 <li> Lyse the infected cells by repeated (3 or more) freeze-thaw cycles using a Dewar flask of liquid nitrogen and a 37 &deg;C water bath. Centrifuge at 2,000 g for 10 min at 4 &deg;C to separate cell debris from the supernatant. </li>
 <li> Prepare a CsCl density gradient centrifuge tube by first pipetting 2.5 ml of 1.25 g/ml CsCl solution into an Ultra Clear centrifuge tube. Next, use a pipette with its tip positioned at the bottom of the tube to slowly add 2.0 ml of 1.4 g/ml CsCl solution, taking care not to disturb the gradient interface. </li>
 <li> Pipette the supernatant from step 3.2 on top of the CsCl density gradient tube, and if necessary add TD buffer to fill the tube. Centrifuge the samples in a swinging bucket rotor at 125,000 g for 1 hr at 15 &deg;C in an ultracentrifuge. </li>
 <li> After completion of ultracentrifugation, the virus particles should be visible as a thick white band at the interface of the two CsCl solutions. Make a small perforation at the bottom of the tube with a sterile needle to allow the solution to drain dropwise.</li>
 <li> Collect the thick white band, which should contain the labeled Ad5 particles, taking care to exclude excess CsCl solution. Avoid collecting cellular proteins which will band above the virus layer. Additionally exclude empty virion capsids which form a lighter layer slightly above the thick white virus band. </li>
 <li> Optionally, perform a final purification on the labeled Ad5 particles. Add the collected fraction from step 3.6 to a 4-ml ultracentrifuge tube and fill the tube with 1.35 g/ml CsCl solution. Centrifuge at 125,000 g for 18 hr at 15 &deg;C and collect the thick white band which forms in the middle of the tube as described in steps 3.5 and 3.6. (Note that if the viral titer is low, a white band may not be visible after the second centrifugation, and it will not be possible to recover the labeled virus). </li>
 <li> For storage of the virus particles over long periods of time, dialysis of the collected virus extract should be performed against virus storage buffer. Store the dialyzed virus samples at -80 &deg;C. </li>
</ol>
4. Ligation of Modified Adenoviral Particles with Alkyne Probes Using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)
<ol>
 <li> To a solution of 50 &mu;l of azide-modified Ad5 in virus storage buffer, add 0.25 &mu;l of SPAAC labeling solution. (Refer to the table of reagents for
a list of venders of commercially available strained alkynes). </li>
 <li> Allow the strain-promoted cycloaddition reaction to proceed overnight at room temperature. If a light-sensitive probe (e.g. TAMRA) is used, cover the reaction vessel with aluminum foil. </li>
 <li> Purify the labeled virus according to step 6. </li>
</ol>
5. Ligation of Modified Adenoviral Particles with Alkyne Probes Using Copper(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) in Deoxygenated Atmosphere
<ol>
 <li> Place 7.2 mg of copper(I) bromide (CuBr) powder in an Eppendorf tube, covered with aluminum foil.</li>
 <li> Pipette 1 ml of DMSO into a second Eppendorf tube. </li>
 <li> Prepare 50 &mu;l of CuAAC labeling solution in a third Eppendorf tube. Cover with aluminum foil if the alkyne probe is light-sensitive (e.g. TAMRA-Alkyne). </li>
 <li> Place the three Eppendorf tubes, uncapped, in a deoxygenated glove bag for 6 hr. All samples should remain inside the glove bag until the completion of the CuAAC reaction. </li>
 <li> Prepare a 50&times; stock solution of CuBr by adding 1 ml of DMSO to 7.2 mg of CuBr powder, both of which have been deoxygenated according to step 5.4. </li>
 <li> Initiate the CuAAC reaction by pipetting 1 &mu;l of this CuBr stock solution to 50 &mu;l of the CuAAC labeling solution, which has been deoxygenated according to step 5.4. Allow the reaction to proceed for 12 hr inside the glove bag. </li>
 <li> Purify the labeled virus according to step 6. </li>
</ol>
6. Purification and Storage of Labeled Virus Particles
<ol>
 <li> Purify the labeled virus samples using Centri-Sep gel filtration spin columns equilibrated with Virus Storage Buffer, following the manufacturer's guidelines.</li>
 <li> Alternatively, purification can be carried out by dialysis against Virus Storage Buffer. </li>
 <li> The labeled virus particles should be stored at -80 &deg;C. </li>
</ol>

7. SDS-PAGE of Labeled Adenovirus &amp; Fluorescent Gel Scanning
<ol>
 <li> Add 20 &mu;l Laemmli sample buffer (2&times;) to 20 &mu;l of purified labeled virus and boil for 10 min. Centrifuge the samples for 30 secs to remove any insoluble precipitates. If residual copper is present after purification, boiling the sample can result in thick streaks of protein on the SDS-PAGE gel, causing difficulty during visualization. In this case, forgo boiling of the sample in this step. </li>
 <li> Attach the gel cassette to the electrophoresis cell and add running buffer. Load the required number of wells with the viral protein samples. Use one well to load pre-stained molecular weight markers. Run the gel for 1 hr at 200 V. Keep the electrophoresis cell covered with aluminum foil during the entire period to reduce photobleaching of the fluorophore label. </li>
 <li> For best resolution and to remove all free fluorophore, allow the tracking dye to run out of the gel during electrophoresis. Stop the electrophoresis and quickly transfer the gel to a fluorescent gel scanner and scan the gel for fluorescence emission at the appropriate wavelength (574 nm for TAMRA). </li>
 <li> The number of dye molecules conjugated to each viral particle can be quantified by measuring the fluorescence of several standard TAMRA concentrations and comparing with the sample from step 7.3. </li>
</ol>
8. Representative Results
The observed results of SDS-PAGE &amp; fluorescent gel scanning of TAMRA-conjugated Ad5 are shown in Figure 3. Although these fluorescent gel scans were performed on samples modified via CuAAC, results should be comparable when probes are ligated via SPAAC. These gel scans indicate that Aha labeling results in modification of the three Ad5 capsid proteins (Hexon, Penton, and Fiber), while sugar labeling modifies only the Fiber capsid protein. Furthermore, an increase in Aha concentration (4 mM vs. 32 mM) results in a greater degree of Aha incorporation, but it also has been shown to decrease infectivity. Prior results7 indicate that for Ad5 produced in 4mM Aha, roughly 5% of exposed methionine sites are TAMRA-modified, with this number increasing to 10% when 32 mM Aha is used. This corresponds to approximately 280 and 510, respectively, dye-labeled sites per Ad5 particle. Analysis of sugar-labeling1 has indicated that roughly 22 of the 36 glycosylation sites on Ad5 are occupied by an azido sugar and subsequently labeled with TAMRA.
If ten tissue culture plates are used to produce azide-modified Ad5 as described in this protocol, 200 - 300 &mu;l of azide-modified virus should be obtained from the white band collected in step 3.6, with a titer of approximately 1.5 &times; 1012 particles / ml. A common difficulty of this procedure is the absence of a viral band at this purification step, which can be attributed to low viral titer. This is generally a result of infecting cells which are either over- or under-confluent, or by washing away cells - which become loosened from the plate after infection - while rinsing during step 1.
<img src="/files/ftp_upload/4246/4246fig1.jpg" alt="Figure 1" /> 
Figure 1. Protocol overview. Azide-containing Aha or GalNAz is first incorporated into adenovirus particles. Ligation with a fluorescent alkyne probe is then performed via "click" chemistry. Finally, SDS-PAGE is used to demonstrate ligation of the fluorescent probe.
<img src="/files/ftp_upload/4246/4246fig2.jpg" alt="Figure 2" /> 
Figure 2. Ligation of alkyne probe onto azide-modified virus via (a) SPAAC, (b) CuAAC under de-oxygenated and (c) oxygenated atmosphere.
<img src="/files/ftp_upload/4246/4246fig3.jpg" alt="Figure 3" /> 
Figure 3. Fluorescent scans of SDS-PAGE of azide-labeled adenovirus type 5 (Ad5) after CuAAC conjugation with TAMRA-Alkyne. (a) Aha-labeled Ad5, using two concentrations of Aha. Labeling appears to occur on adenovirus Hexon, Penton and Fiber capsid proteins, with the extent of labeling increasing with increased Aha concentration. (b) Ac4GalNAz-labeled adenovirus appears to be labeled only on the Fiber capsid protein. 
 <table border="1">
<tbody>
 <tr>
 <td>Solution</td>
 <td>Preparation</td>
 <td>Notes</td>
 </tr>
 <tr>
 <td>Ad5 storage buffer</td>
 <td>5 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM MgCl2, 50 mM NaCl, 25% (v/v) glycerol, and 0.05% (w/v) Bovine Serum Albumin (BSA)</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Ad5 infection buffer</td>
 <td>2% (v/v) bovine calf serum, 1&times; TC solution, Ad5 in storage buffer (volume determined using an MOI of 10 PFU / cell and an infectivity index of 1:20). Bring the solution to the final volume using TD buffer</td>
 <td>Ad5 concentration may be determined via UV absorption.</td>
 </tr>
 <tr>
 <td>TC solution (200&times;)</td>
 <td>136 mM CaCl2, 100 mM MgCl2</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TD buffer</td>
 <td>137 mM NaCl, 5 mM KCl, 0.07 mM Na2HPO4, and 25 mM Tris-HCl, pH 7.5</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>HEK 293 cell growth medium</td>
 <td>Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) bovine calf serum (BCS) and 1% (v/v) Pen Strep</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Cys stock solution (100&times;)</td>
 <td>DMEM (-Met / -Cys) supplemented with 200 mM L-Cysteine</td>
 <td>Prepare fresh and filter with a 0.2-&mu;m sterile syringe filter before use.</td>
 </tr>
 <tr>
 <td>Aha stock solution (25&times;)</td>
 <td>DMEM (-Met / -Cys) supplemented with 100 mM L-Azidohomoalanine</td>
 <td>Prepare fresh and filter with a 0.2-&mu;m sterile syringe filter before use.</td>
 </tr>
 <tr>
 <td>Aha labeling medium</td>
 <td>DMEM (-Met / -Cys) supplemented with 10% BCS, Aha stock solution (1&times;), 2 mM L-Cysteine</td>
 <td>Prepare fresh before use. This corresponds to a 4 mM concentration of Aha, although different concentrations may be used (see Representative Results).</td>
 </tr>
 <tr>
 <td>Met stock solution (25&times;)</td>
 <td>DMEM (-Met / -Cys) supplemented with 100 mM L-Methionine</td>
 <td>Prepare fresh and filter with a 0.2-&mu;m sterile syringe filter before use.</td>
 </tr>
 <tr>
 <td>Met control medium</td>
 <td>DMEM (-Met / -Cys) supplemented with 10% BCS, Met stock solution (1&times;), 2 mM L-Cysteine</td>
 <td>Prepare fresh before use.</td>
 </tr>
 <tr>
 <td>Ac4GalNAz stock solution (1,000&times;)</td>
 <td>50 mM peracetylated N-azidoacetylgalactosamine (Ac4GalNAz) in methanol</td>
 <td>Store at 4 &deg;C. Ac4GalNAz is a metabolic precursor to GlcNAz.</td>
 </tr>
 <tr>
 <td>Azido-sugar labeling medium</td>
 <td>100 &mu;l Ac4GalNAz stock solution, 100 ml HEK 293 cell growth medium</td>
 <td>Allow methanol to evaporate prior to adding HEK 293 cell growth medium.</td>
 </tr>
 <tr>
 <td>Cesium chloride solutions</td>
 <td>CsCl dissolved in TD buffer: 
 1.25 g/ml (18.08 g / 50 ml H2O) 
 1.35 g/ml (25.60 g / 50 ml H2O) 
 1.40 g/ml (31.00 g / 50 ml H2O)</td>
 <td>Density gradients for ultracentrifugation</td>
 </tr>
 <tr>
 <td>Virus storage buffer</td>
 <td>Phosphate buffered saline (PBS), pH 7.2, containing 0.5 mM CaCl2, 0.9 mM MgCl2, and 10% (v/v) glycerol</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>TAMRA-BCN (SPAAC) labeling solution</td>
 <td>TAMRA-BCN in DMSO:
 <ol>
 <li> Use UV absorbance (OD260 Assay) to determine the titer of the virus solution in step 4.1 </li>
 <li> Determine the total number of viral particles in the 50 &mu;l aliquot (NAd5) </li>
 <li> The TAMRA-BCN molarity (in M) is determined using: 
 cBCN = 4 &times; 108 &times; (NAd5 / NAvo) 
 NAvo = 6.022 &times; 1023 
 (so that the reaction mixture prepared in Step 4 contains 100 alkyne probes per virion)</li>
 </ol></td>
 <td>Strain-promoted alkyne probe For SDS-PAGE, a viral titer of approximately 1012 particles / ml is suggested</td>
 </tr>
 <tr>
 <td>TAMRA-Alkyne (CuAAC) labeling solution</td>
 <td>50 &mu;l of azide-modified Ad5 in virus storage buffer (NAd5 determined as above), 1.5 mM bathophenanthroline disulfonate (i.e. 1.5 &times; final CuBr concentration), TAMRA-Alkyne. 
 TAMRA-Alkyne molarity (in M) is determined using: 
 cAlk = 2 &times; 106 &times; (NAd5 / NAvo) 
 (so that the reaction mixture prepared in Step 5 contains 100 alkyne probes per virion)</td>
 <td>Tris buffer is known to be a competitive and inhibitory ligand of copper.8</td>
 </tr>
 <tr>
 <td>Laemmli sample buffer (2&times;)</td>
 <td>125 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, and 0.004% (w/v) bromophenol blue</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Running buffer</td>
 <td>187 mM glycine, 19 mM Tris-HCl, 3.5 mM SDS in H2O</td>
 <td>Store at 4 &deg;C</td>
 </tr>
</tbody>
 </table>
Table 1. Preparation of the buffers and solutions required by this protocol.

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No conflicts of interest declared.

The development of chemoselective and bioorthogonal click reactions, including those of azides, is a rapidly-evolving area of research, and subsequently there is a growing number of these reactions to choose from for bioconjugation applications. We have restricted the scope of this protocol to include only two methods which were chosen because of their usefulness in our own lab and commercial availability of all reagents.
In the first such route - strain-promoted azide-alkyne cycloaddition (SP...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Adenovirus type 5 (Ad5) containing a GFP transgene</td>
 <td>BCBC</td>
 <td>391</td>
 </tr>
 <tr>
 <td>Human Embryonic Kidney (HEK 293) cells</td>
 <td>ATCC</td>
 <td>CRL-1573</td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium (DMEM), High Glucose</td>
 <td>Invitrogen</td>
 <td>11965-092</td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium, no Methionine, no Cysteine (DMEM -Met / -Cys), High Glucose</td>
 <td>Invitrogen</td>
 <td>21013-024</td>
 </tr>
 <tr>
 <td>Bovine Calf Serum</td>
 <td>Invitrogen</td>
 <td>16170-078</td>
 </tr>
 <tr>
 <td>Penicillin - Streptomycin 100&times; Solution (Pen Strep)</td>
 <td>Invitrogen</td>
 <td>15140-122</td>
 </tr>
 <tr>
 <td>0.5% Trypsin-EDTA (10&times;)</td>
 <td>Invitrogen</td>
 <td>15400-054</td>
 </tr>
 <tr>
 <td>100 mm Cell Culture Dish, tissue-culture treated polystyrene</td>
 <td>BD Falcon</td>
 <td>353003</td>
 </tr>
 <tr>
 <td>Cell culture CO2 incubator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biosafety cabinet</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile syringe filter (0.2 &mu;m, cellulose acetate) </td>
 <td>VWR</td>
 <td>28145-477 (NA), 514-0061 (Europe)</td>
 </tr>
 <tr>
 <td>Avanti J-E centrifuge</td>
 <td>Beckman Coulter</td>
 <td>369001</td>
 </tr>
 <tr>
 <td>Conical tubes (50 ml, sterile)</td>
 <td>BD Falcon</td>
 <td>352098</td>
 </tr>
 <tr>
 <td>Tube, Thinwall, Ultra-Clear, 13.2 ml, 14 x 89 mm</td>
 <td>Beckman</td>
 <td>344059</td>
 </tr>
 <tr>
 <td>Ultracentrifuge equipped with an SW 41 and SW 60 rotor</td>
 <td>Beckman</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Eppendorf Biopur Safe-Lock Tubes, 1.5 ml</td>
 <td>Eppendorf</td>
 <td>0030 121.589</td>
 </tr>
 <tr>
 <td>Centrifuge 5418</td>
 <td>Eppendorf</td>
 <td>5418</td>
 </tr>
 <tr>
 <td>Centri-Sep gel filtration spin columns</td>
 <td>Princeton Separations</td>
 <td>CS-901</td>
 </tr>
 <tr>
 <td>Sterile needle, 18 gauge</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nitrogen glove bag (if deoxygenated CuAAC is to be performed)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dewar flask</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Liquid nitrogen</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrophoresis cell</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluorescent gel scanner</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ready Gel Tris-HCl Gel</td>
 <td>Bio-Rad</td>
 <td>161-1105</td>
 </tr>
 <tr>
 <td>L-Azidohomoalanine</td>
 <td>AnaSpec</td>
 <td>63669</td>
 </tr>
 <tr>
 <td>Jena Biosciences</td>
 <td>CLK-AA005</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Peracetylated N-azidoacetylgalactosamine (Ac4GalNAz)</td>
 <td>Invitrogen</td>
 <td>C33365</td>
 </tr>
 <tr>
 <td>Thermo Scientific</td>
 <td>88905</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sigma-Aldrich</td>
 <td>A7480</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bathophenanthroline disulfonic acid (BDA) disodium salt</td>
 <td>MP Biomedicals</td>
 <td>0215011201</td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide (DMSO)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris base (tris(hydroxymethyl)aminomethane)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disodium Phosphate (Na2HPO4)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1M HCl</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-cysteine</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-methionine</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SDS (sodium dodecyl sulfate)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-mercaptoethanol</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bromophenol blue</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cesium Chloride (CsCl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Copper(I) Bromide (CuBr)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Calcium Chloride (CaCl2)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium Chloride (KCl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnesium Chloride (MgCl2)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Chloride (NaCl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="3">&nbsp;</td>
 </tr>
 <tr>
 <td>Alkyne probe for CuAAC*</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TAMRA Alkyne</td>
 <td>Invitrogen</td>
 <td>T10183</td>
 </tr>
 <tr>
 <td colspan="3">&nbsp;</td>
 </tr>
 <tr>
 <td>Strained alkyne probe for SPAAC*</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TAMRA DIBO Alkyne</td>
 <td>Invitrogen</td>
 <td>C10410</td>
 </tr>
</tbody>
</table>
* Notable vendors of click chemistry reagents and kits include Invitrogen, Jena Biosciences, Berry Associates, Sigma-Aldrich, Glen Research, Click Chemistry Tools, and Baseclick. A variety of alkyne dyes and targeting ligands can be found in these vendors' catalogs.

chemoselective modification of viral surfaces via bioorthogonal click chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

chemoselective-modification-viral-surfaces-via-bioorthogonal-click

Research

JoVE Journal

Immunology and Infection

24.0K Görüntüleme.  Stony Brook University. Adenovirüs parçacıklar doğal olmayan bir amino asit analog azidohomoalanine veya azido şeker içermesi için tasarlanmıştır O-GlcNAz. Her bir azid grubu chemoselectively viral yüzey değiştirme aracı olarak "ı" kimyası reaksiyon yoluyla ligate edilir.

Video: Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

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

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

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

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

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...