This work was supported in whole or in part by the National Institutes of Health (R01 AI121315, R01 AI146017, R01 AI148382, R01 AI120860, R37 AI076119, and U54 AI150472). We thank Dr. Raymond F. Schinazi and Sadie Amichai for providing cells infected with influenza A Virus.

1. Seed cells (Suspension vs. Adherent Cells) on coverslips or chamber slides (protocol presented uses coverslips).
<ol>
	<li>Seeding of suspension cells
	<ol>
		<li>Prepare poly-D-lysine (PDL) coated coverslips (facilitates adherence of suspension cells to coverslip) by first incubating coverslips in ethanol (EtOH) for 5 minutes (sterilizes coverslips and removes any residue). Then wash 2x in phosphate buffered saline (PBS) before incubating coverslips in PDL (20 &#181;g/mL) for 30 minutes (min) at room temperature (RT).</li>
		<li>Remove PDL and wash 2x in PBS. Pellet cells (previously infected/treated based on desired imaging conditions) and resuspend 106cells in 50 &#181;L of PBS. Spot 50 &#181;L of cells on glass (PDL-coated) and incubate at RT for 30 min.</li>
	</ol>
	</li>
	<li>Seeding of adherent cells
	<ol>
		<li>Culture cells on sterile coverslip placed in 6-well dish and infect cells with viral particles or treat with compound of interest. Allow cells to reach 50-70% confluency prior to sample preparation for imaging experiments.</li>
	</ol>
	</li>
</ol>
2. Cellular fixation: To preserve cellular morphology for fluorescence imaging studies
NOTE: Keep 4% PFA and PBS at RT for at least 30 minutes before cellular fixation.
<ol>
	<li>Aspirate the cellular media, wash cells on coverslips 3x in PBS, and fix cells in 4% paraformaldehyde (PFA) for 30 min. Aspirate PFA and wash cells in PBS 2x. 
	NOTE: Cells can now be dehydrated and stored, if the experimenter wishes to resume the experiment on another date. Fixed cells can be dehydrated in EtOH prior to storage.
	<ol>
		<li>Remove PBS following wash after cellular fixation and replace with 500 &#181;L of 50% EtOH (v/v in water). Incubate at RT for 5 min.</li>
		<li>Remove 50% EtOH and replace with 500 &#181;L of 70% EtOH. Incubate at RT for 5 min.</li>
		<li>Remove 70% EtOH and replace with 500 &#181;L of 100% EtOH. Incubate at RT for 5 min.</li>
		<li>Remove 100% EtOH and replace with fresh 100% EtOH. 
		NOTE: Dehydrated cells can be stored at -20 &#176;C for 6 months. Seal plates with tape or parafilm prior to storage to prevent evaporation of EtOH.</li>
		<li>Rehydrate cells to move onto multiplexed fluorescence labeling of cells/virus.</li>
		<li>Remove 100% EtOH and replace with 500 &#181;L of 70% EtOH. Incubate at RT for 2 min. 
		NOTE: Do not let cells dry out at any time. Always use enough solution to submerge all the cells.</li>
		<li>Remove 70% EtOH and replace with 500 &#181;L of 50% EtOH. Incubate at RT for 2 min.</li>
		<li>Remove 50% EtOH and replace with 1x PBS. 
		NOTE: Prepare protease dilution, hybridization probes, and wash buffer in advance of protease treatment (Step 4) and target probe application (Step 5). Specific instructions on reagent preparation are presented at the beginning of each respective step.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Other Notes</td>
		</tr>
		<tr>
			<td>Protease solution</td>
			<td>Prepare in 1X PBS</td>
		</tr>
		<tr>
			<td>Target oligonucletodie probe(s)</td>
			<td>Dilute in hybridization buffer</td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>Washes following target hybridization steps</td>
		</tr>
		<tr>
			<td>Hybridization buffer</td>
			<td>Recipe presented in Table 3</td>
		</tr>
	</tbody>
</table>
Table 1: Key reagents in MICDDRP protocol.
3. Cell permeabilization: Increases access into the cell and cellular organelles for entry of large molecules (antibodies, nucleic acid hybridization probes)
<ol>
	<li>Remove 1x PBS following cellular fixation or rehydration and replace with 500 &#181;L of 0.1% (v/v) Tween-20 in 1x PBS. Incubate at RT for 10 min. Replace one by one. Wash once with 500 &#181;L of 1x PBS and add fresh 1x PBS.</li>
</ol>
4. Coverslip immobilization on glass slide and protease treatment to remove nucleic acid binding proteins from fixed viral/cellular nucleic acid to improve hybridization efficiency
NOTE: Prepare the diluted Protease III (see specifics of reagent in Table of Materials) solution during cellular permeabilization. Let protease reach RT for 10 min before permeabilization.
<ol>
	<li>Place a small drop of nail polish on a sterilized glass slide. Dry back (side with no cell layer) and place the edge of coverslip on the nail polish drop, with the side with the adhered cells facing upwards. Add few drops of PBS on the immobilized coverslip to prevent drying.
	<ol>
		<li>Draw a circle (about 3 mm away from the coverslip) around the perimeter of the coverslip now adhered to the slide using hydrophobic barrier pen (water-repellant pen that keeps reagents localized on cells).</li>
	</ol>
	</li>
	<li>Dilute Protease III in 1x PBS (100 &#181;L/coverslip). 
	NOTE: Protease concentration may need to be adjusted depending on differences in cell types, probes, or target nucleic acid(s) through empirical optimization (See Discussion). For most efficient RNA/DNA labeling across different viral systems, we had success with the following dilutions presented in Table 2 below with dilutions ranging from (1 to 2)-(1 to 15) (protease to 1x PBS).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Probe Targets</td>
			<td>Protease Dilution in 1X PBS (Protease III to 1X PBS)</td>
		</tr>
		<tr>
			<td>HIV-1 DNA/RNA</td>
			<td>1 to 5</td>
		</tr>
		<tr>
			<td>HTLV-1 DNA/RNA</td>
			<td>1 to 5</td>
		</tr>
		<tr>
			<td>HBV pgRNA and total HBV RNA</td>
			<td>1 to 15</td>
		</tr>
		<tr>
			<td>IAV RNA</td>
			<td>1 to 15</td>
		</tr>
		<tr>
			<td>ZIKV RNA</td>
			<td>1 to 2</td>
		</tr>
	</tbody>
</table>
Table 2: Protease III dilutions in PBS for viral nucleic acid hybridization.
<ol start="3">
	<li>Decant the 1x PBS on the coverslip following immobilization and apply the diluted Protease III.</li>
	<li>Incubate in a humidified oven at 40 &#176;C for 15 min.</li>
	<li>Decant protease solution and submerge slides in 1x PBS. Agitate with a rocking dish for 2 min at RT. Repeat wash with new 1x PBS.
	<ol>
		<li>For only DNA detection, wash samples three times with nuclease-free water for 2 min each, followed by incubation with 5 mg/mL RNase A diluted in PBS for 30 min at 37 &#176;C.</li>
		<li>Decant RNase A solution, and wash 3x for 2 min with ultrapure water. Continue with ISH of target probe(s). 
		NOTE: Hybridization buffer improves vDNA detection without affecting vRNA staining efficiency for the results presented (Representative Results). Dilute DNA Channel 1 (C1) probes 1:1 with hybridization probe. Dilute RNA C2 and C3 probes in hybridization buffer. The C2 and C3 probes used in our imaging studies are in 50X solutions (1:50; target probe to hybridization buffer).</li>
	</ol>
	</li>
	<li>Prepare hybridization buffer in nuclease-free water following the step-by-step procedure below:
	<ol>
		<li>In a 15 mL tube, add 700 &#181;L of nuclease free-water, 300 &#181;L of 50% (weight/volume (w/v)) dextran sulfate, 300 &#181;L of 5 M NaCl, 125 &#181;L of 200 mM sodium citrate (pH 6.2), and 375 mg (powder) of ethylene carbonate.</li>
		<li>Mix well using a vortex to dissolve ethylene carbonate (ensure all powder is dissolved and clear solution).</li>
		<li>Add 25 &#181;L of 10% (volume/volume (v/v)) Tween-20 and enough nuclease-free water to complete 2.5 mL (2x solution). 
		NOTE: The recipe for the hybridization buffer presented can be found in Table 3 below. Solution is stable for a week. Ensure sufficient mixing of Tween-20 detergent, while being careful to prevent bubbling of solution.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock Concentration</td>
			<td>Additional Notes</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>50% (w/v)</td>
			<td>Very viscous</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>5 M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate (pH 6.2)</td>
			<td>200 mM</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Ethylene carbonate</td>
			<td>NA</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>10% (v/v)</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3: Hybridization buffer list of reagents.
5. Incubation with DNA/RNA target hybridization probes: Target oligonucleotide probes bind to region(s) of interest, creating a molecular scaffold for pre-amplifiers, amplifiers, and fluorescent probes to bind.
NOTE: Warm DNA/RNA probes at 40 &#176;C for 10 min (during Cell Permeabilization) and cool down to RT for at least 10 min, if no RNase-treatment is included. If RNase-treatment or further sample treatment is needed prior to target probe hybridization, warm probes accordingly. Spin down C2 and C3 probes after warming and dilute in hybridization buffer. After dilution, C2 and C3 probes can be briefly warmed. Warm 50x wash buffer (See Table of Materials for more detail) at 40 &#176;C for 10-20 min and dilute to 1x in molecular biology grade water.
<ol>
	<li>Incubate 200 &#181;L of the hybridization buffer at 67 &#176;C for 10 min prior to addition of hybridization probe(s). Dilute probe in hybridization buffer (recipe listed in Table 2). Add 50 &#181;L/coverslip.</li>
	<li>Incubate in humidified oven at 40 &#176;C for 2 hours (h). Decant probes and submerge slides in 1x wash buffer. Agitate by rocking dish for 2 min at RT. Repeat wash with new 1x wash buffer.</li>
</ol>
6. Amplifier (Amp) 1-FL Hybridization: Addition of pre-amplifier that is complementary to the tail sequence of the target DNA/RNA probes (Step 5)
NOTE: Amplifiers should be at RT before use. Get each individual amplifier out of the fridge 30 min before use and leave on the bench at RT.
<ol>
	<li>Remove slides from 1x wash buffer and tap/absorb to remove excess liquid.</li>
	<li>Add 1 drop of Amp 1-FL on the coverslip. Incubate in humidified oven at 40 &#176;C for 30 min.</li>
	<li>Decant Amp 1-FL and submerge in 1x wash buffer. Agitate by rocking dish 2 min at RT. Repeat wash with new 1x wash buffer.</li>
</ol>
7. Amp 2-FL Hybridization: Incubation with signal amplifier with cognate recognition sequence to pre-amplifiers (Amp 1-FL)
<ol>
	<li>Remove slides from 1x wash buffer and tap/absorb to remove excess liquid.</li>
	<li>Add 1 drop of Amp 2-FL on the coverslip. Incubate in humidified oven at 40 &#176;C for 15 min.</li>
	<li>Decant Amp 2-FL and submerge in 1x wash buffer. Agitate by rocking dish 2 min at RT. Repeat wash with new 1x wash buffer.</li>
</ol>
8. Amp 3-FL Hybridization: Incubation with second signal amplifier
<ol>
	<li>Remove slides from 1x wash buffer and tap/absorb to remove excess liquid.</li>
	<li>Add 1 drop of Amp 3-FL on the coverslip. Incubate in humidified oven at 40 &#176;C for 30 min.</li>
	<li>Decant Amp 3-FL and submerge in 1x wash buffer. Agitate by rocking dish 2 min at RT. Repeat wash with new 1x wash buffer.</li>
</ol>
9. Amp 4-FL Hybridization: Fluorescent label and final hybridization step
NOTE: First, see Table 4 to choose the suitable Amp 4 (A,B, or C)- FL based on the channels of the target probe(s). Assess what Amp 4-FL is needed to label DNA/RNA of interest. An example is provided by the table below:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>A</td>
			<td>B</td>
			<td>C</td>
		</tr>
		<tr>
			<td>DNA Channel 1 (C1)</td>
			<td>488</td>
			<td>550</td>
			<td>550</td>
		</tr>
		<tr>
			<td>DNA/RNA Channel 2 (C2)</td>
			<td>550</td>
			<td>488</td>
			<td>647</td>
		</tr>
		<tr>
			<td>RNA Channel 3 (C3)</td>
			<td>647</td>
			<td>647</td>
			<td>488</td>
		</tr>
	</tbody>
</table>
Table 4: Selection of Amp 4 (A, B, or C)-FL fluorescent probe for multiplexed ISH.
NOTE: For multiplexed FISH (multiple targets), choosing the correct Amp 4 (A, B, or C)- FL is critical for properly labeling your target of interest(s). Target probes (Step 5) have different color channels (C1, C2, or C3), which dictate their respective fluorescent label (Alexa 488, Atto 550, or Alexa 647), based on the Amp 4-FL chosen. Examples of choosing fluorophore combinations for multiplexed imaging are provided in the legends of Figure 2 and Figure 3. As an additional example, selection of Amp 4B-FL will selectively label DNA C1 probes with Atto 550 and RNA C3 probes with Alexa 647.
<ol>
	<li>Remove slides from 1x wash buffer and tap/absorb to remove excess liquid.</li>
	<li>Add 1 drop of Amp 4-FL on the coverslip. Incubate in humidified oven at 40 &#176;C for 15 min. 
	NOTE: Following step 9.2, keep samples covered, protected from the light.</li>
	<li>Decant Amp 4-FL and submerge in 1x wash buffer. Agitate by rocking dish for 2 min at RT. Repeat wash with new 1x wash buffer. Wash with 1x PBS (2 min) and store in PBS.</li>
</ol>
10. Protein immunostaining: To label protein(s) of interest
<ol>
	<li>Decant PBS and add 200 &#181;L of blocking buffer (1% w/v BSA, 10% v/v FBS in PBS with 0.1% v/v Tween-20 (PBST)) to the coverslip. Incubate 1 h at RT.</li>
	<li>Decant blocking buffer and apply 200 &#181;L of primary antibody diluted in PBST + 1% w/v BSA. Incubate 1 h at RT.</li>
	<li>Wash the slide twice with PBST for 10 min at RT with shaking.</li>
	<li>Apply secondary antibody of choice for 1 h at RT in PBST + 1% w/v BSA.</li>
	<li>Wash the slide with PBST for 10 min at RT with shaking.</li>
</ol>
11. Nuclear staining: Counter-stain nuclei following immunostaining
<ol>
	<li>Decant PBST and apply DAPI or nuclear stain of choice for 1 min at RT.</li>
	<li>Wash the slide twice with PBS for 10 min at RT with shaking.</li>
</ol>
12. Mounting
<ol>
	<li>Place 1 drop of antifade solution (e.g., Prolong Gold) on new sterile glass slide (First, clean slide with EtOH and let dry to ensure no residues are on glass). With the same tip, spread the antifade solution drop to cover an area approximately the size of the coverslip. 
	NOTE: The antifade solution is very viscous and may be difficult to pipette. Cutting the tip off a 200 &#181;L tip prior to pipetting may mitigate these issues.</li>
	<li>Remove coverslip with cell sample from the slide and submerge in PBS to remove residual nail polish at the back using the forceps and PBS. Dry forceps and back of the coverslip using a Kimwipe.</li>
	<li>Gently imbed coverslip in the drop of antifade solution, placing sample side (side with cell layer) of coverslip on drop).</li>
	<li>Let samples dry overnight.</li>
</ol>
13. Imaging
<ol>
	<li>Image with an epifluorescent microscope.</li>
</ol>

<ol><li>Zheng, Q., Lavis, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+photostable+fluorophores+for+molecular+imaging%5BTitle%5D)+AND+%22Current+Opinion+in+Chemical+Biology%22%5BJournal%5D)">Development of photostable fluorophores for molecular imaging.</a> Current Opinion in Chemical Biology. 39, 32-38 (2017).</li>
<li>Marini, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+architecture+dictates+HIV-1+integration+site+selection%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Nuclear architecture dictates HIV-1 integration site selection.</a> Nature. 521 (7551), 227-231 (2015).</li>
<li>Puray-Chavez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+single-cell+visualization+of+nucleic+acids+and+protein+during+HIV+infection%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Multiplex single-cell visualization of nucleic acids and protein during HIV infection.</a> Nature Communications. 8 (1), 1882(2017).</li>
<li>Ukah, O. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+HIV-1+RNA+Transcription+from+Integrated+HIV-1+DNA+in+Reactivated+Latently+Infected+Cells%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Visualization of HIV-1 RNA Transcription from Integrated HIV-1 DNA in Reactivated Latently Infected Cells.</a> Viruses. 10 (10), (2018).</li>
<li>Liu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+Positive+and+Negative+Sense+Viral+RNA+for+Probing+the+Mechanism+of+Direct-Acting+Antivirals+against+Hepatitis+C+Virus%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus.</a> Viruses. 11 (11), (2019).</li>
<li>Achuthan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capsid-CPSF6+Interaction+Licenses+Nuclear+HIV-1+Trafficking+to+Sites+of+Viral+DNA+Integration%5BTitle%5D)+AND+%22Cell+Host+%26+Microbe%22%5BJournal%5D)">Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites of Viral DNA Integration.</a> Cell Host & Microbe. 24 (3), 392-404 (2018).</li>
<li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNAscope%3A+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed%2C+paraffin-embedded+tissues%5BTitle%5D)+AND+%22Journal+of+Molecular+Diagnostics%22%5BJournal%5D)">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> Journal of Molecular Diagnostics. 14 (1), 22-29 (2012).</li>
<li>Xia, C., Babcock, H. P., Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexed+detection+of+RNA+using+MERFISH+and+branched+DNA+amplification%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Multiplexed detection of RNA using MERFISH and branched DNA amplification.</a> Scientific Reports. 9 (1), 7721(2019).</li>
<li>Player, A. N., Shen, L. P., Kenny, D., Antao, V. P., Kolberg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-copy+gene+detection+using+branched+DNA+%28bDNA%29+in+situ+hybridization%5BTitle%5D)+AND+%22Journal+of+Histochemistry+and+Cytochemistry%22%5BJournal%5D)">Single-copy gene detection using branched DNA (bDNA) in situ hybridization.</a> Journal of Histochemistry and Cytochemistry. 49 (5), 603-612 (2001).</li>
<li>Battich, N., Stoeger, T., Pelkmans, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-based+transcriptomics+in+thousands+of+single+human+cells+at+single-molecule+resolution%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Image-based transcriptomics in thousands of single human cells at single-molecule resolution.</a> Nature Methods. 10 (11), 1127-1133 (2013).</li>
<li>Bushnell, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ProbeDesigner%3A+for+the+design+of+probesets+for+branched+DNA+%28bDNA%29+signal+amplification+assays%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.</a> Bioinformatics. 15 (5), 348-355 (1999).</li>
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<li>Brown, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+nuclear+proteins+together+with+transcribed+and+inactive+genes+in+structurally+preserved+cells%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Visualizing nuclear proteins together with transcribed and inactive genes in structurally preserved cells.</a> Methods. 26 (1), 10-18 (2002).</li>
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</ol>

The authors have nothing to disclose.

Simultaneous visualization of RNA, DNA, and protein often requires extensive optimization. Two commonly used methods are 5-ethynyl-2-deoxyuridine (EdU) labeling and DNA FISH. EdU labeling has been applied to visualize viral DNA and protein simultaneously, as EdU is incorporated in nascent DNA and subsequently labeled with azide-containing fluorescent dyes via click chemistry. EdU labeling can thus be used to monitor native virus replication kinetics of DNA viruses or viruses with DNA templates for replication12. A shortcoming of EdU labeling, however, is that in dividing cells, the replicating genome will incorporate EdU, generating high background and confounding image analysis. DNA FISH can circumvent these issues by directly hybridizing a nucleic acid probe to the respective target regardless of the cell cycle. However, conventional FISH often relied on high temperatures to achieve efficient probe hybridization, hindering immunostaining or even simultaneous RNA staining13. MICDDRP can potentially circumvent these issues providing robust simultaneous fluorescent labeling of DNA, RNA, and protein across a variety of cellular systems.
While we have demonstrated that we can label protein and nucleic acid simultaneously using our MICDDRP protocol, optimization was needed across different systems. The first major parameter that we had to optimize was protease treatment. We varied protease III concentration across the conditions. Optimization of protease treatment was empirical, as we used different dilutions to assess what yielded the greatest hybridization efficiency, without compromising immunostaining efficiency. Appropriate controls were performed side-by-side to assess probe specificity and changes to protein staining efficiency attributed to protease treatment. The next major parameters that needed optimization were probe design and probe hybridization.
Proper design of capture and amplifier probes are critical for achieving the sensitivity and specificity of bDNA technology. Software packages that predict the probability of non-specific hybridization events are available to improve probe design7,11. bDNA probes with the accompanying pre-amplifier, amplifier, and fluorescent label probes can now be commercially purchased to ensure compatibility with bDNA imaging kits. Users can supply manufacturers with sequence information (~300-1000 base pairs) for the target region(s) in the form of a fasta file (text-based format for representing nucleotide sequence). Target probes are generated with &#62; 90% sequence homology to the supplied sequence.
For DNA labeling, we have found that dilution of the probes in the hybridization buffer described in Step 5 of the Protocol improves DNA hybridization. When labeling both DNA and RNA, the RNA probe can be diluted in the hybridization buffer. DNA labeling in the absence of RNase cannot exclude the possibility that the observed nucleic acid includes RNA of the targeted strandedness. Temperature may also have to be adjusted for improving hybridization efficiency. Increasing temperature may affect protein staining efficiency, as increased temperatures may promote protein denaturation, ablating epitope recognition of the primary antibody. In our presented representative data, we have performed ISH at 40 &#176;C.
Compared to conventional DNA FISH, MICDDRP provides an improved procedure for simultaneously labeling DNA, RNA, and protein to visualize via fluorescence microscopy. A potential limitation is that the selection of probe may affect the efficiency of hybridization and ability to quantitatively compare data between probes. This protocol has been effective across diverse cellular and viral systems in our hands with only minor optimization needed across varying conditions. Recent high-profile publications have utilized our approach to study HIV integration site selection14 and HIV reverse transcription kinetics15. Future applications of MICDDRP could include visualization of viral nucleic acids concomitantly with nucleic acid sequences of specific cellular genes and cellular proteins.

While thousands of commercial antibodies are available to specifically label proteins via conventional immunostaining approaches, and while fusion proteins can be engineered with photo-optimized fluorescent tags for tracking multiple proteins in a sample1, microscopic visualization of protein is often not compatible with conventional DNA fluorescence in situ hybridization (FISH)2. Technical limitations in simultaneous visualization of DNA, RNA, and protein using fluorescence-based approaches have hindered in-depth understanding of virus replication. Tracking both viral nucleic acid and protein during the course of infection allows virologists to visualize fundamental processes that underly virus replication and assembly3,4,5,6.
We have developed an imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and Protein (MICDDRP)3, which utilizes branched DNA (bDNA) in situ technology to improve the sensitivity of nucleic acid detection7,8,9. In addition, this method utilizes paired probes for enhanced specificity. bDNA sequence-specific probes use branching preamplifier and amplifier DNAs to produce an intense and localized signal, improving upon previous hybridization methods that relied on targeting repeated regions in the DNA9. Infected cells in a clinical context often do not contain abundant viral genetic material, providing a commodity for a sensitive method for fluorescent nucleic acid detection in diagnostic settings. The commercialization of bDNA technology through approaches such as RNAscope7 and ViewRNA10 have filled this niche. The sensitivity of bDNA fluorescence imaging also has important utility in cell biology, allowing detection of scarce nucleic acid species in cell culture models. The vast improvement of sensitivity makes bDNA-based imaging methods suitable for studying viruses. A potential shortcoming, however, is that these methods focus on visualizing RNA or RNA and protein. All replicating cells and many viruses have DNA genomes or form DNA during their replication cycle, making methods capable of imaging both RNA and DNA, as well as protein, highly desirable.
In the MICDDRP protocol, we perform bDNA FISH for detection of viral nucleic acid using the RNAscope method, with modifications7. One of the major modifications to this protocol is optimization of protease treatment following chemical fixation. Protease treatment facilitates removal of proteins bound to nucleic acid to improve probe hybridization efficiency. Protease treatment is followed by incubation with branched oligonucleotide probe(s). After application of bDNA probe(s), samples are washed and subsequently incubated with signal pre-amplifier and amplifier DNAs. Multiplexed in situ hybridization (ISH), labeling of multiple gene targets, requires target probes with different color channels for spectral differentiation7. Incubation with DNA amplifiers is followed by immunofluorescence (IF).
bDNA ISH imparts improvements in signal-to-noise by amplification of target-specific signals, with a reduction to background noise from non-specific hybridization events7,11. Target probes are designed using software programs publicly available that predict the probability of non-specific hybridization events, as well as calculate melting temperature (Tm) of the probe-target hybrid7,11. Target probes contain an 18- to 25-base region complementary to the target DNA/RNA sequence, a spacer sequence, and a 14-base tail sequence. A pair of target probes, each with a distinct tail sequence, hybridize to a target region (spanning ~50 bases). The two tail sequences form a hybridization site for the pre-amplifier probes, which contain 20 binding sites for the amplifier probes, which, in addition contain 20 binding sites for the label probe. As an example, a one kilobase (kb) region on the nucleic acid molecule is targeted by 20 probe pairs, creating a molecular scaffold for sequential hybridization with the preamplifier, amplifier, and label probe. This can thus lead to a theoretical yield of 8000 fluorescent labels per nucleic acid molecule, enabling detection of single molecules and vast improvements over conventional FISH approaches7 (See Figure 1A for schematic of bDNA signal amplification). To set up probes for multiplexed ISH, each target probe must be in a different color channel (C1, C2, or C3). These target probes with different color channels possess distinct 14-base tail sequences. These tail sequences will bind distinct signal amplifiers with different fluorescent probes, thus enabling facile spectral differentiation across multiple targets. In the presented Protocol, Table 4 in Step 9, provides further information on fluorescently labeling target probes. In addition, Figure 2 and Figure 3 provide examples of how we chose the appropriate Amplifier 4-FL (A, B, or C) (fluorescent probe &#38; the final hybridization step) to achieve specific fluorescent labeling of multiple viral nucleic acid targets following HIV-1 and HTLV-1 infections.
We have demonstrated several applications of simultaneous fluorescence visualization of RNA, DNA, and proteins, observing critical stages of virus replication with high spatiotemporal resolution3,4,5. For example, simultaneous single-cell visualization of viral RNA, cytoplasmic and nuclear DNA, and protein have allowed us to visualize key events during HIV-1 infection, including following RNA containing cores in the cytoplasm prior to nuclear entry and integration of proviral DNA3. In addition, we have applied MICDDRP to characterize the effects of host factors and drug treatment on viral infection and replication4,5. In Ukah et al., we tracked reactivation of HIV-1 transcription in latency cell models treated with different latency-reversing agents to visualize HIV transcription and latency reversal4. In addition, MICDDRP can allow us to visualize phenotypic changes associated with antiviral inhibition attributed to small molecule treatment or host factor restriction. As a proof of concept to the robustness and broad applicability of our approach, we have demonstrated that we can use modifications of our protocol to efficiently label viral nucleic acid to follow infection not only in human immunodeficiency virus (HIV)-1, but also human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZIKV), and influenza A virus (IAV). As the HIV-1 life cycle consists of both viral DNA and RNA species, we have performed the majority of our optimization of MICDDRP following HIV-1 replication kinetics. However, in addition, we have demonstrated that we can track synthesis of different viral RNA transcripts of either or both sense (+) and antisense (-) strandedness in viruses such as ZIKV, IAV, HBV, and HCV to monitor viral transcription and replication3,4,5,6. The studies aim to improve the mechanistic understanding of several viral processes and serve as a guideline to implement this fluorescence imaging technology to a broad range of cellular models.

<table><tbody><tr><td>4% PFA</td><td></td><td></td><td></td></tr><tr><td>50% dextran sulfate</td><td>Amreso</td><td>198</td><td>For DNA hybridization buffer</td></tr><tr><td>50X wash buffer</td><td>ACD Bio</td><td>320058</td><td></td></tr><tr><td>6-well plates</td><td></td><td></td><td></td></tr><tr><td>Amplifier 1-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 2-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 3-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 4-FL</td><td>ACD Bio</td><td></td><td>Consult Amp-4 table in protocol</td></tr><tr><td>Anti-HCV NS5a antibody</td><td>Abcam</td><td>ab13833</td><td>Mouse monoclonal; works with HCV genotypes 1a, 1b, 3, and 4</td></tr><tr><td>Anti-HIV-1 p24 monoclonal antibody</td><td>NIH AIDS Reagent Program</td><td>3537</td><td></td></tr><tr><td>Anti-Mov10 antibody</td><td>Abcam</td><td>ab80613</td><td>Rabbit polyclonal</td></tr><tr><td>Anti-PB1 antibody</td><td>GeneTex</td><td>GTX125923</td><td>Antibody against flu protein</td></tr><tr><td>Bovine serum albumin</td><td></td><td></td><td>Blocking reagent for immunostaining</td></tr><tr><td>Cell media with supplements</td><td></td><td></td><td>Media appropriate for cell model</td></tr><tr><td>Coverslips</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>ACD Bio</td><td></td><td>Nuclear stain (RNAscope kit from ACD Bio)</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (1X PBS)</td><td>Gibco</td><td>14190250</td><td>No calcium and magnesium</td></tr><tr><td>Ethylene carbonate</td><td>Sigma</td><td>E26258</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td></td><td></td><td>Use specific FBS based on what serum secondary antibody was raised in (e.g goat FBS)</td></tr><tr><td>Fisherbrand colorfrost plus microscope slides</td><td>Fisher Scientific</td><td>12-550-17/18/19</td><td>Precleaned</td></tr><tr><td>HCV-GT2a-sense-C2 probe</td><td>ACD Bio</td><td>441371</td><td>HCV(+) sense RNA probe</td></tr><tr><td>HIV-gagpol-C1</td><td>ACD Bio</td><td>317701</td><td>HIV-1 cDNA probe</td></tr><tr><td>HIV-nongagpol-C3</td><td>ACD Bio</td><td>317711-C</td><td>HIV-1 RNA probe</td></tr><tr><td>HybEZ hybridization oven</td><td>ACD Bio</td><td>321710/321720</td><td></td></tr><tr><td>ImmEdge hydrophobic barrier pen</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>Nail polish</td><td></td><td></td><td>For immobolizing coverslip to slide prior to protease treatment</td></tr><tr><td>Nuclease free water</td><td>Ambion</td><td>AM9937</td><td></td></tr><tr><td>Poly-d-lysine (PDL)</td><td></td><td></td><td>Coat coverslips in 20 &amp;micro;g/mL of PDL for 30 minutes</td></tr><tr><td>Probe diluent</td><td>ACD Bio</td><td>300041</td><td>For diluting RNA C2 or C3 probes</td></tr><tr><td>Prolong gold antifade</td><td>Invitrogen</td><td>P36930</td><td></td></tr><tr><td>Protease III</td><td>ACD Bio</td><td>322337</td><td></td></tr><tr><td>RNAscope&amp;reg;&amp;nbsp;Probe- V-Influenza-H1N1-H5N1-NP</td><td>ACD Bio</td><td>436221</td><td></td></tr><tr><td>RNase A</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Secondary antibodies</td><td></td><td></td><td></td></tr><tr><td>Slides</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td></td><td></td><td>For DNA hybridization buffer</td></tr><tr><td>Sodium citrate, pH 6.2</td><td></td><td></td><td>For DNA hybridization buffer</td></tr><tr><td>Tween-20</td><td></td><td></td><td>For DNA hybridization buffer and PBS-T</td></tr><tr><td>V-HBV-GTD</td><td>ACD Bio</td><td>441351</td><td>Total HBV RNA</td></tr><tr><td>V-HBV-GTD-01-C2</td><td>ACD Bio</td><td>465531-C2</td><td>HBV pgRNA probe</td></tr><tr><td>V-HCV-GT2a probe</td><td>ACD Bio</td><td>441361</td><td>HCV(-) sense RNA probe</td></tr><tr><td>V-HTLV-HBZ-sense-C3</td><td>ACD Bio</td><td>495071-C3</td><td>HTLV-1 (-) sense RNA probe targetting HBZ</td></tr><tr><td>V-HTLV1-GAG-C2</td><td>ACD Bio</td><td>495051-C2</td><td>HTLV-1 DNA probe</td></tr><tr><td>V-HTLV1-GAG-POL-sense</td><td>ACD Bio</td><td>495061</td><td>HTLV-1 (+) sense RNA probe</td></tr><tr><td>V-Influenza-H1N1-H5N1-NP</td><td>ACD Bio</td><td>436221</td><td>IAV RNA probe</td></tr><tr><td>V-ZIKA-pp-O2</td><td>ACD Bio</td><td>464531</td><td>Zika(+) sense RNA probe</td></tr><tr><td>V-ZIKA-pp-O2-sense-C2</td><td>ACD Bio</td><td>478731-C2</td><td>Zika(-) sense RNA probe</td></tr></tbody></table>

single-cell multiplexed fluorescence imaging to visualize viral nucleic acids and proteins and monitor hiv, htlv, hbv, hcv, zika virus, and influenza infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

A schematic of MICDDRP is depicted in Figure 1. Labeling of DNA and RNA is followed by immunostaining. The use of branching amplifiers increases signal, allowing detection of single nucleic acid molecules.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61843/61843fig1v5.jpg" /> 
Figure 1: Schematic of MICDDRP and step-by-step workflow.&#160;(A) bDNA signal is amplified via branching preamplifier and amplifier DNAs to enhance detection of viral DNA (1) and RNA species (2). Oligonucleotide probes are hybridized in pairs (ZZ in schematic) to target(s) of interest, creating a scaffold for pre-amplifier (Amp 1-FL, Step 6 in Protocol), amplifier (Amp 2- &#38; 3-FL), and fluorescent probes (Amp 4, Step 9 in Protocol). Consult Table 4 for choosing appropriate Amp 4 (A,B, or C)-FL for multiplexed ISH. Labeling of nucleic acid is followed by immunostaining proteins of interest (3). (B) Thirteen main steps in MICDDRP protocol with estimation of time duration for each respective step. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Application of MICDDRP to study the course of HIV-1 infection has been a useful tool in tracking viral replication kinetics in primary cells. As a proof of concept of this procedure, HIV-1 DNA, RNA, and protein are simultaneously labeled and visualized microscopically at the single-cell level (Figure 2). Two HIV-1 DNA genomes are visualized in a single cell, as they are actively transcribing viral RNA (vRNA). vRNA has been exported through the nuclear pore complex and viral protein is synthesized in the cytoplasm.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61843/61843fig02.jpg" /> 
Figure 2: MICDDRP of primary blood mononuclear cells (PMBCs) infected with HIV-1.&#160;PMBCs infected with HIV-1 (NL4.3) at a multiplicity of infection (MOI) of 2. Cells were fixed 48 hours post infection (hpi). (A) HIV-1 bDNA FISH probe (labeled with ATTO 550; red in Figure). This probe hybridizes to the template (3&#8217;&#60;&#8212;5&#8217;) vDNA strand to prevent crosstalk with sense (+) vRNA. (B) Unspliced HIV-1 RNA (labeled with Alexa 647; green in Figure). The HIV-1 vRNA probe hybridizes to viral transcripts transcribed in the 5&#8217;&#8212;&#62;3&#8217; orientation. (C) Immunostaining of HIV-1 capsid (p24) protein (secondary antibody conjugated to Alexa 488; white in figure). (D) Merged image. Scale bar represents 5 &#181;m. Nuclei were stained with DAPI (blue). To label HIV-1 DNA (DNA C1 probe) with ATTO 550 and HIV-1 RNA (RNA C3 probe) with Alexa 647, respectively, samples were incubated with Amp 4-FL &#8216;B&#8217; (Consult Table 4 in Protocol to choose appropriate channel colors for hybridization probes). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In addition, we have performed dual viral DNA (vDNA) and vRNA staining to follow HTLV-1 infection. For optimization of the nucleic acid labeling, we adhered closely to the vDNA/VRNA staining procedure developed for multiplexed fluorescence imaging of HIV infection. We have demonstrated that we can specifically label HTLV-1 DNA and RNA simultaneously.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61843/61843fig3v2.jpg" /> 
Figure 3: Simultaneous labeling of HTLV-1 vDNA and vRNA in MT-2 cells.&#160;(A) HTLV-1 vDNA (labeled with ATTO 550; red in Figure) (B) Unspliced HTLV-1 sense (+) RNA (RNA C2 probe &#38; labeled with Alexa 488; green in Figure)). (C) HTLV-1 HBZ antisense (-) vRNA (RNA C3 probe &#38; (labeled with Alexa 647; white in Figure)). (D) Merged image. Scale bar represents 20 &#181;m. Nuclei were stained with DAPI (blue). Amp 4-FL &#8216;B&#8217; was used (Consult Table 4 in Protocol) to achieve multiplexed labeling of HTLV-1 (&#8216;+&#8217; &#38; &#8216;-&#8216;) RNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61843/61843fig04.jpg" /> 
Figure 4: Control experiments to assess target probe specificity and background.&#160;Specificity of HIV-1 and HTLV-1 target probes were assessed following infection in lymphocytic cell lines (Uninfected Jurkat T cells, HTLV-1 infected cells (MT2), and HIV-1 infected cells (H9111B)). Scale bars represent 10 &#181;m. (A) Cells were treated with HTLV-1 RNA probes. (B) Cells were treated with HIV-1 (+) RNA probe (C-D). Further demonstration of the specificity of HTLV-1 probes with no cross-reactivity with HIV-1. White arrows in Figure 4C denote HTLV-1 DNA (red). (E-F). vRNA staining (green) +/- RNase-treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To verify the specificity of the hybridization probes and to assess how the ISH labeling method impacts protein staining efficiency and overall background, we perform critical controls to ensure the highest level of rigor and reproducibility for the experiments. As an example, in Figure 4A-D, we verify the specificity of our HIV-1 and HTLV-1 vDNA and vRNA probes, as we show very little to no cross-reactivity between the probe sets across the two viruses. Despite labeling of two retroviruses with the potential for probe cross-reactivity, the HIV-1 probes are only specific to HIV-1 genetic material and not HTLV-1. The same trend is true for the HTLV-1 probes. In addition, in Figure 4F, we show that we can eliminate vRNA staining if we RNase-treat our cells during sample preparation. To assess the possibility of attenuation of protein staining efficiency due to ISH (protease treatment (Step 4 in Protocol &#38; Figure 1B) and hybridization conditions, which can lead to ablation of epitope recognition or increased background signal, we demonstrate that protein staining efficiency for the nuclear speckle marker, sc-35, is comparable across conventional IF approaches and immunostaining during the MICDDRP protocol. In the conventional IF protocol, cells were permeabilized with 0.1% Triton X-100 for 15 minutes, rather than permeabilization with 0.1% Tween-20 for 10 minutes, which is used in MICDDRP protocol (Step 3 in Protocol &#38; workflow in Figure 1B). Protein staining across both conditions (IF vs. MICDDRP) produced a signal several orders of magnitude greater than the control (MICDDRP with no primary antibody), further demonstrating the low background generated following this protocol and preservation of protein epitopes for efficient immunostaining. Image quantification of mean integrated fluorescence intensity of sc-35 signal per cell (Figure 5E) was performed as previously described3. For all imaging results shown, we ensured probe and antibody specificity, as well as assessed any possible perturbations or higher than normal background noise attributed to our ISH approach.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61843/61843fig05v2.jpg" /> 
Figure 5: Comparison of protein staining efficiency following conventional IF vs. MICDDRP.&#160;The cellular protein, sc-35, a biomarker for nuclear speckles, was immunostained in Jurkat T cells. All scale bars represent 10 &#181;m. (A) Uninfected Jurkat T cells underwent the MICDDRP protocol and were treated with HIV-1 DNA/RNA hybridization probes used in Figure 2 &#38; Figure 4 above. During immunostaining, no primary antibody was added. (B). Uninfected Jurkat T cells underwent conventional IF, labeling sc-35 (white). Cells were permeabilized with 0.1% Triton X-100 for 15 minutes. (C). MICDDRP was performed on HIV-infected Jurkat T cells. vRNA is labeled green, vDNA is red, and sc-35 is white. (D). Close-up of vRNA, vDNA, and protein labeling (sc-35) in HIV-infected cell. (E) Quantification of mean integrated fluorescence signal per cell of sc-35 across different immunostaining conditions. The y-axis is on a logarithmic scale. The dotted line represents the background signal from uninfected cells that underwent the MICDDRP protocol where no primary antibody was added. No significant difference in signal following MICDDRP vs. conventional IF. Over 500 cells were sampled for quantification for reach respective condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig05v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This method can also be applied to study RNA viruses that may or may not include DNA templates for viral replication. For instance, strand-specific bDNA probes can be designed to monitor expression of sense (+) or antisense (-) strand RNA and different vRNA species in viruses such as HBV, HCV, IAV, and ZIKV. The visualization of different RNA species during the course of infection can provide insight into the replication kinetics of various viral systems.
Following the time course of HBV infection, we can see that the amount of HBV pre-genomic RNA (pgRNA) and total HBV RNA increases as a function of time. In addition, we simultaneously immunostained a cellular host factor, MOV10 (Figure 6).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61843/61843fig06.jpg" /> 
Figure 6: HBV. Time course of HBV infection of 3E8 cells.&#160;Cells were infected with 300 HBV genomes per cell. Viral replication is shown at three time points (24, 48, and 72 hpi). pgRNA (labeled with ATTO 550; red in Figure), total HBV RNA (labeled with Alexa 647; green in Figure), and MOV10 (secondary antibody conjugated to Alexa 488; white in Figure). Nuclei are stained with DAPI in blue. Scale bar on merged images represent 10 &#181;m. hpi, hours post-infection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Imaging was performed via confocal microscopy using a 60x oil-immersion objective. The excitation/emission bandpass wavelengths used to detect DAPI, Alexa 488, ATTO 550, and Alexa 647 were set to 405/420-480, 488/505-550, 550/560-610, and 647/655-705 nm, respectively (Figure 7, Figure 8 and Figure 9).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61843/61843fig7v2.jpg" /> 
Figure 7: Time course of HCV infection of Huh-7.5.1 cells.&#160;Huh-7.5.1 cells were infected with hepatitis C virus (HCV) Jc1/Gluc2A at an MOI of 0.5. At the time intervals indicated, the cells were fixed and probed sequentially for sense (+) vRNA, antisense (-) vRNA and NS5A (HCV protein). Nuclei were stained with DAPI. Representative merged images from each time-point, showing (+) RNA in green (labeled with Alexa 647) (-) RNA in red (labeled with Atto 555), NS5A in white (secondary goat anti-mouse conjugated to Alexa 488), and nuclei in blue. Scale bars represent 10 &#181;m. The lower images are enlarged cut outs from the corresponding time-point. hpi, hours post-infection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61843/61843fig08.jpg" /> 
Figure 8: Strand-specific bDNA FISH and immunostaining of A549 cells infected with influenza A virus.&#160;A549 cells infected with PR8 Flu A virus were fixed and probed for (A) IAV nucleoprotein (NP) RNA (labeled with Alexa 488; green in Figure), and (B) IAV polymerase protein (PB1) (secondary goat anti-mouse Atto 550; red in Figure) and nuclei were stained with DAPI (blue). (C) Merged image. Scale bar represents 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61843/61843fig9v2.jpg" /> 
Figure 9: Strand-specific bDNA FISH in Zika virus (ZIKV)-infected cells.&#160;Vero cells were infected with ZIKV at a MOI of 0.1. Cells were fixed at 48 hpi. (A) Cells were simultaneously stained for sense (+) vRNA (labeled with Alexa 488; green in Figure) and antisense (-) vRNA (labeled with Atto 550; red in Figure). Nuclei were stained with DAPI. In (A), the white box denotes a region with both (+) and (-) vRNA. The insets present a close-up of (+) vRNA (green) and the scarcer (-) vRNA species (red). (B) sense (+) vRNA (green). (C) antisense (-) vRNA (red). Scale bar in (A) represents 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61843/61843fig9v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infectio

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

single-cell-multiplexed-fluorescence-imaging-to-visualize-viral

Nanyang Technological University

Research

JoVE Journal

Biology

2.7K Views.  Emory University School of Medicine. Hier wordt een protocol gepresenteerd voor een fluorescentiebeeldvormingsbenadering, multiplex immunofluorescent cell-gebaseerde detection van DNA, RNA en protein (MICDDRP), een methode die in staat is tot gelijktijdige fluorescentie eencellige visualisatie van virale eiwitten en nucleïnezuren van verschillende type en gestrandheid. Deze aanpak kan worde...

Video: Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Immunology and Infection

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

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

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

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