This work was supported by the Ministry of Science and Higher Education for research on SARS-CoV-2, and by grants from the National Science Center (grants UMO2017/27/B/NZ6/02488 to K.P. and UMO-2018/30/E/NZ1/00874 to A.K.-P.).

1. Buffer preparation
<ol>
	<li>For 500 mL of 2x PHEM buffer, combine 18.14 g of piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES), 6.5 g of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 3.8 g of ethylene glycol tetraacetic acid (EGTA), and 0.99 g of magnesium sulfate (MgSO4). Make the volume up to ~400 mL with distilled water (dH2O), stir, and adjust the pH to 7.0 using 10 M potassium hydroxide (KOH) or sodium hydroxide (NaOH). Make the final volume up to 500 mL, and then split into 50 mL aliquots. Store at -20 &#176;C until required. 
	NOTE: The buffer will not be clear until the pH reaches 7.0.</li>
	<li>Prepare a stock solution of 37% w/v paraformaldehyde (PFA). For 50 mL, mix 18.5 g of PFA and 35 mL of dH2O in a glass bottle. Place the bottle on a magnetic stirrer with heating. Add 900 &#181;L of 1 M KOH or NaOH, and stir until the solution becomes clear. Allow to cool and transfer to a 50 mL conical centrifuge tube; top up to 50 mL with dH2O. The solution can be stored at -20 &#176;C until required. 
	NOTE: Formaldehyde should be handled in a fume hood while wearing protective gloves and a lab coat.</li>
	<li>Prepare fixation buffer (3.7% PFA buffered with PHEM). For 50 mL, combine 5 mL of 37% PFA solution, 25 mL of 2x PHEM buffer, and 20 mL of dH2O in a 50 mL conical centrifuge tube. Store at -20 &#176;C until required. 
	NOTE: After thawing, the buffer can be stored at 4 &#176;C for up to 3 months.</li>
	<li>Prepare PBST (0.1% Tween-20 in 1x phosphate-buffered saline [PBS]). For 50 mL, add 50 &#181;L of 100% Tween-20 to 50 mL of 1x PBS and mix well. 
	NOTE: The solution can be stored at room temperature (RT).</li>
	<li>Prepare rehydration buffers by combining MeOH/PBST in ratios of 3:1, 1:1, and 1:3 (final volume of 100 mL of each; see Table 1). 
	NOTE: MeOH is very toxic and flammable. As it can damage the optic nerve, it should be handled in a fume hood avoiding any open flames. As mixing MeOH and PBST generates an exothermic reaction, combine the solutions on ice.</li>
	<li>For 1000 mL of 20x SSC, combine 175.3 g of sodium chloride (NaCl) and 88.2 g of sodium citrate in a beaker, and fill up with 800 mL of dH2O. Stir until dissolved, and adjust the pH to 7.2 using NaOH. Add dH2O to a total volume of 1000 mL, and autoclave or filter through a 0.22 &#181;m filter into an autoclaved bottle.</li>
	<li>For 50 mL of 5x SSCT, combine 12.5 mL of 20x SSC buffer with 37.5 mL of dH2O, and add 50 &#181;L of 100% Tween-20. Mix well.</li>
	<li>For 50 mL of 50% 5x SSCT/50% PBST, combine 25 mL of 5x SSCT with 25 mL of PBST.</li>
	<li>For 50 mL of 2x SSC, combine 5 mL of 10x SSC buffer with 45 mL of dH2O. Mix well. 
	&#8203;NOTE: All SSC buffers should be stored at RT in the dark.</li>
</ol>
2. Target definition, probes, and amplifiers
<ol>
	<li>Use the tools available on the manufacturer&#39;s website to design amplifiers and probes. Ensure that the probes are complementary to the reverse DNA strand of the SARS-CoV-2 N gene (Supplementary Figure 1).</li>
	<li>Determine the best probe set size (a set of 20 probes is sufficient for visualization).</li>
	<li>Set the amplifiers used for target RNA detection.
	<ol>
		<li>Use HCR amplifier B1 labeled with Alexa Fluor 647. 
		NOTE: An HCR amplifier comprises metastable HCR hairpins h1 and h2. Multiplexed experiments can be designed using this protocol. If this is planned, choose a different HCR amplifier (B1, B2, &#8230;) for each target RNA to be imaged within the same sample (e.g., amplifier B1 for target 1, amplifier B2 for target 2, &#8230;).</li>
	</ol>
	</li>
</ol>
3. Cell culture and infection with SARS-CoV-2
<ol>
	<li>Culture Vero (monkey kidney epithelial cells) cells in Dulbecco&#39;s modified Eagle medium containing 5% fetal bovine serum.
	<ol>
		<li>Seed 50,000 cells onto coverslips (No. 1, 15 &#215; 15 mm) in a 12-well plate.</li>
	</ol>
	</li>
	<li>Prepare fully differentiated HAE cultures as described18 on permeable Transwell insert supports with a membrane (diameter = 6.5 mm) and culture in bronchial epithelial growth medium until fully confluent.</li>
	<li>Virus infection
	<ol>
		<li>Inoculate cells with SARS-CoV-2 at 1000x median tissue culture infectious dose (TCID50) per mL</li>
		<li>Incubate cells for 2 h at 37 &#176;C.</li>
		<li>Wash cells twice with PBS to remove unbound virus.</li>
		<li>Culture cells for 48 h.</li>
	</ol>
	</li>
</ol>
4. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips
DAY 1
<ol>
	<li>Fixing and permeabilizing cells
	<ol>
		<li>Fix infected cells with 3.7% w/v PFA solution for 1 h at RT.</li>
		<li>Aspirate the 3.7% PFA solution, and wash the cells twice using 1x PBS.</li>
		<li>Permeabilize the cells with PBST solution for 10 min at RT with agitation.</li>
		<li>Aspirate the PBST, and wash the cells twice with 1x PBS.</li>
	</ol>
	</li>
	<li>Detection
	<ol>
		<li>Aspirate the 1x PBS solution, and wash the cells twice with 2x SSC at RT.</li>
		<li>Aspirate the 2x SSC solution, and prehybridize the samples by adding at least 300 &#181;L of probe hybridization buffer. Cover the wells containing the cells, and incubate at 37 &#176;C for 30 min.</li>
		<li>Prepare the probe solution by adding 1.2 pmol of probe mixture to the probe hybridization buffer.
		<ol>
			<li>Use 1.2 &#181;L of the 1 &#181;M probe stock to prepare 300 &#181;L of working stock.</li>
		</ol>
		</li>
		<li>Remove the prehybridization solution, and transfer the coverslips to a humidified chamber.
		<ol>
			<li>Pipette 30-50 &#181;L of probe solution onto parafilm to form individual droplets.</li>
		</ol>
		</li>
		<li>Incubate the samples overnight (12-18 h) at 37 &#176;C. 
		 
		DAY 2</li>
		<li>Transfer the coverslips back into a 12-well plate, and remove excess probe solution by washing for 4 x 5 min with 400 &#181;L of probe wash buffer at 37 &#176;C. 
		&#8203;NOTE: As an alternative to placing 30-50 &#181;l aliquots onto parafilm and under coverslips for incubation, add 300 &#181;L of probe solution directly to coverslips in a 12-well plate. This procedure is simpler, but requires substantial amounts of reagents. Heat the probe wash buffer to 37 &#176;C before use. Calculate the amount of buffer needed, and transfer it to a 15 mL conical centrifuge tube.</li>
		<li>Wash samples for 2 x 5 min with 5x SSCT at RT.</li>
		<li>Replace the 5x SSCT solution with 1x PBS, and store the samples at 4 &#176;C until amplification.</li>
	</ol>
	</li>
	<li>Amplification
	<ol>
		<li>Remove the 1x PBS solution from the wells, and add at least 300 &#181;L of amplification buffer to each well. Incubate the samples for 30 min at RT.</li>
		<li>Prepare each HCR hairpin (h1 and h2) by snap-cooling the desired volume in separate tubes.
		<ol>
			<li>To prepare 300 &#181;L of amplification solution, use 18 pmol of each hairpin (e.g., for 300 &#181;L, use 6 &#181;L of a 3 &#181;M stock hairpin solution).</li>
			<li>Transfer the hairpin solution into tubes.</li>
			<li>Incubate at 95 &#176;C for 90 s.</li>
			<li>Cool to RT for 30 min in the dark.</li>
		</ol>
		</li>
		<li>Prepare a hairpin mixture by adding the snap-cooled &#34;h1&#34; and &#34;h2&#34; hairpins to the amplification buffer.</li>
		<li>Place drops of 30-50 &#181;L of hairpin mixture onto parafilm.</li>
		<li>Incubate the samples overnight (12-18 h) in the dark at RT. 
		 
		DAY 3</li>
		<li>Transfer the coverslips back into the 12 well plate, and remove excess hairpins by washing for 5 x 5 min with 5x SSCT at RT with agitation.</li>
		<li>Aspirate the 5x SSCT buffer, and replace it with 1x PBS. 
		NOTE: If required, use the RNA-FISH samples in a standard immunofluorescence assay, followed by staining of nuclei (see section 5).</li>
	</ol>
	</li>
	<li>Nuclear staining and slide mounting
	<ol>
		<li>Aspirate the 1x PBS solution, and replace it with 4&#8242;,6-diamidino-2-phenylindole (DAPI, 0.2 &#181;g/mL) in 1x PBS solution.</li>
		<li>Incubate the samples for 10 min at RT in the dark.</li>
		<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
		<li>Place two drops of 10 &#181;L each of mounting medium; ensure that the drops are separated sufficiently to allow two coverslips to be placed on a single slide.</li>
		<li>Remove excess liquid by tapping the coverslips on a clean towel, and then place them in antifade mounting medium with the cells facing down.</li>
		<li>Place the mounted samples on a dry, flat surface in the dark and let them cure.</li>
		<li>Following curing, seal the edges of the coverslips with VALAP sealant or nail polish to prevent the samples from drying out.</li>
	</ol>
	</li>
</ol>
5. SARS-CoV-2 RNA-FISH in HAE cultures
DAY 1
<ol start="1">
	<li>Fixing and permeabilizing the HAE culture
	<ol>
		<li>Aspirate the medium, and fix the infected cells using 3.7% PFA solution for 1 h at RT.</li>
		<li>Aspirate the 3.7% PFA solution, and wash the cells twice with 1x PBS.
		<ol>
			<li>Replace the 3.7% PFA solution with 1x PBS under a Transwell insert.</li>
		</ol>
		</li>
		<li>Discard the PBS, and dehydrate the samples using 2 x 5 min washes with 100% MeOH prechilled to -20 &#176;C.</li>
		<li>After the second wash, replace the buffer with fresh, chilled MeOH for permeabilization under the Transwell insert. Store overnight at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
DAY 2
<ol start="2">
	<li>Rehydration 
	&#8203;Rehydrate the samples through a graded series of MeOH/PBST solutions (each for 5 min) on ice: 75% MeOH/25% PBST, 50% MeOH/50% PBST, 25% MeOH/75% PBTS, and 100% PBST (twice).
	<ol>
		<li>Wash the cells for 5 min on ice with 50% 5x SSCT/50% PBST.</li>
		<li>Wash cells for 5 min on ice with 5x SSCT.</li>
		<li>Replace the 5x SSCT buffer with 1x PBS.</li>
	</ol>
	</li>
	<li>Detection
	<ol>
		<li>Incubate the cells (inside the Transwell insert) for 5 min on ice with 100 &#181;L of probe hybridization buffer. Next, transfer the plate to incubator for 30 min at 37 &#176;C (prehybridization). 
		NOTE: The probe hybridization buffer must be pre-heated to 37 &#176;C before use. Calculate the required volume: 100 &#181;L is needed for a single Transwell insert.</li>
		<li>Prepare the probe solution. As 1 mL of probe solution requires 4 pmol of probe, add 4 &#181;L of 1 &#181;M probe stock solution to 1 mL of probe hybridization buffer, and mix well. 
		&#8203;NOTE: For RNA detection, use 100 &#181;L of probe solution per Transwell insert. Leave the probe solution on ice until the end of the prehybridization step.</li>
		<li>Remove the prehybridization solution, and add the probe solution.</li>
		<li>Incubate the cells overnight (12-18 h) at 37 &#176;C. 
		 
		&#8203;DAY 3</li>
		<li>Remove excess probe by washing for 4 x 15 min with 100 &#181;L of probe wash buffer at 37 &#176;C.</li>
		<li>Wash the samples for 2 x 5 min with 5x SSCT at RT.</li>
		<li>Replace the 5x SSCT with 1x PBS, and store the samples at 4 &#176;C until amplification.</li>
	</ol>
	</li>
	<li>Amplification
	<ol>
		<li>Preamplify the samples by incubating them with amplification buffer for 30 min at RT.</li>
		<li>Prepare each hairpin by snap-cooling the desired volumes in separate tubes.
		<ol>
			<li>To prepare 500 &#181;L of amplification solution, use 30 pmol of each hairpin (e.g., for 500 &#181;L, use 10 &#181;L of 3 &#181;M stock hairpin solution).</li>
			<li>Transfer the hairpin solution to the tubes.</li>
			<li>Incubate at 95 &#176;C for 90 s.</li>
			<li>Cool to RT for 30 min in the dark.</li>
		</ol>
		</li>
		<li>Prepare the hairpin solution by adding all snap-cooled hairpins to 500 &#181;L of amplification buffer at RT.</li>
		<li>Remove the preamplification solution, and add the complete hairpin solution.</li>
		<li>Incubate the samples overnight (12-18 h) at RT in the dark.</li>
		<li>Remove excess hairpins by washing with 5x SSCT at RT as follows: 2 x 5 min, 2 x 15 min, and 1 x 5 min.</li>
		<li>Replace the 5x SSCT solution with 1x PBS, and store at 4 &#176;C for not more than 2-3 days or proceed directly to nuclear staining. 
		&#8203;NOTE: If required, use the RNA-FISH samples in a standard immunofluorescence assay, followed by nuclear staining (see section 6).</li>
	</ol>
	</li>
	<li>Nuclear staining and slide mounting
	<ol>
		<li>Aspirate the 1x PBS solution, and replace it with DAPI solution (0.2 &#181;g/mL) in 1x PBS.</li>
		<li>Incubate the samples for 10 min at RT in the dark.</li>
		<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
		<li>Place the cut-out membrane from the Transwell inserts onto 10 &#181;L of antifade mounting medium with the cells facing up, and add extra mounting medium (5 &#181;L) to the membrane.</li>
		<li>Cover the membranes with coverslips.</li>
		<li>Cure the mounted samples on a dry, flat surface in the dark.</li>
		<li>Following curing, seal the edges of the coverslips with VALAP sealant or nail polish to prevent the samples from drying out.</li>
	</ol>
	</li>
</ol>
6. Immunofluorescence analysis of Vero cells and HAE cultures
NOTE: Perform the immunofluorescence assay on day 3 for cell lines or day 4 for HAE cultures. Use a different approach for each model. All differences are indicated clearly.
<ol>
	<li>Aspirate the 1x PBS solution from the wells.</li>
	<li>Block the samples by incubation with 1% w/v bovine serum albumin in PBST solution for 30 min at 37 &#176;C.</li>
	<li>Prepare primary antibodies by preparing appropriate dilutions in blocking solution, and incubate.
	<ol>
		<li>For Vero cells on coverslips:
		<ol>
			<li>Place drops (30-50 &#181;L) of antibody solution onto parafilm in a humidity chamber.</li>
			<li>Place coverslips onto the antibody drops with the cells facing down.</li>
		</ol>
		</li>
		<li>For HAE, replace the blocking agent in the inserts with antibody solution (100 &#181;L), and incubate the samples in a humidified chamber. 
		NOTE: Adjust the time and temperature for each incubation with primary antibody. Typical parameters are 2&#160;h at RT or overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Wash the samples for 3 x 5 min with PBST.
	<ol>
		<li>For cells on coverslips, transfer to a 12-well plate and add PBST solution.</li>
		<li>For HAE cultures, replace the antibody solution with PBST solution.</li>
	</ol>
	</li>
	<li>Check the light sources and filters available for the confocal microscope.</li>
	<li>Choose secondary antibodies according to the host species. Choose the fluorophore parameters (excitation and emission wavelengths, spectrum width, and excitation efficiency according to the available light source). 
	NOTE: Spectral parameters can be modeled using online tools (see Table of Materials).</li>
	<li>Prepare appropriate secondary antibody solutions by diluting them with blocking solution.</li>
	<li>Incubate with secondary antibodies (as in 6.3), but incubate for 1 h at 37 &#176;C.</li>
	<li>Wash the samples as in step 6.4.</li>
	<li>Nuclear staining and slide mounting
	<ol>
		<li>For cells on coverslips
		<ol>
			<li>Aspirate the PBST, and replace it with DAPI (0.2 &#181;g/mL) in 1x PBS solution.</li>
			<li>Incubate the samples for 10 min at RT in the dark.</li>
			<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
			<li>Place the coverslips onto drops of 10 &#181;L of mounting medium with the cells facing down.</li>
			<li>Seal the coverslips with nail polish.</li>
		</ol>
		</li>
		<li>For HAE cultures
		<ol>
			<li>Aspirate the 1x PBST, and replace it with DAPI (0.2 &#181;g/mL) in 1x PBS solution.</li>
			<li>Incubate the samples for 10 min at RT in the dark.</li>
			<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
			<li>Place the cut-out membranes onto drops of 10 &#181;L of mounting medium with the cells facing up, and add extra (5 &#181;L) mounting medium to the membrane.</li>
			<li>Cover the membranes with coverslips.</li>
			<li>Seal the coverslips with nail polish.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Confocal microscopy
<ol>
	<li>Define the tracks by specifying the fluorophores used.</li>
	<li>Choose the scanning mode and speed.</li>
	<li>Adjust the laser power, gain, and offset values for each fluorophore by comparing them with respective negative controls: for virus, mock-infected cells; for cellular proteins, samples stained with isotype control antibodies from an appropriate host.</li>
	<li>To acquire a 3D image, activate z-stack mode, and set the top and bottom limits. Set the step size/number. 
	NOTE: For more details on coronavirus imaging, see19.</li>
</ol>

<ol>
	<li>Choi, H. M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Third-generation+in+situ+hybridization+chain+reaction:+multiplexed,+quantitative,+sensitive,+versatile,+robust.">Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.</a> Development. 145, (2018).</li><li>Milewska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+severe+acute+respiratory+syndrome+coronavirus+2+in+human+respiratory+epithelium.">Replication of severe acute respiratory syndrome coronavirus 2 in human respiratory epithelium.</a> Journal of Virology. 94, (2020).</li><li>Banach, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+airway+epithelial+cell+culture+to+identify+new+respiratory+viruses:+coronavirus+NL63+as+a+model.">Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model.</a> Journal of Virological Methods. 156 (1-2), 19-26 (2009).</li><li>Milewska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+human+coronavirus+NL63+into+the+cell.">Entry of human coronavirus NL63 into the cell.</a> Journal of Virology. 92, (2018).</li><li>El Baba, R., Herbein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+epigenomic+networks+entailed+in+coronavirus+infections+and+COVID-19.">Management of epigenomic networks entailed in coronavirus infections and COVID-19.</a> Clinical Epigenetics. 12, 118 (2020).</li><li>Pinto, B. G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACE2+expression+is+increased+in+the+lungs+of+patients+with+comorbidities+associated+with+severe+COVID-19.">ACE2 expression is increased in the lungs of patients with comorbidities associated with severe COVID-19.</a> Journal of Infectious Diseases. 222 (4), 556-563 (2020).</li><li>Verdecchia, P., Cavallini, C., Spanevello, A., Angeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pivotal+link+between+ACE2+deficiency+and+SARS-CoV-2+infection.">The pivotal link between ACE2 deficiency and SARS-CoV-2 infection.</a> European Journal of Internal Medicine. 76, 14-20 (2020).</li><li>Gordon, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SARS-CoV-2+protein+interaction+map+reveals+targets+for+drug+repurposing.">A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.</a> Nature. 583, 459-468 (2020).</li><li>Ferron, F., Decroly, E., Selisko, B., Canard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viral+RNA+capping+machinery+as+a+target+for+antiviral+drugs.">The viral RNA capping machinery as a target for antiviral drugs.</a> Antiviral Research. 96 (1), 21-31 (2012).</li><li>Mehta, S., Jeffrey, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+receptors+and+signaling:+epigenetic+factors+in+the+regulation+of+innate+immunity.">Beyond receptors and signaling: epigenetic factors in the regulation of innate immunity.</a> Immunology and Cell Biology. 93 (3), 233-244 (2015).</li><li>Poppe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NF-kappaB-dependent+and+-independent+transcriptome+and+chromatin+landscapes+of+human+coronavirus+229E-infected+cells.">The NF-kappaB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells.</a> PLoS Pathogens. 13, 1006286 (2017).</li><li>Schafer, A., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+landscape+during+coronavirus+infection.">Epigenetic landscape during coronavirus infection.</a> Pathogens. 6 (1), 8 (2017).</li><li>Carletti, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+priming+of+cell+intrinsic+innate+antiviral+signaling+by+the+unfolded+protein+response.">Viral priming of cell intrinsic innate antiviral signaling by the unfolded protein response.</a> Nature Communications. 10, 3889 (2019).</li><li>Heil, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+recognition+of+single-stranded+RNA+via+toll-like+receptor+7+and+8.">Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.</a> Science. 303 (5663), 1526-1529 (2004).</li><li>Sze, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+restriction+factor+SAMHD1+limits+human+T+cell+leukemia+virus+type+1+infection+of+monocytes+via+STING-mediated+apoptosis.">Host restriction factor SAMHD1 limits human T cell leukemia virus type 1 infection of monocytes via STING-mediated apoptosis.</a> Cell Host and Microbe. 14 (4), 422-434 (2013).</li><li>Kikkert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+evasion+by+human+respiratory+RNA+viruses.">Innate immune evasion by human respiratory RNA viruses.</a> Journal of Innate Immunity. 12, 4-20 (2020).</li><li>Kennedy, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranscriptional+m(6)A+editing+of+HIV-1+mRNAs+enhances+viral+gene+expression.">Posttranscriptional m(6)A editing of HIV-1 mRNAs enhances viral gene expression.</a> Cell Host and Microbe. 22 (6), 830 (2017).</li><li>Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R., Randell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Human Cell Culture Protocols in Methods in Molecular Medicine. 107, 183-206 (2005).</li><li>Milewska, A., Owczarek, K., Szczepanski, A., Pyrc, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+coronavirus+entry+into+cells.">Visualizing coronavirus entry into cells.</a> Methods in Molecular Biology. 2203, 241-261 (2020).</li></ol>

The authors have no conflicts of interest to declare.

Immuno-RNA-FISH is a reliable method for double-staining of RNA and cellular proteins. Here, a modified immuno-RNA-FISH protocol has been described that allows detection of SARS-CoV-2 RNA and cellular proteins in cell lines and HAE cultures. This protocol can be adapted for use in different cell models including cell monolayers or specific tissues. The method relies on the concept of an HCR initiated by appropriate probe localization. Next, the use of split-initiator probes to begin amplification of the signal by fluores...

SARS-CoV-2 is a novel human betacoronavirus that emerged at the end of 2019, causing an unprecedented pandemic a few months later. Because the virus is new to science, much of its biology and its impact on host cells remain unknown. Therefore, mapping the virus-cell and -tissue tropism during infection is important if its basic biological characteristics and its effects on the host are to be understood. Several techniques are used to examine virus-host interplay including biochemical, biological, and physical assays. In situ hybridization is a common method that employs labeled complementary DNA, RNA, or modified nucleic acid probes, which localize to specific DNA or RNA sequences in a cell or tissue.
A new RNA fluorescent in situ hybridization (RNA-FISH) method has been developed that incorporates modifications to increase sensitivity by amplifying the signal-to-noise ratio via an HCR1. HCR allows the study of RNA localization at a single-cell level. Owing to its high specificity, sensitivity, and resolution, this method is useful not only for basic science studies, but also for applicatory projects, e.g., diagnostics. Recently, the feasibility of this method was demonstrated for detecting SARS-CoV-2 RNA localized to ciliated cells within fully differentiated 3D human airway epithelium (HAE) cultures2. HAE cultures constitute one of the most advanced tools used to study viral infection in the context of the &#34;natural infection&#34; microenvironment3,4.
Several reports on human coronaviruses (HCoV), including SARS-CoV-2, highlight the importance of epigenetic modifications with respect to HCoV infection and pathophysiology [reviewed in 5], e.g., the methylation pattern of the gene encoding the angiotensin-converting enzyme 2 (ACE-2) receptor6,7. Interestingly, mass-spectrometric screening identified several epigenetic factors that interact with the SARS-CoV-2 proteome8. More specifically, nonstructural protein 5 (NSP5) binds to the epigenetic regulator, histone deacetylase 2, and the catalytically inactive NSP5 (C145A) interacts with tRNA methyltransferase 1 (24). Additionally, NSP16 methyltransferase activity is blocked by the methyltransferase inhibitor, sinefungin9. However, the exact role of these epigenetic factors in COVID-19 remains unclear. Replication of HCoV takes place in the cytoplasm of the infected cell, and triggers inflammatory responses that are regulated by epigenetic modifications10.
For instance, HCoV-229E fine-tunes nuclear factor-kappa B signaling and profoundly reprograms the host cellular chromatin landscape by increasing acetylation of H3K36 and H4K5 in certain regions11. The Middle East respiratory syndrome-related coronavirus infection increases levels of H3K27me3 and depletes H3K4me3 at the promoter regions of subsets of specific interferon-sensitive genes12. Additionally, viral RNA triggers cell immune responses, as demonstrated for flaviviruses13, retroviruses14,15, and coronaviruses16. The epigenetic markers on viral RNA may play a role in recognition by cellular sensors, as shown for m7A methylation of human immunodeficiency virus-1 RNA17. However, questions remain: What is the impact of SARS-CoV-2 RNA on the immune response, and are epigenetic marks involved?
Here, an optimized RNA-FISH method coupled with immunofluorescence analysis of cell lines and 3D tissues (fully differentiated HAE) has been described. Although cytological methods, such as FISH and immunofluorescence, are used widely, this new-generation in situ hybridization method based on HCR has never been used for virus detection (except in a recent publication)2. In general, immunostaining and FISH require the following steps: permeabilization to enable penetration of probes or antibodies; fixation in which cellular material is fixed and preserved; detection in which antibodies or nucleic acid probes are applied; and finally, mounting of the samples for visualization.
Although existing protocols share these general features, they vary markedly with respect to the parameters involved. Here, an optimized, simple, immuno-RNA-FISH protocol has been described to detect SARS-CoV-2 RNA in HAE cultures and Vero cells. The technique comprises the following steps: (1) fixation of cells with paraformaldehyde; (2) permeabilization with detergent or methanol (MeOH); (3) rehydration through a graded series of MeOH solutions (HAE cultures only); (4) detection; (5) amplification using HCR technology to detect SARS-CoV-2 RNA; (6) immunostaining; and (7) imaging under a confocal microscope.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Confocal Microscope LSM 880</td>
			<td >ZEISS </td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Grant Bio, Mini Rocker- Shaker</td>
			<td >Fisher Scientific</td>
			<td>12965501</td>
			<td></td>
		</tr>
		<tr >
			<td >Incubator Galaxy170R</td>
			<td >New Brunswick</td>
			<td>CO170R-230-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermomixer Comfort</td>
			<td >Eppendorf</td>
			<td>5355 000 011</td>
			<td></td>
		</tr>
		<tr >
			<td>Materials</td>
			<td></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >15 mm x 15 mm NO. 1 coverslips</td>
			<td >LabSolute</td>
			<td >7695022</td>
			<td ></td>
		</tr>
		<tr >
			<td >1.5 mL tubes</td>
			<td >FL-MEDICAL</td>
			<td >5.350.023.053</td>
			<td ></td>
		</tr>
		<tr >
			<td >12-well plate</td>
			<td >TTP</td>
			<td >92412</td>
			<td ></td>
		</tr>
		<tr >
			<td >Conical centrifuge tube</td>
			<td >Sarstedt</td>
			<td >5.332.547.254</td>
			<td ></td>
		</tr>
		<tr >
			<td >parafilm</td>
			<td >Sigma</td>
			<td >P7793-1EA</td>
			<td ></td>
		</tr>
		<tr >
			<td >serological pipets</td>
			<td >VWR Collection</td>
			<td >612-5523P, 612-5827P</td>
			<td ></td>
		</tr>
		<tr >
			<td >slide glass</td>
			<td >PTH CHEMLAND</td>
			<td >04-296.202.03</td>
			<td ></td>
		</tr>
		<tr >
			<td >Transwell ThinCerts</td>
			<td >Grainer bio-one</td>
			<td >665641</td>
			<td ></td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Alexa fluorophore 488-conjugated secondary antibodies </td>
			<td>Invitrogen</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >&#946;5-tubulin </td>
			<td>Santa Cruz Biotechnology</td>
			<td > sc-134234</td>
			<td ></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Thermo Scientific</td>
			<td >D1306</td>
			<td ></td>
		</tr>
		<tr >
			<td >Disodium phosphate</td>
			<td >Sigma</td>
			<td >S51136-500G</td>
			<td ></td>
		</tr>
		<tr >
			<td >EGTA</td>
			<td >BioShop</td>
			<td >EGT101.25</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR Amplification Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BAM01522</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HCR amplifier B1-h1 Alexa Fluor 647</td>
			<td >Molecular Instruments, Inc.</td>
			<td >S013922</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR amplifier B1-h2 Alexa Fluor 647</td>
			<td >Molecular Instruments, Inc.</td>
			<td >S012522</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR Probe Hybridization Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BPH03821</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HCR probe set for SARS-CoV-2 Ncapsid</td>
			<td >Molecular Instruments, Inc.</td>
			<td >PRE134</td>
			<td></td>
		</tr>
		<tr >
			<td >HCR Probe Wash Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BPW01522</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td >BioShop</td>
			<td >HEP001.100</td>
			<td ></td>
		</tr>
		<tr >
			<td >Magnesium sulfate heptahydrate</td>
			<td >Sigma</td>
			<td >63138-250G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Methanol</td>
			<td >Sigma</td>
			<td >32213-1L-M</td>
			<td ></td>
		</tr>
		<tr >
			<td >Monopotassium phosphate</td>
			<td >Sigma</td>
			<td >P5655-100G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Sigma</td>
			<td >P6148-1KG</td>
			<td ></td>
		</tr>
		<tr >
			<td >PIPES</td>
			<td >BioShop</td>
			<td >PIP666.100</td>
			<td ></td>
		</tr>
		<tr >
			<td >Potassium Chloride</td>
			<td >Sigma</td>
			<td >P5405-250G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Prolong Diamond Antifade Mounting Medium</td>
			<td >Invitrogen</td>
			<td >P36970</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td >BioShop</td>
			<td >SOD001.5</td>
			<td ></td>
		</tr>
		<tr >
			<td >Trisodium Citrate 2-hydrate</td>
			<td >POCH</td>
			<td > 6132-04-3</td>
			<td ></td>
		</tr>
		<tr >
			<td >Tween-20</td>
			<td >BioShop</td>
			<td >TWN580.500</td>
			<td ></td>
		</tr>
		<tr >
			<td ></td>
			<td >Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence Spectraviewer</td>
			<td ></td>
			<td></td>
			<td>Modeling spectral parameters</td>
		</tr>
		<tr >
			<td >ImageJ Fiji</td>
			<td ></td>
			<td ></td>
			<td >Acquiring and processing z-stack images</td>
		</tr>
	</tbody>
</table>

visualization of sars-cov-2 using immuno rna-fluorescence in situ hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

The immuno-RNA-FISH protocol described in this manuscript was carried out using two cellular systems: a Vero cell line and a 3D HAE culture. The major steps for both cellular models are shown in Table 2. The RNA-FISH protocol for visualization of SARS-CoV-2 in HAE cultures includes steps that are typical for tissue samples, i.e., permeabilization with 100% MeOH and rehydration through a graded series of MeOH-PBS and 0.1% Tween solutions. Immunofluorescence was performed a...

Watch this Scientific Journal Video about Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization at JoVE.com

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

visualization-sars-cov-2-using-immuno-rna-fluorescence-situ

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JoVE Journal

Immunology and Infection

5.9K Views.  Jagiellonian University. Here, we describe a simple method that combines RNA fluorescence in situ hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

Video: Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...