This study has the following funding sources: National Institutes of Health (NIH) R01AI 169537 (S.R.W. and N.A.C.), NIH R01AI 140442 (S.R.W.), VA Merit Review CX001717 (N.A.C.), VA Merit Review BX005432 (S.R.W. and N.A.C.),&#160; Penn Center for Research on Coronaviruses and other Emerging Pathogens (S.R.W.), Laffey-McHugh Foundation (S.R.W. and N.A.C.), T32 AI055400 (CJO), T32 AI007324 (AF).

The use of nasal specimens was approved by the University of Pennsylvania Institutional Review Board (protocol # 800614) and the Philadelphia VA Institutional Review Board (protocol # 00781).
1. Infection of nasal ALI cultures
NOTE: Acquisition of clinical specimens, as well as growth and differentiation of nasal ALI cultures, is outside the scope of this paper. Specific methods for culturing primary nasal epithelial cells can be found in recently pub...

<ol>
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R., Regamey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+airway+epithelium:+Soldier+in+the+fight+against+respiratory+viruses.">The airway epithelium: Soldier in the fight against respiratory viruses.</a> Clinical Microbiology Reviews. 24 (1), 210-229 (2011).</li><li>Farzal, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+simulated+nebulized+and+spray+particle+deposition+in+chronic+rhinosinusitis+patients.">Comparative study of simulated nebulized and spray particle deposition in chronic rhinosinusitis patients.</a> International Forum of Allergy and Rhinology. 9 (7), 746-758 (2019).</li><li>Gaeckle, N. T., Pragman, A. A., Pendleton, K. M., Baldomero, A. K., Criner, G. 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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=Delayed+induction+of+type+I+and+III+interferons+mediates+nasal+epithelial+cell+permissiveness+to+SARS-CoV-2.">Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2.</a> Nature Communications. 12 (1), 7092 (2021).</li><li>Sungnak, W., 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=SARS-CoV-2+entry+factors+are+highly+expressed+in+nasal+epithelial+cells+together+with+innate+immune+genes.">SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes.</a> Nature Medicine. 26 (5), 681-687 (2020).</li><li>Li, Y., 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=SARS-CoV-2+induces+double-stranded+RNA-mediated+innate+immune+responses+in+respiratory+epithelial-derived+cells+and+cardiomyocytes.">SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (16), 2022643118 (2021).</li><li>Comar, C. 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The methods detailed here describe a primary epithelial culture system in which patient-derived nasal epithelial cells are grown at an air-liquid interface and applied to the study of HCoV-host interactions. Once differentiated, these nasal ALI cultures recapitulate many features of the in vivo nasal epithelium, including a heterogeneous cellular population with ciliated, goblet, and basal cells represented, as well as intact mucociliary function with robustly beating cilia and mucus secretion. This heterogeneou...

Seven human coronaviruses (HCoVs) have been identified to date and cause a range of respiratory diseases2. The common or seasonal HCoVs (HCoV-NL63, -229E, -OC43, and -HKU1) are typically associated with upper respiratory tract pathology and cause an estimated 10%-30% of common cold cases annually. Though this is the typical clinical phenotype associated with the common HCoVs, these viruses can cause more significant lower respiratory tract disease in at-risk populations, including children, older adults, and immunocompromised individuals3,4. Three pathogenic HCoVs have emerged and caused significant public health emergencies in the last 20 years, including severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. Lethal HCoVs are associated with more severe respiratory tract pathology, which is clearly illustrated by the &#62;34% case-fatality rate associated with MERS-CoV cases (894 deaths from over 2,500 cases since its emergence in 2012)5,6. It is important to note that the lethal HCoVs also cause a range of respiratory tract diseases, from asymptomatic infections to lethal pneumonia, as seen with the ongoing COVID-19 pandemic7.
HCoVs, like other respiratory pathogens, enter the respiratory tract and establish a productive infection in the nasal epithelium8. Spread to the lower airway is thought to be associated with aspiration from the oral/nasal cavity to the lung, where HCoVs cause more significant lower respiratory tract pathology9,10,11. Thus, the nose serves as the initial portal for viral entry and is the primary barrier to infection with its robust mucociliary clearance machinery and unique innate immune mechanisms aimed at preventing further viral spread to the lower airway12,13. For example, nasal epithelial cells have been reported to express higher than average basal levels of antiviral interferons and interferon-stimulated genes, indicating that nasal cells may be primed for early responses to respiratory viruses14,15,16.
We have previously utilized patient-derived primary nasal epithelial cells grown at an air-liquid interface (ALI) to model HCoV-host interactions in the nose, where HCoV infections begin. Nasal ALI cultures are permissive to both pathogenic (SARS-CoV-2 and MERS-CoV) and common HCoVs (HCoV-NL63 and HCoV-229E) and offer various advantages over traditional airway epithelial cell lines such as A549 (a lung adenocarcinoma cell line)16,17. After differentiation, nasal ALI cultures contain a heterogeneous cellular population and exhibit many of the functions expected of the in vivo nasal epithelium, such as mucociliary clearance machinery18. Nasal cells also offer advantages over lower airway culture systems (such as human bronchial epithelial cells, HBECs), as the acquisition of nasal epithelial cells via cytologic brushing is significantly less invasive compared with using techniques such as bronchoscopy for attaining HBECs19,20,21.
This paper describes methods for utilizing this nasal ALI culture system to characterize HCoV-host interactions in the nasal epithelium. We have applied these methods in recently published works to compare SARS-CoV-2, MERS-CoV, HCoV-NL63, and HCoV-229E1,16,17. Though these methods and representative results emphasize the study of HCoVs in this nasal cell model, the system is highly adaptable to other HCoVs, as well as other respiratory pathogens. Further, these methods can be applied more broadly to other ALI culture systems in order to investigate viral replication and cellular tropism, as well as cytotoxicity and innate immune induction following infection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor secondary antibodies (488, 594, 647)</td>
			<td>Invitrogen</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete mini EDTA-free protease inhibitor</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytotoxicity detection kit</td>
			<td>Roche</td>
			<td>11644793001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Media)</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#39;s Phosphate Buffered Saline)</td>
			<td>Gibco</td>
			<td>14190136</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS + calcium + magnesium</td>
			<td>Gibco</td>
			<td>14040-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Endohm-6G measurement chamber</td>
			<td>World Precision Instruments</td>
			<td>ENDOHM-6G</td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial cell adhesion marker (EpCAM; CD326)</td>
			<td>eBiosciences</td>
			<td>14-9326-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial Volt/Ohm (TEER) Meter (EVOM)</td>
			<td>World Precision Instruments</td>
			<td>300523</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW software for imaging</td>
			<td>Olympus</td>
			<td>Version 4.02</td>
			<td></td>
		</tr>
		<tr>
			<td>HCoV-NL63 (Human coronavirus, NL63)</td>
			<td>BEI Resources</td>
			<td>NR-470</td>
			<td></td>
		</tr>
		<tr>
			<td>HCoV-NL63 nucleocapsid antibody</td>
			<td>Sino Biological</td>
			<td>40641-V07E</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer (4x)</td>
			<td>BIO-RAD</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>LLC-MK2 cells</td>
			<td>ATCC</td>
			<td>CCL-7</td>
			<td>To titrate HCoV-NL63</td>
		</tr>
		<tr>
			<td>MERS-CoV (Human coronavirus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), EMC/2012)</td>
			<td>BEI Resources&#160;</td>
			<td>NR-44260</td>
			<td></td>
		</tr>
		<tr>
			<td>MERS-CoV nucleocapsid antibody</td>
			<td>Sino Biological</td>
			<td>40068-MM10</td>
			<td></td>
		</tr>
		<tr>
			<td>MUC5AC antibody</td>
			<td>Sigma-Aldrich</td>
			<td>AMAB91539</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Fluoview confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 647 stain</td>
			<td>Abcam</td>
			<td>ab176759</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosStop easy pack (phosphatase inhibitors)&#160;</td>
			<td>Roche</td>
			<td>PHOSS-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader&#160;</td>
			<td>Perkin Elmer</td>
			<td>HH34000000</td>
			<td>Any plate reader or ELISA reader is sufficient; must be able to read absorbance at 492 nm</td>
		</tr>
		<tr>
			<td>RIPA buffer (50 mM Tris pH 8; 150 mM NaCl; 0.5% deoxycholate; 0.1% SDS; 1% NP40)</td>
			<td>Thermo Fisher</td>
			<td>89990</td>
			<td>Can prep in-house or purchase</td>
		</tr>
		<tr>
			<td>RNeasy Plus Kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (SARS-Related Coronavirus 2, Isolate USA-WA1/2020)</td>
			<td>BEI Resources</td>
			<td>NR-52281</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 nucleocapsid antibody</td>
			<td>Genetex</td>
			<td>GTX135357</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Fisher Scientific</td>
			<td>BP151100</td>
			<td></td>
		</tr>
		<tr>
			<td>Type IV &#946;- tubulin antibody</td>
			<td>Abcam</td>
			<td>ab11315</td>
			<td></td>
		</tr>
		<tr>
			<td>VeroCCL81 cells</td>
			<td>ATCC</td>
			<td>CCL-81</td>
			<td>To titrate MERS-CoV</td>
		</tr>
		<tr>
			<td>VeroE6 cells</td>
			<td>ATCC</td>
			<td>CRL-1586</td>
			<td>To titrate SARS-CoV-2</td>
		</tr>
	</tbody>
</table>

infection of primary nasal epithelial cells grown at an air-liquid interface to characterize human coronavirus-host interactions

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public health crises in the past 20 years. Four additional HCoVs cause a significant portion of common cold cases each year (HCoV-NL63, -229E, -OC43, and -HKU1), highlighting the importance of studying these viruses in physiologically relevant systems. HCoVs enter the respiratory tract and establish infection in the nasal epithelium, the primary site encountered by all respiratory pathogens. We use a primary nasal epithelial culture system in which patient-derived nasal samples are grown at an air-liquid interface (ALI) to study host-pathogen interactions at this important sentinel site. These cultures recapitulate many features of the in vivo airway, including&#160;the cell types present, ciliary function, and mucus production. We describe methods to characterize viral replication, host cell tropism, virus-induced cytotoxicity, and innate immune induction in nasal ALI cultures following HCoV infection, using recent work comparing lethal and seasonal HCoVs as an example1. An increased understanding of host-pathogen interactions in the nose has the potential to provide novel targets for antiviral therapeutics against HCoVs and other respiratory viruses that will likely emerge in the future.

The representative figures are partially adapted from data that can be found in the manuscript Otter et al.1. Nasal ALI cultures derived from four or six donors were infected with one of four HCoVs (SARS-CoV-2, MERS-CoV, HCoV-NL63, and HCoV-229E) according to the protocols described above, and the average apically shed viral titers for each virus are depicted in Figure 1A. While all four of these HCoVs replicate productively in nasal ALI cultures, SARS-CoV-2 and HCoV-...

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Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions

The nasal epithelium is the primary barrier site encountered by all respiratory pathogens. Here, we outline methods to use primary nasal epithelial cells grown as air-liquid interface (ALI) cultures to characterize human coronavirus-host interactions in a physiologically relevant system.

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public ...

The protocols described here address key questions such as replication kinetics, host cell tropism, innate immune induction, and cytotoxicity induction in the nasal epithelium during infections with human coronaviruses. This protocol enables the study of human coronavirus host interactions in an organoid microenvironment that closely recapitulates features of the in vivo nasal epithelium, including its heterogeneous cellular population and mucociliary functions. This technique can be applied to other ALI culture systems such as those derived from the lower airway.Additionally, the system can be used to mimic clinical disease states like asthmatic or cystic fibrosis airways. Grow and differentiate the nasal air-liquid interface, or ALI cultures, in transwells having a basal and an apical chamber. Expand the dissociated human sinonasal cells under submerged condition, following which, remove the media from the apical chamber to allow differentiation of the cultures in an air-liquid interface.On the day prior to infection, wash the ALI cultures apically with phosphate-buffered saline, or PBS. To do so, add 200 microliters of warmed PBS into the apical compartment of each transwell and incubate the plate at 37 degrees Celsius for five minutes. Aspirate the PBS and repeat the washing process twice.Then, replace the basal medium with 500 microliters of fresh medium per well and equilibrate the culture overnight at the intended infection temperature. On the next day, dilute the virus in serum-free DMEM to achieve the desired multiplicity of infection in a total inoculum volume of 50 microliters. Add the viral inoculum apically to the culture and place it back in the incubator for one hour.Every 15 minutes during incubation, gently rock the plate forward and backward as well as side to side by holding it firmly with both hands to ensure a uniform adsorption of the viral inoculum. After incubation, aspirate the viral inoculum and ensure its complete removal by washing each infected culture three times with PBS as described previously. If desired, collect the third PBS wash to confirm adequate removal of the input virus.Replace the basal medium on the infected ALIs every 72 hours. At predetermined time points after the infection, add 200 microliters of PBS to the apical chamber of each infected transwell. Pipette the PBS up and down five times to ensure maximum apically-shed virus collection, and transfer the entire volume into a microcentrifuge tube to collect the apical surface liquid, or ASL sample.Next, serially dilute the sample to quantify the concentration of viral particles in the ASL using a standard viral plaque assay. As outlined on the screen, select the cell type depending on the human coronavirus, or HCoV, being titrated. If desired, confirm the absence of basally-released virus by plaque assay of undiluted basal medium collected at various time points post-infection.To measure TEER on nasal ALI cultures, clean, equilibrate, and blank the EVOM instrument per the manufacturer's instructions. Use an empty transwell with no nasal cells for blanking and record the blank measurement. To wash residual basal medium, transfer each infected transwell to a pre-labeled, clean, 24-well plate containing 500 microliters of PBS with calcium and magnesium in each well.Then, add 200 microliters of PBS containing calcium and magnesium to the apical compartment of each transwell. Add one milliliter of PBS containing calcium and magnesium to the Endohm-6 measurement chamber. Place each transwell into the Endohm-6 measurement chamber using forceps and close the chamber by replacing the lid.Ensure that the apical electrode is submerged in the 200 microliters of PBS in the apical compartment. The basal electrode remains at the bottom of the chamber. Once the EVOM reading stabilizes, record the raw TEER measurement.If titering is desired, collect the ASL sample after TEER measurement. Convert the raw TEER readings to final measurements in ohms per centimeter squared using the equation shown on the screen. For human coronaviruses, or HCoVs, assess the TEER at 24-or 48-hour intervals post-infection.Ensure to include a negative control at each time point. For the medium or background control, use PBS with the same composition as the one used for collecting the ASL sample. For the positive control, treat the control ALA cultures apically with 200 microliters of 2%Triton X-100.After incubating for 10 to 15 minutes, collect the entire volume as a Triton sealing sample, following the plate layout consisting of Triton-positive control samples, mock background control samples, PBS control samples, and experimental samples. Load the lactate dehydrogenase, or LDH plate, in triplicate. Finally, calculate the percentage cytotoxicity relative to the Triton sealing value using the displayed equation, The average apically-shed viral titers from nasal ALI cultures infected with four HCoVs revealed that each of the viruses replicated productively, though SARS-CoV-2 and HCoV-229E replicated most efficiently.MERS-CoV intracellular and apically-shed viral titers were approximately the same at 48 hours post-infection, or HPI. Co-staining of the infected cultures with antibodies specific to viral antigens and markers for ciliated or goblet cells showed that SARS-CoV-2 and HCoV-NL63 primarily infected ciliated cells, but MERS-CoV predominantly infected non-ciliated goblet cells. The difference in TEER from baseline to a given post-infection time point or delta-TEER for both SARS-CoV-2 and HCoV-NL63 infections were negative at 192 HPI, unlike MERS-CoV infection, which resulted in no significant change in TEER.Negative delta-TEER values indicate damage to epithelial barrier integrity. HCoV-229E caused epithelial barrier dysfunction at an earlier time point of 96 HPI, but recovered to mock levels later in the infection. TEER traces depicting raw TEER data for each infected transwell over time allowed for the visualization of TEER trends over the course of infection.Quantification of apically-released LDH during infection revealed that SARS-CoV-2, HCoV-NL63, and HCoV-229E caused significant cytotoxicity in nasal cultures, while MERS-CoV did not. It's crucial to understand viral replication kinetics in nasal cultures, as these determine the optimal time points for other downstream analysis such as cytotoxicity and innate immune induction. TEER measurements are most informative if taken sequentially on the same cultures, both prior to infection and then at various times post-infection in order to monitor changes in TEER over the course of infection.Our current research applies RNA sequencing approaches to the described system to understand transcriptional changes during human coronavirus infections, as well as ELISA techniques to measure cytokine and other protein production.

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Research

JoVE Journal

Immunology and Infection

1.0K visualizações.  University of Pennsylvania. The protocols described here address key questions such as replication kinetics, host cell tropism, innate immune induction, and cytotoxicity induction in the nasal epithelium during infections with human coronaviruses. This protocol enables the study of human coronavirus host interactions in an organoid microenvironment that closely recapitulates features of the in vivo nasal epithelium, including its heterogeneous cellular population and mucociliary functions. This technique can be applied ...