AOP-Wiki

<!DOCTYPE html>
<html lang="en">
<div id="title">
<h2>AOP ID and Title:</h2>
<div class="title">AOP 394: SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</div>
Short Title: SARS-CoV-2 causes anosmia
</div>
<h2>Graphical Representation</h2>
~~<img src="https://aopwiki.org/system/dragonfly/production/2022/01/05/8mqcnf1d35_Figure_wiki.png" height="500" width="700" alt=""/>~~
<img src="https://www.aopwiki.org/system/dragonfly/production/2022/01/05/8mqcnf1d35_Figure_wiki.png" height="500" width="700" alt=""/>
<div id="authors">
<h2>Authors</h2>
Francesca De Bernardi, Surat Saravan, Amalia Munoz, Magda Sachana, Laure-Alix Clerbaux and Sandra Coecke
</div>
<div id="status">
<h2>Status</h2>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Author status</th>
<th scope="col">OECD status</th>
<th scope="col">OECD project</th>
<th scope="col">SAAOP status</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Under development: Not open for comment. Do not cite</td>
<td>Under Development</td>
<td>1.96</td>
<td>Included in OECD Work Plan</td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
</div>
<div id="background">
<h3>Background</h3>
AOP394 is developed as a part of the <a href="https://www.ciao-covid.net/">CIAO project</a>, "Modelling the Pathogenesis of COVID-19 Using the Adverse Outcome Pathway (AOP)". The overall goal is to organize and understand the vast amount of data that is constantly evolving as a result of the COVID-19 pandemic and identify uncertainties and knowledge gaps that may be missing using the AOP framework. Many AOPs were developed in the CIAO project, each AOP focusing on a specific element of the SARS-COV-2 virus responses in humans.
AOP394 focuses on the short-term olfactory disfunction (anosmia) following binding of SARS-CoV-2 to <a href="https://bioregistry.io/genecards:ACE2">ACE2 </a>receptors in the sustencular cells of the olfactory epithelium.
</div>
<div id="aop_summary">
<h2>Summary of the AOP</h2>
<h3>Events</h3>
<h3>Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sequence</th>
<th scope="col">Type</th>
<th scope="col">Event ID</th>
<th scope="col">Title</th>
<th scope="col">Short name</th>
</tr>
</thead>
<tbody>
<tr>
<td></td>
<td>MIE</td>
<td>1739</td>
<td><a href="/events/1739">Binding to ACE2</a></td>
<td>Binding to ACE2</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>KE</td>
<td>1738</td>
<td><a href="/events/1738">SARS-CoV-2 cell entry </a></td>
<td>SARS-CoV-2 cell entry </td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1847</td>
<td><a href="/events/1847">Increased SARS-CoV-2 production</a></td>
<td>SARS-CoV-2 production</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1870</td>
<td><a href="/events/1870">Sustentacular cells, decrease </a></td>
<td>Sustentacular cells, decrease </td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1871</td>
<td><a href="/events/1871">olfactory sensory neurons, decrease </a></td>
<td>olfacotry neurons, decrease</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1872</td>
<td><a href="/events/1872">Olfactory epithelium degeneration</a></td>
<td>Olfactory epithelium degeneration</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>AO</td>
<td>1873</td>
<td><a href="/events/1873">impaired olfactory function (anosmia) </a></td>
<td>anosmia</td>
</tr>
</tbody>
</table>
</div>
<h3>Key Event Relationships</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Upstream Event</th>
<th scope="col">Relationship Type</th>
<th scope="col">Downstream Event</th>
<th scope="col">Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/relationships/2056">Binding to ACE2</a></td>
<td>adjacent</td>
<td>SARS-CoV-2 cell entry </td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2310">SARS-CoV-2 cell entry </a></td>
<td>adjacent</td>
<td>Increased SARS-CoV-2 production</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2545">Increased SARS-CoV-2 production</a></td>
<td>adjacent</td>
<td>Sustentacular cells, decrease </td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2362">Sustentacular cells, decrease </a></td>
<td>adjacent</td>
<td>olfactory sensory neurons, decrease </td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2363">olfactory sensory neurons, decrease </a></td>
<td>adjacent</td>
<td>Olfactory epithelium degeneration</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2364">Olfactory epithelium degeneration</a></td>
<td>adjacent</td>
<td>impaired olfactory function (anosmia) </td>
<td></td>
<td></td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2365">Sustentacular cells, decrease </a></td>
<td>non-adjacent</td>
<td>impaired olfactory function (anosmia) </td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2366">olfactory sensory neurons, decrease </a></td>
<td>non-adjacent</td>
<td>impaired olfactory function (anosmia) </td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="overall_assessment">
<h2>Overall Assessment of the AOP</h2>
</div>
<div id="considerations_for_potential_applicaitons">
</div>
<div id="references">
<h2>References</h2>
</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of MIEs in this AOP</h3>
<h4><a href="/events/1739">Event: 1739: Binding to ACE2</a></h4>
<h5>Short Name: Binding to ACE2</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>receptor binding</td>
<td>angiotensin-converting enzyme 2</td>
<td>occurrence</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/320">Aop:320 - Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortality</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/374">Aop:374 - Binding of Sars-CoV-2 spike protein to ACE 2 receptors expressed on brain cells (neuronal and non-neuronal) leads to neuroinflammation resulting in encephalitis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/381">Aop:381 - Binding of viral S-glycoprotein to ACE2 receptor leading to dysgeusia</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/385">Aop:385 - Viral spike protein interaction with ACE2 leads to microvascular dysfunction, via ACE2 dysregulation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/395">Aop:395 - Binding of Sars-CoV-2 spike protein to ACE 2 receptors expressed on pericytes leads to disseminated intravascular coagulation resulting in cerebrovascular disease (stroke)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/406">Aop:406 - SARS-CoV-2 infection leading to hyperinflammation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/407">Aop:407 - SARS-CoV-2 infection leading to pyroptosis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/426">Aop:426 - SARS-CoV-2 spike protein binding to ACE2 receptors expressed on pericytes leads to endothelial cell dysfunction, microvascular injury and myocardial infarction. </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/427">Aop:427 - ACE2 downregulation following SARS-CoV-2 infection triggers dysregulation of RAAS and can lead to heart failure.</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/422">Aop:422 - Binding of SARS-CoV-2 to ACE2 in enterocytes leads to intestinal barrier disruption</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/428">Aop:428 - Binding of S-protein to ACE2 in enterocytes induces ACE2 dysregulation leading to gut dysbiosis </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/430">Aop:430 - Binding of SARS-CoV-2 to ACE2 leads to viral infection proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/379">Aop:379 - Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/468">Aop:468 - Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Sars-CoV-2</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>cell</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>organ</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Homo sapiens</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Mustela lutreola</td>
<td>Mustela lutreola</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9666" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Felis catus</td>
<td>Felis catus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9685" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Panthera tigris</td>
<td>Panthera tigris</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9694" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Canis familiaris</td>
<td>Canis lupus familiaris</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9615" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult, reproductively mature</td>
<td>High</td>
</tr>
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
~~The KE is applicable to broad species/life stage/sex. The binding of ACE2 occure in the cells which express ACE2. ~~
The KE is applicable to broad species/life stage/sex. The binding of ACE2 occurs in the cells which express ACE2.
<h4>Key Event Description</h4>
Angiotensin-converting enzyme 2 (<a href="https://www.genecards.org/cgi-bin/carddisp.pl?gene=ACE2">ACE2</a>) is an enzyme that can be found either attached to the membrane of the cells (mACE2) in many tissues and in a soluble form form (sACE2).
A table on ACE2 expression levels according to tissues (Kim et al.)
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:medium none; height:806px; width:1049px">
<tbody>
<tr>
<td style="background-color:#a6a6a6; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:146px">
</td>
<td style="background-color:#a6a6a6; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:146px">
Sample size
</td>
<td style="background-color:#a6a6a6; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:147px">
ACE2 mean expression
</td>
<td style="background-color:#a6a6a6; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:147px">
Standard deviation of expression
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Intestine
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
51
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
9.50
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
1.183
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Kidney
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
129
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
9.20
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
2.410
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Stomach
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
35
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
8.25
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
3.715
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Bile duct
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
9
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
7.23
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
1.163
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Liver
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
50
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
6.86
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
1.351
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Oral cavity
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
32
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
6.23
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
1.271
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Lung
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
110
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
5.83
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
0.710
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Thyroid
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
59
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
5.65
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
0.646
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Esophagus
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
11
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
5.31
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
1.552
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Bladder
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
19
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
5.10
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
1.809
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Breast
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
113
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
4.61
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
0.961
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Uterus
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
25
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
4.37
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
1.125
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
Protaste
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
52
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
4.35
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
1.905
</td>
</tr>
</tbody>
</table>
ACE2 receptors in the brain (endothelial, neuronal and glial cells):
The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010). The neurons and glial cells, like astrocytes and microglia also express ACE-2.
In the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020). Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018).
ACE2 receptors in the intestines
The highest levels of ACE2 are found at the luminal surface of the enterocytes, the differentiated epithelial cells in the small intestine, lower levels in the crypt cells and in the colon (Liang et al, 2020; Hashimoto et al., 2012, Fairweather et al. 2012; Kowalczuk et al. 2008).
<h4>How it is Measured or Detected</h4>
In vitro methods supporting interaction between ACE2 and SARS-CoV-2 spike protein
Several reports using surface plasmon resonance (SPR) or biolayer interferometry binding (BLI) approaches. to study the interaction between recombinant ACE2 and S proteins have determined a dissociation constant (Kd) for SARS-CoV S and SARS-CoV-2 S as follow,
<table cellspacing="0" class="Table" style="border-collapse:collapse; width:568px">
<tbody>
<tr>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:28px; width:176px">
Reference
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:102px">
ACE2 protein
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:140px">
SARS-CoV S
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:151px">
SARS-CoV2 S
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:128px">
Method
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:156px">
Measured Kd
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
doi:<a class="id-link" href="https://doi.org/10.1126/science.abb2507" rel="noopener" target="_blank">10.1126/science.abb2507</a>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
1–615 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
306–577 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
SPR
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
325.8 nM
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
1–1208 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
14.7 nM
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
doi:<a class="id-link" href="https://doi.org/10.1001/jama.2020.3786" rel="noopener" target="_blank">10.1001/jama.2020.3786</a>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
19–615 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
306–527 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
SPR
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
408.7 nM
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
319–541 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
133.3 nM
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
<a href="https://elifesciences.org/articles/61390#bib67" style="color:blue; text-decoration:underline">Lan et al., 2020</a>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
19–615 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
306–527 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
SPR
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
31.6 nM
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
319–541 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
4.7 nM
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
doi:<a class="id-link" href="https://doi.org/10.1016/j.cell.2020.02.058" rel="noopener" target="_blank">10.1016/j.cell.2020.02.058</a>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
1–614 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
306–575 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
BLI
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
1.2 nM
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
328–533 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
5 nM
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
doi:<a class="id-link" href="https://doi.org/10.1126/science.abb2507" rel="noopener" target="_blank">10.1126/science.abb2507</a>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
1–615 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
306–577 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
BLI
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
13.7 nM
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
319–591 aa
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
34.6 nM
</td>
</tr>
</tbody>
</table>
Pseudo typed vesicular stomatitis virus expressing SARS-CoV-2 S (VSV-SARS-S2) expression system can be used efficiently infects cell lines, with Calu-3 human lung adenocarcinoma epithelial cell line, CaCo-2 human colorectal adenocarcinoma colon epithelial cell line and Vero African grey monkey kidney epithelial cell line being the most permissive (Hoffmann et al., 2020; Ou et al., 2020).  It can be measured using a wide variety of assays targeting different biological phases of infection and altered cell membrane permeability and cell organelle signaling pathway. Other assay measured alteration in the levels of permissive cell lines all express ACE2 or hACE2-expressing 293T cell (e.g. pNUO1-hACE2, pFUSE-hIgG1-Fc2), as previously demonstrated by indirect immunofluorescence (IF) or by immunoblotting are associated with ELISA(W Tai et al., nature 2020). To prioritize the identified potential KEs for selection and to select a KE to serve as a case study, further in-silico data that ACE2 binds to SARS-CoV-2 S is necessary for virus entry. The above analysis outlined can be used evidence-based assessment of molecular evidence as a MIE.
<h4>References</h4>
de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.
Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.
Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.
Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.
Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.
Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.
Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.
Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468
Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.
Xia, H. and Lazartigues, E.  Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010  Rep. 12 (3), 170– 175
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/1738">Event: 1738: SARS-CoV-2 cell entry </a></h4>
<h5>Short Name: SARS-CoV-2 cell entry </h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>membrane fusion</td>
<td>transmembrane protease serine 2</td>
<td>occurrence</td>
</tr>
<tr>
<td>endocytosis involved in viral entry into host cell</td>
<td>cathepsin L1 (human)</td>
<td>occurrence</td>
</tr>
<tr>
<td>viral entry into host cell</td>
<td>viral genome</td>
<td>occurrence</td>
</tr>
<tr>
<td>viral entry into host cell</td>
<td>viral protein</td>
<td>occurrence</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/320">Aop:320 - Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/379">Aop:379 - Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/395">Aop:395 - Binding of Sars-CoV-2 spike protein to ACE 2 receptors expressed on pericytes leads to disseminated intravascular coagulation resulting in cerebrovascular disease (stroke)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/406">Aop:406 - SARS-CoV-2 infection leading to hyperinflammation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/407">Aop:407 - SARS-CoV-2 infection leading to pyroptosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/426">Aop:426 - SARS-CoV-2 spike protein binding to ACE2 receptors expressed on pericytes leads to endothelial cell dysfunction, microvascular injury and myocardial infarction. </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/422">Aop:422 - Binding of SARS-CoV-2 to ACE2 in enterocytes leads to intestinal barrier disruption</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/430">Aop:430 - Binding of SARS-CoV-2 to ACE2 leads to viral infection proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/468">Aop:468 - Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Sars-CoV-2</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Cellular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>cell</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>organ</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Homo sapiens</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Manis javanica</td>
<td>Manis javanica</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9974" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Canis familiaris</td>
<td>Canis lupus familiaris</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9615" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Macaca fascicularis</td>
<td>Macaca fascicularis</td>
<td>Not Specified</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9541" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Mesocricetus auratus</td>
<td>Mesocricetus auratus</td>
<td>Not Specified</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10036" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Mustela putorius furo</td>
<td>Mustela putorius furo</td>
<td>Not Specified</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9669" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Felis catus</td>
<td>Felis catus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9685" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Mustela lutreola</td>
<td>Mustela lutreola</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9666" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Neovison vison</td>
<td>Neovison vison</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=452646" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Panthera tigris</td>
<td>Panthera tigris</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9694" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
TMPRSS2 vertebrates (Lam et al., 2020)
NRP1 in human & rodents (but also present in monkey and other vertebrates (Lu and Meng, 2015)
The ability for SARS-CoV-2 to use multiple host pathways for viral entry, means that it is critical to map which viral entry pathway is prevalent in specific cell types. This is key for understanding coronavirus biology, but also use informed decisions to select cells for cell-based genetic and small-molecule screens and to interpret data. In fact, a combination of protease inhibitors that block both TRMPSS2 and cathepsin L is the most efficient combination to block coronavirus infection (Yamamoto et al., 2020, Shang et al., 2020, Shirato et al., 2018). In accordance, SARS-CoV-2 entry processes are highly dependent on endocytosis and endocytic maturation in cells that do not express TMPRSS2, such as VeroE6 or 293T cells (Murgolo et al., 2021, Kang et al., 2020, Mirabelli et al., 2020, Riva et al., 2020). However, even in these cells, heterologous expression of TMPRSS2 abrogates the pharmacological blockade of cathepsin inhibitors (Kawase et al., 2012, Hoffmann et al., 2020a). Treatment of SARS-CoV-2 with trypsin enables viral cell surface entry, even when TMPRSS2 is absent. Moreover, TMPRSS2 is more efficient to promote viral entry than cathepsins (Lamers et al., 2020), as when both factors are present,d cathepsin inhibitors are less effective than TMPRSS2 inhibitors (Hoffmann et al., 2020b). Therefore it is critical to map which cells contain the different types of proteases.
In summary, TMPRSS2 appears to be expressed in a wide range of healthy adult organs, but in restricted cell types, including:
<ul>
<li>AT2 and clara cells of lungs</li>
<li>sinusoidal endothelium, and hepatocyte of the liver, </li>
<li>endocrine cells of the prostate, </li>
<li>goblet cells , and enterocytes of the small intestine, </li>
<li>intercalated cells, and the proximal tubular of the kidney.</li>
<li>Ciliated, secretory and suprabasal of nasal</li>
<li>spermatogonial stem cells of testes</li>
<li>cyto tropoblast and peri vascular cells of placenta</li>
<li>The nasal epithelium expresses various combinations of factors that, in principle, could facilitate SARS-CoV-2 infection, but it also expresses robust basal levels of RFs, which may act as a strong protective barrier in this tissue.</li>
</ul>
There is a shift in TMPRSS2 regulation during nasal epithelium differentiation in young individuals that is not occurring in old individuals (Lin et al., 1999, Lucas et al., 2008, Singh et al., 2020).
Only a small minority of human respiratory and intestinal cells have genes that express both ACE2 and TMPRSS2. Amongst the ones that do, three main cell types were identified: A) lung cells called type II pneumocytes (which help maintain air sacs, known as alveoli); B) intestinal cells called enterocytes, which help the body absorb nutrients; and C) goblet cells in the nasal passage, which secrete mucus (Ziegler et al., 2020).
The clinical manifestations of COVID‐19 include not only complications from acute myocardial injury, elevated liver enzymes, and acute kidney injury in patients presenting to hospitals, but also gastrointestinal symptoms in community patients experiencing milder forms of the disease (Madjid et al., 2020, Pan et al., 2020).
NRP-1:
All life stages
The expression of isoforms 1 (NRP1) and 2 (NRP2) does not seem to overlap. Isoform 1 is expressed by the blood vessels of different tissues. In the developing embryo it is found predominantly in the nervous system. In adult tissues, it is highly expressed in heart and placenta; moderately in lung, liver, skeletal muscle, kidney and pancreas; and low in adult brain. Isoform 2 is found in liver hepatocytes, kidney distal and proximal tubules. Expressed in colon and 234 other tissues with Low tissue specificity (UniProtKB).
The expression of NRP1 protein in gastric cancer was not related to gender or age (Cao et al., 2020).
Sex Applicability:
TMPRSS2:
Androgen receptors (ARs) play a key role in the transcription of TMPRSS2 (Fig. 1). This may explain the predominance of males to COVID-19 infection, fatality, and severity because males tend to have a higher expression and activation of ARs than females, which is due to the presence of dihydrotestosterone (DHT).
Regulation of COVID-19 severity and fatality by sex hormones. Females have aromatase, the enzyme that converts androgen substrates into estrogen. On the other hand, males have steroid 5α reductase, the enzyme that is responsible for the conversion of testosterone into dihydrotestosterone (DHT). In case of males, DHT activates androgen receptor (AR) that binds to the androgen response element (ARE) present in the promoter of TMPRSS2 gene, leading to its transcription. This ultimately results into enhanced processing of viral spike protein for greater entry and spread of SARS-CoV-2 into host cells. On the other hand,in females, estrogen activates estrogen receptor (ER), which binds to the estrogen response element (ERE) present in the promoter of eNOS gene to drive its transcription and catalyze the formation of nitric oxide (NO) from L-arginine. This NO is involved in vasodilation as well as inhibition of viral replication.
NRP-1:
For more information difference of NRP1 expression between male and female see <a href="https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue">https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue</a>.
The expression of NRP1 protein in gastric cancer was not related to gender, age. The expression of NRP1 protein in gastric cancer is closely correlated to clinical stage, tumor size, TNM stage, differentiation, and lymph node metastasis (Cao et al., 2020).
SARS-CoV-2 Spike protein co-opts VEGF-A/Neuropilin-1 receptor signalling to induce analgesia had same results on both male and female rodents (Moutal et al., 2020).
<h4>Key Event Description</h4>
Coronavirus is recognized by the binding of S protein on the viral surface and angiotensin-converting enzyme 2 (ACE2) receptor on the cellular membrane, followed by viral entry via processing of S protein by transmembrane serine protease 2 (TMPRSS2) (Hoffmann et al., 2020b). ACE2 is expressed on epithelial cells of the lung and intestine, and also can be found in the heart, kidney, adipose, and male and female reproductive tissues (Lukassen et al., 2020, Lamers et al., 2020, Chen et al., 2020, Jing et al., 2020, Subramanian et al., 2020).
SARS-CoV-2 is an enveloped virus characterized by displaying spike proteins at the viral surface (Juraszek et al., 2021). Spike is critical for viral entry (Hoffmann et al., 2020b) and is the primary target of vaccines and therapeutic strategies, as this protein is the immunodominant target for antibodies (Yuan et al., 2020, Ju et al., 2020, Robbiani et al., 2020, Premkumar et al., 2020, Liu et al., 2020). Spike is composed of S1 and S2 subdomains. S1 contains the N-terminal (NTD) and receptor-binding (RBD) domains, and the S2 contains the fusion peptide (FP), heptad repeat 1 (HR1) and HR2, the transmembrane (TM) and cytoplasmic domains (CD) (Lan et al., 2020). S1 leads to the recognition of the angiotensin-converting enzyme 2 (ACE2) receptor and S2 is involved in membrane fusion (Hoffmann et al., 2020b, Letko et al., 2020, Shang et al., 2020).
Upon binding to ACE2, the spike protein needs to be activated (or primed) through proteolytic cleavage (by a host protease) to allow membrane fusion. Fusion is a key step in viral entry as it is the way to release SARS-CoV-2 genetic material inside the cell. Cleavage happens between its spike’s S1 and S2 domains, liberating S2 that inserts its N-terminal domain into a host cell membrane and mediates membrane fusion (Millet and Whittaker, 2018). Many proteases were identified to activate coronaviruses including furin, cathepsin L, trypsin-like serine proteases TMPRSS2, TMPRSS4, TMPRSS11, and human airway trypsin-like protease (HATs). These may operate at four different stages of the<a href="https://www.wikipathways.org/index.php/Pathway:WP4846"> virus infection cycle</a>: (a) pro-protein convertases (e.g., furin) during virus packaging in virus-producing cells, (b) extracellular proteases (e.g., elastase) after virus release into extracellular space, (c) cell surface proteases [e.g., type II transmembrane serine protease (TMPRSS2)] after virus attachment to virus-targeting cells, and (d ) lysosomal proteases (e.g., cathepsin L) after virus endocytosis in virus-targeting cells (Li, 2016). SARS-CoV-2 lipidic envelope may fuse with two distinct membrane types, depending on the host protease(s) responsible for cleaving the spike protein: (i) cell surface following activation by serine proteases such as TMPRSS2 and furin (Hoffmann et al., 2020b); or (ii) endocytic pathway within the endosomal–lysosomal compartments including processing by lysosomal cathepsin L (Yang and Shen, 2020). These flexibility for host cell factors mediating viral entry, highlights that the availability of factors existing in a cell type dictates the mechanism of viral entry (Kawase et al., 2012). When TMPRSS2 (or other serine proteases such as TMPRSS4 (Zang et al., 2020) or human airway trypsin-like protease [HAT] (Bestle et al., 2020a)) is expressed, fusion of the virus with the cell surface membrane is preferred (Shirato et al., 2018), while in their absence, the virus can penetrate the cell by endocytosis (Kawase et al., 2012). A third factor has also been shown to facilitate SARS-CoV-2 entry in cells that have ACE2 and even promote, although to very low levels, SARS-CoV-2 entry in cells that lack ACE2 and TMPRSS2 which is the neuropilin-1 (NRP-1) (Cantuti-Castelvetri et al., 2020). This key event deals with SARS-CoV-2 entry in host cells and is divided in three categories: TMPRSS2, capthesin L and NRP-1.
TMPRSS2 Spike cleavage:
TMPRSS2 (transmembrane serine protease 2, (<a href="https://www.ncbi.nlm.nih.gov/gene/7113" style="color:blue; text-decoration:underline">https://www.ncbi.nlm.nih.gov/gene/7113</a>) is a cell-surface protease (Hartenian et al., 2020) that facilitates entry of viruses into host cells by proteolytically cleaving and activating viral envelope glycoproteins. Viruses found to use this protein for cell entry include Influenza virus and the human coronaviruses HCoV-229E, MERS-CoV, SARS-CoV and SARS-CoV-2 (COVID-19 virus).
TMPRSS2 is a membrane bound serine protease also known as epitheliasin. TMPRSS2 belongs to the S1A class of serine proteases alongside proteins such as factor Xa and trypsin. Whilst there is evidence that TMPRSS2 autoclaves to generate a secreted protease, its physiological function has not been clearly identified. However, it is known to play a crucial role in facilitating entry of coronavirus particles into cells by cleaving the spike protein (Huggins, 2020).
After ACE2 receptor binding, SARS-CoV-2 S proteins can be subsequently cleaved and activated by host cell-surface protease at the S1/S2 and S2’ sites, generating the subunits S1 and S2 that remain non-covalently linked. Cleavage leads to activation of the S2 domain that drives fusion of the viral and host membranes (Hartenian et al., 2020, Walls et al., 2016). For other coronaviruses, processing of spike was proposed to be sequential with S1/S2 cleavage preceding that of S2. Cleavage at S1/S2 may be crucial for inducing conformational changes required for receptor binding or exposure of the S2 site to host proteases.
The S1/S2 site of SARS-CoV-2 S protein contains an insertion of four amino acids providing a minimal furin cleavage site (RRAR685↓) (that is absent in SARS-CoV). Interestingly, the furin cleavage site has been implicated in increased viral pathogensis (Bestle et al., 2020b, Huggins, 2020). Processing of the spike protein by furin at the S1/S2 cleavage site is thought to occur following viral replication in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) (Hasan et al., 2020). The spike S2’ cleavage site of SARS-CoV-2 possesses a paired dibasic motif with a single KR segment (KR815↓) (as SARS-CoV) that is recognized by trypsin-like serine proteases such as TMPRSS2. The current data support a model for SARS-CoV-2 entry in which furin-mediated cleavage at the S1/S2 site pre-primes spike during biogenesis, facilitating the activation for membrane fusion by a second cleavage event at S2’ by TMPRSS2 following ACE2 binding (Bestle et al., 2020b, Johnson et al., 2020).
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:166px">
Virus
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:233px">
S1/S2 site
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:236px">
S2’ site
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:166px">
SARS-CoV-2
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:233px">
TNSPRRAR|SVA
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:236px">
PSKPSKR|SFIEDL
</td>
</tr>
<tr>
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SARS-CoV
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:233px">
S----LLR|STS
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:236px">
PLKPTKR|SFIEDL
</td>
</tr>
</tbody>
</table>
Camostat mesylate, an inhibitor of TMPRSS2, blocks SARS-CoV-2 infection of lung cells like Calu-3 cells but not Huh7.5 and Vero E6 cells. Cell entry was assessed using a viral isolate and viral pseudotypes (artificial viruses) expressing the COVID-19 spike (S) protein. The ability of the viral pseudotypes (expressing S protein from SARS-CoV and SARS-CoV-2) to enter human and animal cell lines was demonstrated, showing that SARS-CoV-2 can enter similar cell lines as SARS-CoV. Amino acid analysis and cell culture experiments showed that, like SARS-CoV, SARS-CoV-2 spike protein binds to human and bat angiotensin-converting enzyme 2 (ACE2) and uses a cellular protease TMPRSS2 for priming. Priming activates the spike protein to facilitate viral fusion and entry into cells. Cell culture experiments were performed using immortalized cell lines and primary human lung cells (Hoffmann et al., 2020b, Rahman et al., 2020).
Spike binding to neuropilin-1:
Neuropilin-1 (NRP1) is a transmembrane glycoprotein that serves as a cell surface receptor for semaphorins and various ligands involved in angiogenesis in vertebrates. NRP1 is expressed in neurons, blood vessels (endothelial cells), immune cells and many other cell types in the mammalian body (maternal fetal interface) and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function (Raimondi et al., 2016).  NRP1 is well described as a co-receptor for members of the class 3 semaphorins (SEMA3) or vascular endothelial growth factors (VEGFs) (Gelfand et al., 2014). Structurally, NRP1 comprises seven sub-domains, of which the first five are extracellular; two CUB domains (a1 and a2), two coagulation factor V/VIII domains (FV/VIII; b1 and b2) and a meprin, A5 μ-phosphatase domain (MAM; c). NRP1 contains only a short cytosolic tail with a PDZ-binding domain lacking internal signaling activity. The different ligand families bind to different sites of NRP1; SEMA3A binding requires the first three sub-domains of NRP1 (a1, a2, and b1), whereas binding of VEGF-A requires the b1 and b2 domains (Muhl et al., 2017). Additional studies conducted by means of in silico computational technology to identify and validate inhibitors of the interaction between NRP1 and SARS-CoV-2 Spike protein are reported in (Perez-Miller et al., 2020).  Represents a schematic picture of VEGF-A triggered phosphorylation of VEGF-R2. Screening of NRP-1/VEGF-A165 inhibitors by in-cell Western (Perez-Miller et al., 2020).v NRP1 acts as a co-receptor for SARS-CoV-2.
NRP1 is a receptor for furin-cleaved SARS-CoV-2 spike peptide (Cantuti-Castelvetri et al., 2020, Daly et al., 2020, Johnson et al., 2020). Blockade of NRP1 reduces infectivity and entry, and alteration of the furin site leads to loss of NRP1 dependence, reduced replication in Calu3, augmented replication in Vero E6, and attenuated disease in a hamster pathogenesis disease model (Johnson et al., 2020). In fact, a small sequence of amino acids was found that appeared to mimic a protein sequence found in human proteins that interact with NRP1. The spike protein of SARS-CoV-2 binding with NRP1 aids viral infection of human cells. This was confirmed by applying a range of structural and biochemical approaches to establish that the spike protein of SARS-CoV-2 does indeed bind to NRP1. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic RRAR C-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors. It was reported that the S1 CendR motif directly bound NRP1 by X-ray crystallography and biochemical approaches. Blocking this interaction using RNAi or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture (Daly et al., 2020).
NRP1, known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was revealed by a monoclonal blocking antibody against NRP1. It was found that a SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells faced the nasal cavity (Cantuti-Castelvetri et al., 2020). Furthermore, it has been found that NRP1 is a new potential SARS‑CoV‑2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID‑19.  Preclinical studies have suggested that NRP1, a transmembrane receptor that lacks a cytosolic protein kinase domain and exhibits high expression in the respiratory and olfactory epithelium, may also be implicated in COVID‑19 by enhancing the entry of SARS‑CoV‑2 into the brain through the olfactory epithelium. NRP1 is also expressed in the CNS, including olfactory‑related regions such as the olfactory tubercles and paraolfactory gyri. Supporting the potential role of NRP1 as an additional SARS‑CoV‑2 infection mediator implicated in the neurologic manifestations of COVID‑19. Accordingly, the neurotropism of SARS‑CoV‑2 via NRP1‑expressing cells in the CNS merits further investigation (Davies et al., 2020).
Up-regulation of NRP1 protein in diabetic kidney cells hints at its importance in a population at risk of severe COVID-19. Involvement of NRP-1 in immune function is compelling, given the role of an exaggerated immune response in disease severity and deaths due to COVID-19. NRP-1 has been suggested to be an immune checkpoint of T cell memory. It is unknown whether involvement and up-regulation of NRP-1 in COVID-19 may translate into disease outcome and long-term consequences, including possible immune dysfunction (Mayi et al., 2021).
The main feature of NRP1 co-receptor is to form complexes with multiple other receptors. Hence, there is a competition between receptors to complex with NRP-1, which may determine their abilities both quantitatively and qualitatively to transduce signals. It is tempting to hypothesize that the occupancy of NRP-1 with one receptor may thus decrease its availability for virus entry. Recent proteomics work has shown that NRP-1 can form a complex with the α7 nicotinic receptor in mice. Both receptors are expressed in the human nasal and pulmonary epithelium (Mayi et al., 2021).
NRP1, is highly expressed in the respiratory and olfactory epithelium; it is also expressed in the CNS, including olfactory related regions such as the olfactory tubercles and paraolfactory gyri (Davies et al., 2020).
More information on tissue distribution and protein expression of NRP1 can be found in https://www.proteinatlas.org/ENSG000000992 50-NRP1
Spike entry via lysosomal cathepsins and endocytosis:
Evidence shows the role of TMPRSS2 and other serine proteases in activating the coronavirus spike protein for plasma membrane fusion. However, studies using various cell culture systems showed that SARS-CoV2 could enter cells via an alternative endosomal–lysosomal pathway. Evidence came from studies demonstrating that lysosomotropic agents reduced SARS-CoV replication in cells lacking TMPRSS2 and other studies, using highly potent and specific small-molecule cathepsin inhibitors, to understand the role of cathepsins in processing and activating the spike for membrane fusion, mainly of cathepsin L (one of the 11 cathepsins) (Rossi et al., 2004, Simmons et al., 2005). SARS-CoV-2 and other coronaviruses can establish infection through endosomal entry in commonly used in vitro cell culture systems. Of relevance, inhibitors of the endosomal pathway, as the cathepsin inhibitor Z-FA-FMK and PIKfyve inhibitor apilimod, blocked viral entry in Huh7.5 and Vero E6 cells but not Calu-3 cells.
Viral entry leads to delivery of virion proteins and translation of viral proteins immediately:
Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [Kim D, et al., 2020]. The virus contains a 29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively that contain cis-acting secondary RNA structures essential for RNA synthesis [Huston N. C. et al., 2021]. The genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [Budzilowicx C.J., et al., 1985]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [Snijder E. J., et al., 2003]. Preceding each ORF there are other TRSs called the body TRS (TRS B). The 5′ two-thirds of the genome contains two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [Di H., et al., 2018]. Genome expression starts with the translation of two large ORFs of the 5’ two-thirds: ORF1a of 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a programmed (- 1) ribosomal frameshifting [Snijder E. J., et al., 2003], yielding pp1a and pp1ab. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a, nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain [Sacco M. D. et al., 2020]. The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication and transcription complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [Klein S., et al., 2021, Snijder E. J., et al., 2020, V'Kovski P. , et al., 2021]. This association leads to replication factories or organelles, that are originate new membranous structures that are observed by electron mciroscopy . They are a feature of all coronaviridae and the site of viral replication and transcription hidden from innate immune molecules.
<h4>How it is Measured or Detected</h4>
SARS-CoV2 entry can be determined by many different ways:
1) quantitative RT-PCR specific to the subgenomic mRNA of the N transcript, in cells manipulated with host factors that express of not TMPRSS2, cathepsinL, neuropilin-1, hACE2 [Glowacka I, et al. (2011)], or exogenous addition of HAT or furin.
2) using spike-pseudotyped viral particles expressing GFP/luciferase/bgalactosidase and comparing with vesicular stomatitis virus G seudotyped particles expressing the same reporter analysed in manipulated cultured with cell lines, followed by determining fluorescence, biolumincescence, luciferase activity in cell lysates  [Hoffmann M, et al. (2020)].
TMPRSS2:
TMPRSS2 gene expression can be measured by RNAseq and microarray (Baughn et al., 2020).
Expression levels of TMPRSS2 can be measured by RNA in situ hybridization (RNA-ISH) (Qiao et al., 2020)
NRP-1:
Several methods have been identified in the literature for measuring and detecting NRP1 receptor binding. Briefly described:
<ol>
<li>X-ray crystallography  and biochemical approaches help to show that the S1 CendR motif directly bound NRP1 (1).  Binding of the S1 fragment to NRP1 was assessed and ability of SARS-CoV-2 to use NRP1 to infect cells was measured in angiotensin-converting enzyme-2 (ACE-2)-expressing cell lines by knocking out NRP1 expression, blocking NRP1 with 3 different anti-NRP1 monoclonal antibodies, or using NRP1 small molecule antagonists (Centers for Disease Control and Prevention, 2020, Daly et al., 2020).</li>
</ol>
Key findings (Centers for Disease Control and Prevention, 2020, Daly et al., 2020):
• The S1 fragment of the cleaved SARS-CoV-2 spike protein binds to the cell surface receptor neuropilin-1 (NRP1).
• SARS-CoV-2 utilizes NRP1 for cell entry as evidenced by decreased infectivity of cells in the presence of: NRP1 deletion (p <0.01). Three different anti-NRP1 monoclonal antibodies (p <0.001). Selective NRP1 antagonist, EG00229 (p <0.01).
<ol start="2">
<li>Cell lines were modified to express ACE2 and TMPRSS2, the two known SARS-CoV-2 host factors, and NRP1 to assess the contribution of NRP1 to infection. Autopsy specimens from multiple airway sites were stained with antibodies against SARS-CoV-2 proteins, ACE2, and NRP1, to visualize co-localization of proteins (6, 15).</li>
</ol>
Key findings (Cantuti-Castelvetri et al., 2020, Centers for Disease Control and Prevention, 2020):
• Infectivity of cells expressing angiotensin converting enzyme-2 (ACE2, receptor for SARS-CoV-2), transmembrane protease serine-2 (TSS2, primes the Spike [S] protein), and neuropilin-1 (NRP1) with pseudovirus expressing the SARS-CoV-2 S1 protein was approximately 3-fold higher than in cells expressing either ACE2 or TSS2 alone (p<0.05).
• Analysis of autopsy tissue from COVID-19 patients showed co-localization of the SARS-CoV-2 spike (S) protein and NRP1 in olfactory and respiratory epithelium.
Virtual screen of nearly 0.5 million compounds against the NRP-1 CendR site, resulting in nearly 1,000 hits. A pharmacophore model was derived from the identified ligands, considering both steric and electronic requirements. Preparation of receptor protein and grid for virtual screening, docking of known NRP-1 targeting compounds, ELISA based NRP1-VEGF-A165 protein binding assay; more details on methodology in the referenced paper (Perez-Miller et al., 2020)
<h4>References</h4>
BAUGHN, L. B., SHARMA, N., ELHAIK, E., SEKULIC, A., BRYCE, A. H. & FONSECA, R. 2020. Targeting TMPRSS2 in SARS-CoV-2 Infection. Mayo Clin Proc, 95, 1989-1999.
BESTLE, D., HEINDL, M. R., LIMBURG, H., VAN LAM VAN, T., PILGRAM, O., MOULTON, H., STEIN, D. A., HARDES, K., EICKMANN, M., DOLNIK, O., ROHDE, C., KLENK, H.-D., GARTEN, W., STEINMETZER, T. & BÖTTCHER-FRIEBERTSHÄUSER, E. 2020a. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. Life Science Alliance, 3.
BESTLE, D., HEINDL, M. R., LIMBURG, H., VAN LAM VAN, T., PILGRAM, O., MOULTON, H., STEIN, D. A., HARDES, K., EICKMANN, M., DOLNIK, O., ROHDE, C., KLENK, H. D., GARTEN, W., STEINMETZER, T. & BOTTCHER-FRIEBERTSHAUSER, E. 2020b. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. Life Sci Alliance, 3.
BUDZILOWICZ, C.J., WILCZYNSKI, S.P., AND WEISS, S.R. (1985). Three intergenic regions of coronavirus mouse hepatitis virus strain A59 genome RNA contain a common nucleotide sequence that is homologous to the 3' end of the viral mRNA leader sequence. J Virol 53, 834-840.
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<h4><a href="/events/1847">Event: 1847: Increased SARS-CoV-2 production</a></h4>
<h5>Short Name: SARS-CoV-2 production</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>viral RNA genome replication</td>
<td>viral RNA-directed RNA polymerase complex</td>
<td>increased</td>
</tr>
<tr>
<td>positive stranded viral RNA replication</td>
<td>viral RNA-directed RNA polymerase complex</td>
<td>increased</td>
</tr>
<tr>
<td>viral RNA genome packaging</td>
<td>viral assembly compartment</td>
<td>increased</td>
</tr>
<tr>
<td>mRNA transcription</td>
<td>ssRNA viral genome</td>
<td>increased</td>
</tr>
<tr>
<td>viral translation</td>
<td>ssRNA viral genome</td>
<td>increased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/379">Aop:379 - Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/320">Aop:320 - Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/406">Aop:406 - SARS-CoV-2 infection leading to hyperinflammation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/407">Aop:407 - SARS-CoV-2 infection leading to pyroptosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/422">Aop:422 - Binding of SARS-CoV-2 to ACE2 in enterocytes leads to intestinal barrier disruption</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/430">Aop:430 - Binding of SARS-CoV-2 to ACE2 leads to viral infection proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/468">Aop:468 - Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Sars-CoV-2</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Cellular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>cell</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>organ</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Homo sapiens</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Mus musculus</td>
<td>Mus musculus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Mustela putorius furo</td>
<td>Mustela putorius furo</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9669" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Broad mammalian host range has been demonstrated based on spike protein tropism for and binding to ACE2 [Conceicao et al. 2020; Wu et al. 2020] and cross-species ACE2 structural analysis [Damas et al. 2020]. No literature has been found on primary translation and molecular interactions of nsps in non-human host cells, but it is assumed to occur if the virus replicates in other species.
Very broad mammalian tropism: human, bat, cat, dog, civet, ferret, horse, pig, sheep, goat, water buffalo, cattle, rabbit, hamster, mouse
<h4>Key Event Description</h4>
This KE1847 "Increase coronavirus production" deals with how the genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is translated, replicated, and transcribed in detail, and how the genomic RNA (gRNA) is packaged, and the virions are assembled and released from the cell.
Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [D. Kim et al.]. The virus contains a 29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively [E. J. Snijder et al.] that contain cis-acting secondary RNA structures essential for RNA synthesis [N. C. Huston et al.]. The genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [C.J. Budzilowicx et al.]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [E. J. Snijder et al.]. Preceding each ORF there are other TRSs called the body TRS (TRS B). The 5′ two-thirds of the genome contains two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [H. Di et al.]. Genome expression starts with the translation of two large ORFs of the 5’ two-thirds: ORF1a of 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a programmed (- 1) ribosomal frameshifting [E. J. Snider et al.], yielding pp1a and pp1ab. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a, nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain [M. D. Sacco et al.]. The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication and transcription complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [S. Klein et al., E. J. Snijder et al., P. V'Kovski, et al.]. Besides replication, which yields the positive-sense gRNA, the replicase also mediates transcription leading to the synthesis of a nested set of subgenomic (sg) mRNAs to express all ORFs downstream of ORF1b that encode structural and accessory viral proteins. These localize to the 3′ one-third of the genome, as stated above, and result in a 3′ coterminal nested set of 7–9 mRNAs that share ~70–90 nucleotide (nt) in the 5′ leader and that is identical to the 5′ end of the genome [P. Liu, and J. Leibowitz]. sgRNAs encode conserved structural proteins (spike protein [S], envelope protein [E], membrane protein [M], and nucleocapsid protein [N]), and several accessory proteins. SARS-CoV-2 is known to have at least six accessory proteins (3a, 6, 7a, 7b, 8, and 10). Overall the virus is predicted to express 29 proteins [D. Kim et al.]. The gRNA is packaged by the structural proteins to assemble progeny virions.
Viral translation:
SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of ~30 kb, sequence orientation in a 5’ to 3’ direction typical of positive sense and reflective of the resulting mRNA [D. Kim et al.]. The SARS-CoV-2 genome contains a 5’-untranslated region (UTR; 265 bp), ORF1ab (21,289 bp) holding two overlapping open reading frames (13,217 bp and 21,289 bp, respectively) that encode two polyproteins [D. Kim et al.]. Other elements of the genome include are shown below [V. B. O'Leary et al.]. The first event upon cell entry is the primary translation of the ORF1a and ORF1b gRNA to produce non-structural proteins (nsps).
This is completely dependent on the translation machinery of the host cell. Due to fewer rare “slow-codons”, SARS-CoV-2 may have a higher protein translational rate, and therefore higher infectivity compared to other coronavirus groups [V. B. O'Leary et al.]. The ORF1a produces polypeptide 1a (pp1a, 440–500 kDa) that is cleaved into nsp-1 through nsp-11. A -1 ribosome frameshift occurs immediately upstream of the ORF1a stop codon, to allow translation through ORF1b, yielding 740–810 kDa polypeptide pp1ab, which is cleaved into 15 nsps [D. Kim et al.]. Two overlapping ORFs, ORF1a and ORF1b, generate continuous polypeptides, which are cleaved into a total of 16 so-called nsps [Y Finkel et al.]. Functionally, there are five proteins from pp1ab (nsp-12 through nsp-16) as nsp-1-11 are duplications of the proteins in pp1a due to the ORF overlap. The pp1a is approximately 1.4–2.2 times more expressed than pp1ab. After translation, the polyproteins are cleaved by viral proteases nsp3 and nsp5. Nsp5 protease can be referred to as 3C-like protease (3CLpro) or as main protease (Mpro), as it cleaves the majority of the polyprotein cleavage sites. [H.A. Hussein et al.] Nsp1 cleavage is quick and nsp1 associates with host cell ribosomes and results in host cellular shutdown, suppressing host gene expression [M. Thoms et al.]. Fifteen proteins, nsp2–16 constitute the viral RTC. They are targeted to defined subcellular locations and establish a network with host cell factors. Nsp2–11 remodel host membrane architecture, mediate host immune evasion and provide cofactors for replication, whilst nsp12–16 contain the core enzymatic functions involved in RNA synthesis, modification and proofreading [P. V'Kovski et al.].  nsp-7 and nsp-8 form a complex priming the RNA-dependent RNA polymerase (RdRp or RTC) - nsp-12. nsp14 provides a 3′–5′ exonuclease activity providing RNA proofreading function. Nsp-10 composes the RNA capping machinery nsp-9. nsp13 provides the RNA 5′-triphosphatase activity. Nsp-14 is a N7-methyltransferase and nsp-16 the 2′-O-methyltransferase. Many of the nsps have multiple functions and many viral proteins are involved in innate immunity inhibition. Nsp-3 is involved in vesicle formation along with nsp-4 and nsp-6 where viral replication occurs. Interactions between SARS-CoV-2 proteins and human RNAs thwart the IFN response upon infection: nsp-16 binds to U1 and U2 splicing RNAs to suppress global mRNA splicing; nsp-1 binds to 40S ribosomal RNA in the mRNA entry channel of the ribosome to inhibit host mRNA translation; nsp-8 and nsp-9 bind to the 7SL RNA to block protein trafficking to the cell membrane [A. K. Banerjee et al.]. Xia et al. [H. Xia et al.] found that nsp-6 and nsp-13 antagonize IFN-I production via distinct mechanisms: nsp-6 binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, and nsp-13 binds and blocks TBK1 phosphorylation.
Viral transcription and replication:
Viral transcription and replication occur at the viral replication organelle (RO) [E. J. Snijder et al.]. The RO is specifically formed during infection by reshaping ER and other membranes, giving rise to small spherular invaginations, and large vesiculotubular clusters, consisting of single- and/or double-membrane vesicles (DMV), convoluted membranes (CM) and double-membrane spherules invaginating from the ER  [S. Klein et al., E. J. Snijder et al.]. There is some evidence that DMV accommodate viral replication which is based on radiolabelling viral RNA with nucleoside precursor ([5-3[H]uridine) and detection by EM autoradiography [E. J. Snijder et al.].
Viral replicative proteins and specific host factors are recruited to ROs [E. J. Snijder et al.]. RNA viral genome is transcribed into messenger RNA by the viral RTC [P. Ahlquist et al.]. Viral RTC act in combination with other viral and host factors involved in selecting template RNAs, elongating RNA synthesis, differentiating genomic RNA replication from mRNA transcription, modifying product RNAs with 5’ caps or 3’ polyadenylate [P. Ahlquist et al.]. Positive-sense (messenger-sense) RNA viruses replicate their genomes through negative-strand RNA intermediates [M. Schwartz et al.]. The intermediates comprise full-length negative-sense complementary copies of the genome, which functions as templates for the generation of new positive-sense gRNA, and a nested set of sg mRNAs that lead to the expression of proteins encoded in all ORFs downstream of ORF1b. The transcription of coronaviruses is a discontinuous process that produces nested 3′ and 5′ co-terminal sgRNAs. Of note, the synthesis of sg mRNAs is not exclusive to the order Nidovirales but a discontinuous minus-strand synthesis strategy to produce a nested set of 3′ co-terminal sg mRNAs with a common 5′ leader in infected cells are unique features of the coronaviruses and arteriviruses [W. A. Miller and G. Koev.]. Of note, the produced genomic RNA represents a small fraction of the total vRNA [N. S. Ogando et al.].
The discontinuous minus-strand synthesis of a set of nested sg mRNAs happens during the synthesis of the negative-strand RNA, by an interruption mechanism of the RTC as it reads the TRS-B preceding each gene in the 3′ one-third of the viral genome [I. Sola, F. Almazan et al., I. Sola, J. L. Moreno, et al.]. The synthesis of the negative-strand RNA stops and is re-initiated at the TRS-L of the genome sequence close from the 5′ end of the genome [H. Di et al.]. Therefore, the mechanism by which the leader sequence is added to the 5' end requires that the RTC switches template by a jumping mechanism. This interruption process involves the interaction between complementary TRSs of the nascent negative-strand RNA TRS-B and the positive-strand gRNA at the positive-sense TRS-L. The TRS-B site has a 7-8 bp conserved core sequence (CS) that facilitates RTC template switching as it hybridizes with a near complementary CS in the TRS-L [I. Sola, F. Almazan et al. I. Sola, J. L. Moreno, et al.]. Upon re-initiation of RNA synthesis at the TRS-L region, a negative-strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs. This means that all sg mRNAs as well as the genomic RNA share a common 5' sequence, named leader sequence [X. Zhang et al.]. This programmed template switching leads to the generation of sg mRNAs with identical 5' and 3' sequences, but alternative central regions corresponding to the beginning of each structural ORF [I. Sola et al. 2015, S. G. Sawicki et al., Y. Yang et al.]. Of note, the existence of TRSs also raises the possibility that these sites are at the highest risk of recombining through TRS-B mediated template switching [Y. Yang]. The set of sg mRNAs is then translated to yield 29 identified different proteins [F. Wu et al.], although many papers have identified additional ORFs [D. Kim et al.. Y. Finkel et al., A. Vandelli et al.]. The translation of the linear single-stranded RNA conducts to the generation of the following proteome: 4 are structural proteins, S, N, M, and E; 16 proteins nsp: the first 11 are encoded in ORF1a whereas the last 5 are encoded in ORF1ab. In addition, 9 accessory proteins named ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10 have been identified [F. Wu et al.]. At the beginning of infection, there is the predominant expression of the nsp that result from ORF1a and ORF1ab, however, at 5 hpi, the proteins encoded by the 5′ last third are found in higher amounts, and the nucleoprotein is the protein found in higher levels [Y. Finkel et al.].
Viral assembly:
The final step of viral production requires virion assembly and this process is not well explored for SARS-CoV-2. For example, the role of the structural proteins of SARS-CoV-2 in virus assembly and budding in not known. In general, all beta-coronavirus structural proteins assemble at the endoplasmic reticulum (ER)-to-Golgi compartment [J. R. Cohen et al., A. Perrier et al.] and viral assembly requires two steps: Genome packaging which is a process in which the SARS-CoV-2 gRNA must be coated by the viral protein nucleoprotein (N) protein, forming viral ribonucleoprotein (vRNPs) complexes, before being selectively packaged into progeny virions [P. V'Kovski et al.], a step in which vRNPs bud into the lumen of the ER and the ER-Golgi intermediate compartment (ERGIC) [N. S. Ogando et al.]. This results in viral particles enveloped with host membranes containing viral M, E, and S transmembrane structural proteins that need to be released from the cell.
SARS-CoV-2 gRNA packaging involves the N protein. The N protein of human coronaviruses is highly expressed in infected cells. It is considered a multifunctional protein, promoting efficient sub-genomic viral RNA transcription, viral replication, virion assembly, and interacting with multiple host proteins [P. V'Kovski et al., D. E. Gordon et al., R. McBride, and M. van Zyl, B. C.]. In relation to transcription and replication, the N protein could provide a cooperative mechanism to increase protein and RNA concentrations at specific localizations S. Alberti, and S. Carra, S. F. Banani et al.], and this way organize viral transcription. Five studies have shown that N protein undergoes liquid-liquid phase separation (LLPS) in vitro [A. Savastano et al., H. Chen et al., C. Iserman et al., T. M. Perdikari et al., J. Cubuk et al.], dependent on its C-terminal domain (CTD) [H. Chen et al.]. It has been hypothesised that N could be involved in replication close to the ER and in packaging of gRNA into vRNPs near the ERGIC where genome assembly is thought to take place [A. Savastano et al.], but so far this is still speculative. Phosphorylation of N could adjust the physical properties of condensates differentially in ways that could accommodate the two different functions of N: transcription and progeny genome assembly [A. Savastano et al., C. Iserman et al., C. R. Carlson et al.].
The ERGIC constitutes the main assembly site of coronaviruses [S. Klein et al., E. J. Snijder et al., L. Mendonca et al.] and budding events have been seen by EM studies. For SARS-CoV-2, virus-budding was mainly clustered in regions with a high vesicle density and close to ER- and Golgi-like membrane arrangements [S. Klein et al., E. J. Snijder et al., L. Mendonca et al.]. The ectodomain of S trimers were found facing the ERGIC lumen and not induce membrane curvature on its own, therefore proposing that vRNPs and spike trimers [S. Klein et al.].
Finally, it has been shown that SARS-CoV-2 virions de novo formed traffic to lysosomes for unconventional egress by Arl8b-dependent lysosomal exocytosis [S. Ghosh et al.]. This process results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation [S. Ghosh et al.].
<h4>How it is Measured or Detected</h4>
Viral translation:
SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation [Schubert et al. 2020]
<ul>
<li>Sucrose pelleting binding assay to verify Nsp1–40S complex formation</li>
<li>In vivo translation assay</li>
<li>Transient expression of FLAG-Nsp1 in HeLa cells and puromycin incorporation assay</li>
</ul>
SARS-CoV-2 disrupts splicing, translation, and protein trafficking [Banerjee et al. 2020]
<ul>
<li>SARS-CoV-2 viral protein binding to RNA</li>
<li>Interferon stimulation experiments</li>
<li>Splicing assessment experiments</li>
<li>IRF7-GFP splicing reporter, 5EU RNA labeling, capture of biotinylated 5EU labeled RNA</li>
</ul>
Membrane SUnSET assay for transport of plasma membrane proteins to the cell surface
Viral transcription:
The mRNA transcripts are detected by the real-time reverse transcription-PCR (RT-PCR) assay. Several methods targeting the mRNA transcripts have been developed, which includes the RT-PCR assays targeting RdRp/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 [Chan et al.]. RT-PCR assays detecting SARS-CoV-2 RNA in saliva include quantitative RT-PCR (RT-qPCR), direct RT-qPCR, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) [Nagura-Ikeda M, et al.]. The viral mRNAs are reverse-transcribed with RT, followed by the amplification by PCR.
Viral replication:
viral replication is measured by RT-qPCR in infected cells, formation of liquid organelles is assessed in vitro reconstitution systems and in infected cells. Labelling by radioactive nucleosides.
Viral production:
Plaque assays, infectivity assays, RT-qPCR to detect viral RNA in released virions, sequencing to detect mutations in the genome, electron microscopy.
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41.       L. Mendonca et al., SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging. bioRxiv,  (2020).
42.       S. Ghosh et al., beta-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway. Cell 183, 1520-1535 e1514 (2020).
43.       Schubert, K., Karousis, E.D., Jomaa, A. et al. SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nat Struct Mol Biol 27, 959–966 (2020).
44.       Chan, Jasper Fuk-Woo et al. Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens. J Clin Microbiol. 2020:58(5)e00310-20. doi:10.1128/JCM.00310-20
45.       Nagura-Ikeda M, Imai K, Tabata S, et al. Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19. J Clin Microbiol. 2020;58(9):e01438-20. doi:10.1128/JCM.01438-20
46.       Conceicao C, Thakur N, Human S, Kelly JT, Logan L, Bialy D, et al. (2020) The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins. PLoS Biol 18(12): e3001016. https://doi.org/10.1371/journal.pbio.3001016
47.       Damas J, Hughes GM, Keough KC, Painter CA, Persky NS, Corbo M, Hiller M, Koepfli KP, Pfenning AR, Zhao H, Genereux DP, Swofford R, Pollard KS, Ryder OA, Nweeia MT, Lindblad-Toh K, Teeling EC, Karlsson EK, Lewin HA. Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates. Proc Natl Acad Sci U S A. 2020 Sep 8;117(36):22311-22322. doi: 10.1073/pnas.2010146117. Epub 2020 Aug 21. PMID: 32826334; PMCID: PMC7486773.
<h4><a href="/events/1870">Event: 1870: Sustentacular cells, decrease </a></h4>
<h5>Short Name: Sustentacular cells, decrease </h5>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Cellular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>sustentacular cell</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>nose epithelium</td></tr>
</tbody>
</table>
</div>
<h4>Key Event Description</h4>
Biological state: The sustentacular cells are one of the major non neuronal cellular constituents of the olfactory epithelium (OE). The olfactory sustentacular cells are believed to be partly epithelial and partly glial (Liang, 2020).
Biological compartment: The sustentacular cells are located in the OE.
General role in the biology: The sustentacular cells provide mechanical strength to the olfactory epithelium, generate the olfactory binding protein, and support the other cells with nutrients (Choi and Goldstein, 2018). Sustentacular cells are responsible for the maintenance of the ion and water balance within the olfactory epithelium (Suzuki et al., 2000). Sustentacular cells provide support to olfactory sensory neurons by maintaining ion balance (Vogalis, 2005).
Sustentacular cells have been proposed to be involved in peripheral processing of odorants in multiple ways:
<ul>
<li>appear to endocytose (clear) the odorant-binding proteins after signal transduction at the neurons’ cilia to allow the next round of odorant receptor binding, thereby increasing sensitivity (Heydel et al., 2013; Strotmann and Breer 2011). </li>
<li>express multiple CYP450-family monooxygenases, which hydroxylate and help to remove toxic volatiles (Heydel et al., 2013). </li>
<li>supply neuronal cilia with some of the glucose required to meet the high energy demands of the olfactory transduction cascade (Cooper et al., 2020; Villar et al., 2017). </li>
<li> maintain the structural integrity of the olfactory epithelium (Bryche et al., 2020; Jia et al., 2010). </li>
<li>are closely associated, both metabolically and functionally, with olfactory neurons and with odorant signal transduction. (Butowt and von Bartheld, 2020)</li>
</ul>
<h4>References</h4>
Butowt, R., and von Bartheld, C. S. (2020). Anosmia in COVID-19: underlying mechanisms and assessment of an olfactory route to brain infection. Neuroscientist doi: 10.1177/1073858420956905
Bryche B, St Albin A, Murri S, Lacôte S, Pulido C, Ar Gouilh M, Lesellier S, Servat A, Wasniewski M, Picard-Meyer E, Monchatre-Leroy E, Volmer R, Rampin O, Le Goffic R, Marianneau P, Meunier N. Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters. Brain Behav Immun. 2020 Oct;89:579-586. doi: 10.1016/j.bbi.2020.06.032. Epub 2020 Jul 3. PMID: 32629042; PMCID: PMC7332942.
Torabi A., Mohammadbagheri E., Akbari Dilmaghani N., Bayat A.H., Fathi M., Vakili K., Alizadeh R., Rezaeimirghaed O., Hajiesmaeili M., Ramezani M., Simani L., Aliaghaei A. Proinflammatory cytokines in the olfactory mucosa result in COVID-19 induced anosmia. ACS Chem. Neurosci. 2020
<h4><a href="/events/1871">Event: 1871: olfactory sensory neurons, decrease </a></h4>
<h5>Short Name: olfacotry neurons, decrease</h5>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Cellular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>ciliated olfactory receptor neuron</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>nose epithelium</td></tr>
</tbody>
</table>
</div>
<h4>Key Event Description</h4>
Biological state: The olfactory sensory neurons (OSNs) together with a number nonneuronal cells form the olfactory epithelium (OE). They are bipolar neuronal cells. The dendritic end of the OSNs is exposed to the nasal mucus, whereas their axonal pole is synaptically connected to the olfactory bulb of the central nervous system (CNS) (Liang, 2020). OSNs in the OE could be differentiated into hundreds of subsets, depending on the phenotype of odorant receptor proteins.
Biological compartment: The OSNs are located in the olfactory epithelium (OE).
General role in the biology: The OSNs are the OE’s parenchymal cells responsible for olfactory reception and transduction. The OSNs project their axons via the olfactory nerve onto the olfactory bulb. The OSNs are undoubtedly the OE’s parenchymal cells responsible for olfactory reception and transduction.
<h4>References</h4>
Liang F. Sustentacular Cell Enwrapment of Olfactory Receptor Neuronal Dendrites: An Update. Genes (Basel). 2020 Apr 30;11(5):493. doi: 10.3390/genes11050493. PMID: 32365880; PMCID: PMC7291085.
<h4><a href="/events/1872">Event: 1872: Olfactory epithelium degeneration</a></h4>
<h5>Short Name: Olfactory epithelium degeneration</h5>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>olfactory epithelium</td></tr>
</tbody>
</table>
</div>
<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/1873">Event: 1873: impaired olfactory function (anosmia) </a></h4>
<h5>Short Name: anosmia</h5>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>AdverseOutcome</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Organ</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>nose epithelium</td></tr>
</tbody>
</table>
</div>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2056">Relationship: 2056: Binding to ACE2 leads to SARS-CoV-2 cell entry </a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/320">Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortality</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/394">SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/406">SARS-CoV-2 infection leading to hyperinflammation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/422">Binding of SARS-CoV-2 to ACE2 in enterocytes leads to intestinal barrier disruption</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/430">Binding of SARS-CoV-2 to ACE2 leads to viral infection proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/379">Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/468">Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Homo sapiens</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
This KER applies to humans in all life stages and all sexes.
<h4>Key Event Relationship Description</h4>
This KER deals with the evidence supporting the individual weight that the surface protein of SARS-CoV-2 spike needs to bind:ACE2, and of being cleaved in two different sites, for viral entry to occur. Viral entry is essential for initiating a cascade of events leading to COVID19.
<h4>Evidence Supporting this KER</h4>
Binding of SARS-CoV-2 S protein to ACE2 receptors present in the brain (endothelial, neuronal and glial cells) :
The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers, and this may in part explain the susceptibility of many COVID-19 patients to severe respiratory distress (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010). The neurons and glial cells, like astrocytes and microglia also express ACE-2, thus highlighting the vulnerability of the nervous system to SARS-CoV-2 infection. Additionally, they also express transmembrane serine protease 2 (TMPRSS2) and furin, which facilitate virus entry into the host (Jakhmola et al. 2020).
Once inside the brain, the virus can infect the neural cells, astrocytes, and microglia. These cells express ACE-2, thus initiating the viral budding cycle followed by neuronal damage and inflammation (Jakhmola et al. 2020). Specifically in the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020).
Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018).
Binding of S protein to ACE2 receptors present in the intestines
Biological Plausibility
Upon binding of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to angiotensin-converting enzyme 2 (ACE2) on the surface of the host cells, SARS-CoV-2 enters inside the cells with an internalization mechanism.
Empirical Evidence
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptor (Nature 579, 270–273, 2020 ; J. Virol. 94, e00127-20; Nature 588, 327–330). The SARS-CoV-2 surface spike (S) protein mediates the binding to the receptor and requires 2 cleavage steps for viral entry to occur, as follows. The spike protein contains 1273 aminoacids divided into two subunits, S1 and S2. The subunits are cleaved by furin-like enzymes, as spike of sars-cov-2 contains an insertion 680SPRRAR↓SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. In addition, there is a second cleavage site 808PSKPSKR|SFIEDL822 just before the fusion peptide that needs to occur for viral entry. The S1 subunit contains a receptor-binding domain (RBD) encompassing the receptor-binding motif (RBM) that binds ACE2. The S2 contains a fusion peptide (FP), that penetrates into cell membranes and mediates fusion between the viral and host membranes to release viral proteins and genome.
Uncertainties and Inconsistencies
When TMPRSS2 is not available, spike it is hypothethised that the virus may use alternative proteases to get in the cells either by fusion with the plasma membrane or entry via endosomes and fusion with endocytic membranes at low pH, when proteases for priming become active, but evidence is less robust.
When TMPRSS2 is not available, it is hypothesized that the virus may use alternative proteases to get in the cells either by fusion with the plasma membrane or entry via endosomes and fusion with endocytic membranes at low pH when proteases for priming become active, but the evidence is less robust (Jackson et al, 2022).
<h4>Quantitative Understanding of the Linkage</h4>
Known modulating factors
<div>
<table border="1" bordercolor="#ccc" cellpadding="5" cellspacing="0" class="table table-bordered table-fullwidth" style="border-collapse:collapse">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td>
Chemicals
(weak evidence)
</td>
<td>
PFAS (PFOS, PFOA, PFNA, PFHxS, and GenX)
</td>
<td>
Short-term (10 days), high dose (20 mg/kg/day) exposure to PFOA leads to about 1.6 fold upregulation of the pulmonary mRNA level of Ace2 and to about 1.5 upregulation of the pulmonary mRNA level of Tmprss2 in CD1 mice. [1]
Long-term (12 weeks) of an environmentally relevant PFAS mixture (PFOS, PFOA, PFNA, PFHxS, and GenX; each in 2 mg/l concentration) exposure leads to downregulation of pulmonary mRNA expression of Ace2 2.5-fold in C57BL/6 J male mice. A similar decreasing trend was observed in PFAS-exposed male mice for Tmprss2. [2]
</td>
<td>
1. doi: <a href="http://10.1016/j.toxrep.2021.11.014">10.1016/j.toxrep.2021.11.014</a>
2.  doi: <a href="http://10.1016/j.taap.2022.116284">10.1016/j.taap.2022.116284</a>
</td>
</tr>
<tr>
<td colspan="1" rowspan="2">
Sex
(strong evidence)
</td>
<td>female sex (XX chromosomes)</td>
<td>
ACE2 localizes to the X sex chromosome and displays a sex-dependent expression profile with higher expression in female than in male tissues [1,2]. Estradiol inhibits TMPRSS2, needed to facilitate SARS-CoV-2 entry into the cell [3]. Estrogen therapy has been shown to mitigate endoplasmic reticulum stress induced by SARS-CoV-2 invasion through activation of cellular unfold protein response and regulation of inositol triphosphate (IP3) and phospholipase C [4].
Different studies have also illustrated that estradiol increases the expression of ADAM17, leading to high-circulating soluble ACE2 potentially neutralizing SARS-CoV-2 and preventing its binding to mACE2. [5] Thus, Estradiol might reduce SARS-CoV-2 infectivity through modulation of cellular ACE2/TMPRSS2/ADAM17 axis expression.
</td>
<td>
1. doi:<a href="http://10.1177/1933719115597760"> 10.1177/1933719115597760</a>
2. doi:<a href="http://10.1016/j.mce.2015.11.004"> 10.1016/j.mce.2015.11.004</a>
3. doi: <a href="http://10.1007/s11033-021-06390-1">10.1007/s11033-021-06390-1</a>
4.doi: <a href="http://10.1016/j.mehy.2020.110148">10.1016/j.mehy.2020.110148</a>
5. doi: <a href="http://10.2217/pgs-2020-0092">10.2217/pgs-2020-0092</a>
</td>
</tr>
<tr>
<td>
Male sex (XY chromosomes)
</td>
<td>
Androgen receptors (ARs) play a key role in increasing transcription of TMPRSS2. This may explain the predominance of males to COVID-19 fatality and severity. [6]
</td>
<td>
6. doi: <a href="http://10.1073/pnas.2021450118">10.1073/pnas.2021450118 </a>
</td>
</tr>
<tr>
<td>Age</td>
<td>Young/old people</td>
<td>ACE2 protein expression is increased with aging in several tissues [1], including lungs and particularly in patients requiring mechanical ventilation [2]. During aging, telomere dysfunction activates a DNA damage response leading to higher ACE2 expression. Thus, telomere shortening could contribute to make elderly more susceptible to SARS-CoV-2 infection [3].</td>
<td>
1. doi: <a href="https://doi.org/10.1016/j.exger.2021.111507" style="color:blue; text-decoration:underline">10.1016/j.exger.2021.111507</a>
2. doi: <a href="https://doi.org/10.1371/journal.pone.0247060" style="color:blue; text-decoration:underline">10.1371/journal.pone.0247060</a>
3. doi: <a href="https://doi.org/10.15252/embr.202153658" style="color: blue; text-decoration-line: underline;">10.15252/embr.202153658</a>
</td>
</tr>
<tr>
<td rowspan="2">Lipids</td>
<td>Atherogenic dyslipidemia</td>
<td>
Lipids, as important structural components of cellular and sub-cellular membranes, are crucial in the infection process [1]. Changes in intracellular cholesterol alter cell membrane composition, impacting structures such as lipid rafts, which accommodate many cell-surface receptors [2], including ACE2 and TMPRSS2 [3, 4].
In COVID-19. In an in vitro study, the depletion of membrane-bound cholesterol in ACE2-expressing cells led to a reduced infectivity of SARS-CoV [3]. In vitro, higher cellular cholesterol increased uptake of SARS-CoV-2 S protein; this effect was decreased with Methyl-beta-cyclodextrin, a compound which extracts cholesterol from cell membranes [5]. HDL scavenger receptor B type 1 (SR-B1), a receptor found in pulmonary and many other cells, could facilitate ACE2-dependent entry of SARS-CoV-2 [6].
</td>
<td rowspan="2">
1. doi: 10.1001/jama.2020.12839
2. doi: <a href="https://doi.org/10.3389/fcell.2020.618296" style="color:blue; text-decoration:underline">10.3389/fcell.2020.618296</a>
3. doi: <a href="https://doi.org/10.1016/j.bbrc.2008.02.023" style="color:blue; text-decoration:underline" target="_blank" title="Persistent link using digital object identifier">10.1016/j.bbrc.2008.02.023</a>
4. doi: <a href="https://doi.org/10.1096/fj.202000654R" style="color:blue; text-decoration:underline">10.1096/fj.202000654R</a>
5. doi: 10.1101/2020.05.09.086249
6.doi: 10.1038/s42255-020-00324-0
7.doi: <a href="https://doi.org/10.1016/j.bbalip.2020.158849" style="color:blue; text-decoration:underline" target="_blank" title="Persistent link using digital object identifier">10.1016/j.bbalip.2020.158849</a>
8.doi: <a href="https://doi.org/10.1016/j.obmed.2020.100283" style="color:blue; text-decoration:underline" target="_blank" title="Persistent link using digital object identifier">10.1016/j.obmed.2020.100283</a>
9. doi: <a href="https://doi.org/10.3390/ijms21103544" style="color:blue; text-decoration:underline">10.3390/ijms21103544</a>
10.doi: <a href="http://10.1101/2020.04.16.20068528">10.1101/2020.04.16.20068528</a>
</td>
</tr>
<tr>
<td>Obesity</td>
<td>
In COVID-19. ACE2 is highly expressed in adipose tissue, thus excess adiposity may drive more infection (8). Obese patients have more adipose tissue and therefore more ACE2-expressing cells [9]. SARS-CoV-2 dysregulates lipid metabolism in the host and the effect of such dysregulated lipogenesis on the regulation of ACE2, specifically in obesity [10]. Lung epithelial cells infected with SARS-CoV-2 showed upregulation of genes associated with lipid metabolism [11], including the SOC3 gene. A mouse model of diet-induced obesity showed higher Ace2 expression in the lungs, which negatively correlated with the expression sterol response element binding proteins 1 and 2 (SREBP) genes. Suppression of Srebp1 showed a significant increase in Ace2 expression in the lung. Lipids, including fatty acids, could interact directly with SARS-CoV-2 influencing spike configuration and modifying the affinity for ACE2 and thus its infectivity [12]. The dysregulated lipogenesis and the subsequently high ACE2 expression in obese patients might be one mechanism underlying the increased risk for severe complications [10].
</td>
</tr>
<tr>
<td>
Vitamin D
(moderate evidence)
</td>
<td>
Vitamin D deficiency
</td>
<td>
Vitamin D administration enhanced mRNA expression of VDR and ACE2 in a rat model of acute lung injury [1]. In particular, vitamin D upregulates the soluble ACE2 form [2]. Thus, low vitamin D status may impair the trapping protective mechanism of soluble ACE2 [3]. Furthermore, vitamin D deficiency has been shown to reduce the expression of antimicrobial peptides (-defensin, cathelicidin), which act against enveloped viruses [4,5].
In COVID-19. Decreased sACE2 and cellular viral defense might be some mechanisms explaining how low vitamin D modulate SARS-CoV-2 infectibility.
</td>
<td>
1. doi: <a href="http://10.1016/j.injury.2016.09.025">10.1016/j.injury.2016.09.025</a>
2. doi: <a href="http://10.1152/ajplung.00071.2009">10.1152/ajplung.00071.2009</a>
3. doi: <a href="http://10.3390/ijms22105251">10.3390/ijms22105251</a>
4. doi:<a href="http://10.1007/s11154-021-09679-5"> 10.1007/s11154-021-09679-5</a>
5. doi: <a href="http://10.1080/14787210.2021.1941871">10.1080/14787210.2021.1941871</a>
</td>
</tr>
<tr>
<td>
Gut microbiota
</td>
<td>
Gut dysbiosis (alteration of gut microbiota)
</td>
<td>
<div>
<div>
<div>
Some evidence shows that gut microbiota influences Ace2 expression in the gut. Colonic Ace2 expression decreased significantly upon microbial colonization in mice and rats [1,2]. Coprobacillus enrichment was associated with severe COVID-19 in patients [3] and was shown to upregulate colonic ACE2 in mice [4]. The abundance of Bacteroides species was associated with reduced ACE2 expression in the murine gut [4] and negatively correlated with fecal SARS-CoV-2 load [3,5]. Thus, gut dysbiosis might lead to higher levels of ACE2 in the gut, potentially increasing the ability of SARS-CoV-2 to enter enterocytes.
</div>
</div>
</div>
</td>
<td>
1. doi: <a href="http://10.1080/19490976.2021.1984105">10.1080/19490976.2021.1984105</a>
2. doi: <a href="http://10.1161/HYPERTENSIONAHA.120.15360">10.1161/HYPERTENSIONAHA.120.15360</a>
3.doi: <a href="http://10.1053/j.gastro.2020.05.048">10.1053/j.gastro.2020.05.048</a>
4.doi: <a href="http://10.1016/j.cell.2017.01.022">10.1016/j.cell.2017.01.022</a>
5. doi: <a href="http://10.1016/j.tifs.2020.12.009">10.1016/j.tifs.2020.12.009</a>
</td>
</tr>
<tr>
<td>Genetic factors</td>
<td> </td>
<td>
Polymorphisms inducing amino acid residue changes of ACE2 in the binding interface would influence affinity for the viral S protein. Evidence exists that K353 and K31 in hACE2, the main hotspots that form hydrogen bonds with the main chain of N501 and Q493 in receptor-binding motif respectively, play a role in tightly binding to the S protein of SARS-CoV-2 [1]. Around the twenty natural ACE2 variants, three alleles of 17 variants were found to affect the attachment stability [2]. Thus, the ACE2 variants modulating the interaction between the virus and the host have been reported to be rare, consistently with the overall low appearance of ACE2 polymorphisms. In this context, it is key to approach both the ACE2 genotypes and the clinical descriptions of the phenotypes in a population-wide manner, in order to better understand how ACE2 variations are relevant in the susceptibility for SARS-CoV-2 infection [3]. In addition, since ACE2 is X-linked, the rare variants that enhance SARS-CoV-2 binding are expected to increase susceptibility to COVID-19 in males [4]. On the other hand, the X-chromosome inactivation of the female causes a “mosaic pattern”, which might be an advantage for females in terms of reduced viral binding [5]. TMPRSS2 single-nucleotide polymorphisms (SNPs) were associated with a frequent “European haplotype” [6], which not observed in Asians, is suggested to upregulate TMPRSS2 gene expression in an androgen-specific way. Thus, there is a need for in vitro validation studies to assess the involvements of population-specific SNPs of both ACE2 and TMPRSS2 in susceptibility toward SARS-CoV-2 infection. The occurrence of a pandemic is related to the genetics of the infecting agent. In the case of SARS-CoV-2, through genomic surveillance it is possible to track the spread of SARS-CoV-2 lineages and variants, and to monitor changes to its genetic code that can influence viral entry and production. Consequently, genomic surveillance is crucial to understand how mutations occurring on SARS-CoV-2 genome influence and drive the pandemic [7]. For example, a recent study [8] highlights that through genomic surveillance it is possible to trace co-infections by distinct SARS-CoV-2 genotypes, which are expected to have a different impact on factors modulating COVID-19. Genomic surveillance of SARS-CoV-2 is able to reveal tremendous genomic diversity [9], and coupled with language models and machine learning approaches, contributes to predicting the impact of mutations (such as those occurring in the spike protein), and thus can better address challenging aspects, like an estimation of the efficacy of therapeutic treatments [10].
</td>
<td>
[1] doi: 10.1080/07391102.2020.1796809
[2] doi: 10.1002/jmv.26126
[3] doi: 10.1038/s42003-021-02030-3
[4] doi: 10.1101/2020.04.05.026633
[5] doi: 10.3390/ijms21103474
[6] doi: 10.18632/aging.103415
[7] doi: 10.1038/s41588-022-01033-y
[8] doi: 10.1038/s41598-022-13113-4
[9] doi:10.1371/journal.pone.0262573
[10] doi: 10.3389/fgene.2022.858252
</td>
</tr>
<tr>
<td>Therapeutic intervention against COVID-19</td>
<td>
Casirivimab, Imdevimab
and Sotrivimab</td>
<td>
Are monoclonal antibodies designed to recognize and attach to two
different sites of the Receptor-Binding Domain (RBD) of the S protein of SARS-CoV-2,
blocking the virus to enter cells [1,2,3].</td>
<td>
1) 10.1056/NEJMoa2035002
2) EMA Starts Rolling Review of REGN-COV2 Antibody Combination (Casirivimab / Imdevimab). EMA 2021. Available online:
https://www.ema.europa.eu/en/news/ema-starts-rolling-review-regn-cov2-antibody-combination-casirivimab-imdevimab
(accessed on 12 May 2022)
3) EMA Starts Rolling Review of Sotrovimab (VIR-7831) for COVID-19. EMA 2021. Available online:
https://www.ema.europa.eu/en/news/ema-starts-rolling-review-sotrovimab-vir-7831-covid-19 (accessed on 12 May 2022)</td>
</tr>
<tr>
<td> </td>
<td>Heparin</td>
<td>
Interacts directly with viral
particles and has been shown to bind to the SARS-CoV-2 S1 Spike RBD, causing significant
protein architecture alteration, impacting infectivity [1,2].</td>
<td>
1) 10.3389/fmed.2021.615333
2) 10.1055/s-0040-1721319
</td>
</tr>
<tr>
<td>Air pollution</td>
<td> </td>
<td>
Air pollution induces Increased expression of ACE2 which may result in increased viral entry and coronavirus production.
Increased ACE2 expression has been reported in the respiratory system in response to air pollution exposure (1-4). Increased expression may affect susceptibility to SARS-CoV-2 infection. Similarly, some constituents of air pollution (PM, ozone) have been reported to increase the expression of TMPRSS2 (3, 5-6).
</td>
<td>
1) https://doi.org/10.1186/s12989-015-0094-4
2) 10.1016/j.burns.2015.04.010
3) 10.1016/j.envres.2021.110722
4) 10.3390/ijerph17155573
5) 10.1186/s12989-021-00404-3
6) https://doi.org/10.1038/s41598-022-04906-8
</td>
</tr>
<tr>
<td>Pre-existing heart failure</td>
<td> </td>
<td>
ACE2 mRNA and protein levels, as well as enzymatic activity, were shown to be upregulated in explanted hearts from patients with end-stage HF, as well as in the HF rat model [1-3].
Myocytes, fibroblasts, vascular smooth muscle cells, pericytes [4] and endothelial cells of the coronaries [5] express ACE2, while myocytes in patients suffering from heart disease exhibit higher ACE2 expression [6].
Pericytes - the mural cells lining microvasculature, interacting with endothelial cells notably to maintain microvascular stability - exhibited the strongest ACE2 expression in HF patients [7], rendering these cells involved in the coronary vasculature of the myocardium, more susceptible to infection.
Furthermore, SARS-CoV-2 infects and replicates in pericytes, and a decrease in their numbers follows [8].
Patients with pre-existing HF showed increased ACE2 levels in myocytes and pericytes, having thereby higher risk of heart injury [7, 9].
In addition, sACE2 levels are higher in HF patients [10, 11] and sACE2 activity is increased in HF [12].
In contrast to a protective role of sACE2, it has been proposed that viral binding to circulating sACE2 forms SARS-CoV-2/sACE2 complexes, which might mediate infection of cells in distal tissues [13]; hence, pre-existing HF might disseminate SARS-CoV-2 infection.
Interestingly, the increase in sACE2 activity is associated with HF with reduced ejection fraction (HFrEF) but not with HF with preserved ejection fraction (HFpEF), suggesting (i) a rather complex role of HF in regulating ACE2-mediated infection by SARS-CoV-2 [10] and (ii) the potential of sACE2 activity to be used as a biomarker to distinguish between the two HF types.
Lastly, it is noteworthy that Khoury et al. provided evidence in a different direction, by showing that ADAM17 and TMPRSS2 [14] expression levels are downregulated in a HF rat model, thus potentially conferring a protective role against infection by SARS-CoV-2 in HF [3].
</td>
<td>
1: <a href="https://doi.org/10.1186/1741-7015-2-19" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1186/1741-7015-2-19</a>
2: <a href="https://doi.org/10.1161/01.CIR.0000094734.67990.99" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/01.CIR.0000094734.67990.99</a>
3: <a href="https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16310#:~:text=https%3A//doi.org/10.1111/jcmm.16310" style="color:#0563c1; text-decoration:underline">https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16310#:~:text=https%3A//doi.org/10.1111/jcmm.16310</a>
4: <a href="https://doi.org/10.1161/CIRCULATIONAHA.120.047911" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/CIRCULATIONAHA.120.047911</a>
5: <a href="https://doi.org/10.1152/ajpheart.00331.2008" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/ajpheart.00331.2008</a>
6: <a href="https://doi.org/10.1093/eurheartj/ehaa311" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/eurheartj/ehaa311</a>
7: <a href="https://doi.org/10.1093/cvr/cvaa078" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/cvr/cvaa078</a>
8: <a href="https://doi.org/10.21203/rs.3.rs-105963/v1" style="color:#0563c1; text-decoration:underline">https://doi.org/10.21203/rs.3.rs-105963/v1</a>
9: <a href="https://doi.org/10.1016/j.jacbts.2020.06.007" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.jacbts.2020.06.007</a>
10: <a href="https://doi.org/10.1177/1470320316668435" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1177/1470320316668435</a>
11: <a href="https://doi.org/10.1093/eurheartj/ehaa697" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/eurheartj/ehaa697</a>
12: <a href="https://doi.org/10.1002/jmv.27144" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1002/jmv.27144</a>
13: <a href="https://doi.org/10.1002/rmv.2213" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1002/rmv.2213</a>
14: <a href="https://doi.org/10.1016/j.cell.2020.02.052" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.cell.2020.02.052</a>
</td>
</tr>
<tr>
<td>Diet</td>
~~<td>Chemicals in foods affect ACE3 expression</td>~~
<td>Chemicals in foods affect ACE2 expression</td>
<td>
<ul>
<li>Geranium and lemon oils were found to reduce in vitro ACE2 activity and expression, as well as ACE2 and TMPRSS2 mRNA levels [207].</li>
<li>Several molecular modelling and docking studies indicate the potential for compounds found in garlic [208], turmeric (curcumin) [209], thyme and oregano (carvacrol) [210], green tea [211] and other plant foods (quercetin) [212] to inhibit binding of SARS-CoV-2.</li>
<li>Pelargonidin, found in red and black berries, was shown to dose-dependently block SARS-CoV-2 binding to ACE2, reduce SARS-CoV-2 replication in vitro and reduce ACE2 expression [213].</li>
<li>Quercetin and related compounds inhibit recombinant human ACE2 activity [214] at physiologically relevant concentrations in vitro.</li>
<li>In a human crossover study, 30-day supplementation with resveratrol decreased ACE2 in adipose tissue [216], potentially attenuating an increased risk for infection and viral replication in humans with obesity. In vitro, resveratrol inhibited the replication of SARS-CoV-2 [217].</li>
</ul>
</td>
<td>
<ul>
<li>207: <a href="http://doi.org/10.3390/plants9060770">http://doi.org/10.3390/plants9060770</a> </li>
<li>208: <a href="http://doi.org/10.1021/acsomega.0c00772">http://doi.org/10.1021/acsomega.0c00772</a></li>
<li>209: <a href="http://doi.org/10.1007/s13337-020-00598-8">http://doi.org/10.1007/s13337-020-00598-8</a></li>
<li>210:<a href="http:// http://doi.org/10.1080/07391102.2020.1772112"> http://doi.org/10.1080/07391102.2020.1772112</a></li>
<li>211: <a href="http://doi.org/10.1080/07391102.2020.1779818">http://doi.org/10.1080/07391102.2020.1779818</a></li>
<li>212: <a href="http://doi.org/10.18632/aging.103001">http://doi.org/10.18632/aging.103001</a></li>
<li>213: <a href="http://doi.org/10.1016/j.bcp.2021.114564">http://doi.org/10.1016/j.bcp.2021.114564</a></li>
<li>214: <a href="http://doi.org/10.1021/acs.jafc.0c05064">http://doi.org/10.1021/acs.jafc.0c05064</a></li>
<li>216: <a href="http://doi.org/10.1080/21623945.2021.1965315">http://doi.org/10.1080/21623945.2021.1965315</a></li>
<li>217: <a href="http://doi.org/10.1002/ptr.6916">http://doi.org/10.1002/ptr.6916</a></li>
</ul>
</td>
</tr>
</tbody>
</table>
</div>
<h4>References</h4>
COVID19 References related to CNS:
de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.
Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.
Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.
Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.
Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.
Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.
Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.
Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468
Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.
Xia, H. and Lazartigues, E.  Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010  Rep. 12 (3), 170– 175
References in general:
Benton DJ, Wrobel AG, Xu P, Roustan C, Martin SR, Rosenthal PB, Skehel JJ, Gamblin SJ. Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion. Nature. 2020 Dec;588(7837):327-330. doi: 10.1038/s41586-020-2772-0.
Jackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol. 2022 Jan;23(1):3-20. doi: 10.1038/s41580-021-00418-x.
Wan Y, Shang J, Graham R, Baric RS, Li F. Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. J Virol. 2020 Mar 17;94(7):e00127-20. doi: 10.1128/JVI.00127-20.
Zhou, P., Yang, XL., Wang, XG. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020). doi: 10.1038/s41586-020-2012-7
</div>
<div>
<h4><a href="/relationships/2310">Relationship: 2310: SARS-CoV-2 cell entry leads to SARS-CoV-2 production</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/406">SARS-CoV-2 infection leading to hyperinflammation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/394">SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Quantitative Understanding of the Linkage</h4>
Known modulating factors
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td colspan="1" rowspan="2">
Sex
</td>
<td>
female sex (XX chromosomes)
</td>
<td>
ACE2 localizes to the X sex chromosome and displays a sex-dependent expression profile with higher expression in female than in male tissues [1,2]. Estradiol inhibits TMPRSS2, needed to facilitate SARS-CoV-2 entry into the cell [3]. Estrogen therapy has been shown to mitigate endoplasmic reticulum stress induced by SARS-CoV-2 invasion through activation of cellular unfold protein response and regulation of inositol triphosphate (IP3) and phospholipase C [4]. Different studies have also illustrated that estradiol increases the expression of ADAM17, leading to high-circulating soluble ACE2 potentially neutralizing SARS-CoV-2 and preventing its binding to mACE2 [5]. Thus, estradiol might reduce SARS-CoV-2 infectivity through modulation of cellular ACE2/TMPRSS2/ADAM17 axis expression.
</td>
<td>
1. doi: <a href="http://10.1177/1933719115597760">10.1177/1933719115597760</a>
2. doi: <a href="http://10.1016/j.mce.2015.11.004">10.1016/j.mce.2015.11.004</a>
3) doi: <a href="http://10.1007/s11033-021-06390-1">10.1007/s11033-021-06390-1</a>
4) doi: <a href="http://10.1016/j.mehy.2020.110148">10.1016/j.mehy.2020.110148</a>
5) doi: <a href="http://10.2217/pgs-2020-0092">10.2217/pgs-2020-0092</a>
</td>
</tr>
<tr>
<td>Male sex (XY chromosomes)</td>
<td>Androgen receptors (ARs) play a key role in increasing transcription of TMPRSS2. This may explain the predominance of males to COVID-19 fatality and severity. [6]</td>
<td>
6) doi: <a href="http://10.1073/pnas.2021450118">10.1073/pnas.2021450118 </a>
</td>
</tr>
<tr>
<td colspan="1">Age</td>
<td>Old people</td>
<td>ACE2 protein expression is increased with aging in several tissues [1], including lungs and particularly in patients requiring mechanical ventilation [2]. During aging, telomere dysfunction activates a DNA damage response leading to higher ACE2 expression. Thus, telomere shortening could contribute to make elderly more susceptible to SARS-CoV-2 infection [3].</td>
<td>
1. <a href="https://10.1016/j.exger.2021.111507" style="color:blue; text-decoration:underline">10.1016/j.exger.2021.111507</a>
2. <a href="https://doi.org/10.1371/journal.pone.0247060" style="color:blue; text-decoration:underline">10.1371/journal.pone.0247060</a>
3. <a href="https://doi.org/10.15252/embr.202153658" style="color:blue; text-decoration:underline">10.15252/embr.202153658</a>
</td>
</tr>
<tr>
<td colspan="1">
Vitamin D
(high evidence)
</td>
<td>Vitamin D deficiency</td>
<td>
Vitamin D administration enhanced mRNA expression of VDR and ACE2 in a rat model of acute lung injury [1]. In particular, vitamin D upregulates the sACE2 form [2]. Thus, low vitamin D status may impair the trapping protective mechanism of sACE2 [3]. Furthermore, vitamin D deficiency has been shown to reduce the expression of antimicrobial peptides (_-defensin, cathelicidin), which act against enveloped viruses [4,5]. Decreased sACE2 and cellular viral defense might be some mechanisms explaining how low vitamin D modulate SARS-CoV-2 infectibility.
</td>
<td>
1. doi: <a href="http://10.1016/j.injury.2016.09.025">10.1016/j.injury.2016.09.025</a>
2. doi: <a href="http://10.1152/ajplung.00071.2009">10.1152/ajplung.00071.2009</a>
3. doi: <a href="http://10.3390/ijms22105251">10.3390/ijms22105251</a>
4. doi: <a href="http://10.1007/s11154-021-09679-5">10.1007/s11154-021-09679-5</a>
5. doi: <a href="http://10.1080/14787210.2021.1941871">10.1080/14787210.2021.1941871</a>
</td>
</tr>
<tr>
<td colspan="1">
Gut microbiota
</td>
<td>
Gut dysbiosis (alteration of gut microbiota)
</td>
<td>
<div>
<div>
<div>
The human gut expresses high levels of ACE2 [1-3], and SARS-CoV-2 infection of human enterocytes in vitro is supported by strong evidence [4-6]. However human healthy gut may not be permeable to viral entry due notably to the protective multi-layers of the intestinal barrier including the mucus layer [5]. The colonic mucus barrier is shaped by the composition of the gut microbiota [7]. Thus, individuals with altered mucosal barrier (gut dysbiosis) might be more vulnerable to gastrointestinal SARS-CoV-2 infection [8]. Further research is needed to acquire a comprehensive understanding of the experimental and clinical conditions under which SARS-CoV-2 productively infects enterocytes.
</div>
</div>
</div>
</td>
<td>
1. doi: <a href="http://10.1002/path.1570">10.1002/path.1570</a>
2. doi: <a href="http://10.1038/s41575-021-00416-6">10.1038/s41575-021-00416-6</a>
3.doi: <a href="http://10.3390/genes1160645">10.3390/genes1160645</a>
4.doi: <a href="http://10.1126/science.abc.1669">10.1126/science.abc.1669</a>
5.doi: <a href="http://10.1126/sciimmunol.abc.3582">10.1126/sciimmunol.abc.3582</a>
6. doi: <a href="http://10.1038/s41467-021-25729-7">10.1038/s41467-021-25729-7</a>
7.doi: <a href="http://10.15252/embr.20139263">10.15252/embr.20139263</a>
8.doi: <a href="http://10.3390/jcm11195691">10.3390/jcm11195691</a>
</td>
</tr>
<tr>
<td colspan="1">Therapeutic intervention against COVID-19.</td>
<td>Remdesivir</td>
<td>
Is a prodrug of adenosine analogue, which binds to the viral RNA-dependent RNA polymerase (RdRp) and inhibits viral replication inside cells through premature termination of RNA transcription [1 – 4].
</td>
<td>
1) (EMA), E.M.A. Veklury. 2021.
Available online: https://www.ema.europa.eu/en/medicines/human/EPAR/veklury (accessed on 12 May 2022)
2) 10.1038/s41467-020-20542-0
3) 10.1016/j.cegh.2020.07.011
4) 10.1038/s41586-020-2423-5
</td>
</tr>
<tr>
<td colspan="1"> </td>
<td>Molnupiravir</td>
<td>
Is an isopropyl ester prodrug, which is cleaved in plasma by host esterases to an active nucleoside analog b-D-N4-hydroxycytidine (NHC) [1]. After entering host cells, it is intracellularly transformed into its active form, β-DN4- hydroxycytidine-triphosphate (NHC triphosphate) [2,3]. This then targets the RdRp, which is virally encoded and competitively inhibits the cytidine and uridine triphosphates and incorporates Molnupiravir instead. The RdRp (RNA-dependent RNA polymerase) enzyme of SARS-CoV-2 uses the NHC triphosphate as a substrate instead of the cytidine and uridine triphosphates and then incorporates either A or G in the RdRp active centres, forming stable complexes, thus escaping proof reading by the synthesis of a mutated RNA [4,5]. As a result, the virus can no longer reproduce. This mechanism of action (the accumulation of mutations) is referred to as viral error catastrophe [2].
</td>
<td>
1) 10.1016/j.trsl.2019.12.002
2) 10.1126/science.abb7498
3) 10.1128/AAC.00766-18
4) 10.1038/s41594-021-00651-0
5) 10.1016/j.jbc.2021.100770
</td>
</tr>
<tr>
<td colspan="1"> </td>
<td>Nirmatrelvir (formerly PF-07321332, Paxlovid™)</td>
<td>
Is an orally bioavailable 3C-like protease (3CL PRO) inhibitor that is the subject of phase 1 clinical trial NCT04756531 and the phase 2/3 clinical trials (NCT04960202 and NCT05011513, NCT04756531, NCT04960202 and NCT05011513). A 3CLpro antagonist will be highly specific to SARSCoV-2 and will have minimal side effects because 3Clpro shares no homology with human proteases [1,2]. The SARS-CoV-2 genome encodes two polyproteins (pp1a and pp1ab) and four structural proteins [3,4]. The polyproteins are cleaved by the critical SARSCoV-2 main protease (Mpro, also referred to as 3CL protease) at eleven different sites to yield shorter, non-structural proteins [5,6]. Without the activity of the 3CL PRO, nonstructural proteins cannot be released to perform their functions, inhibiting viral replication [7–9].
</td>
<td>
1) 10.1126/science.abb4489
2) 10.1007/s13238-013-2841-3
3) 10.1038/s41586-020-2008-3
4) 10.1038/s41586-020-2012-7
5) 10.1021/acs.jmedchem.5b01461
6) 10.1038/s41586-020-2223-y
7) 10.1007/s10930-020-09933-w
8) 10.1073/pnas.1601327113
9) 10.1002/med.21783
</td>
</tr>
<tr>
<td colspan="1">
<pre>
Air pollution</pre>
</td>
<td> </td>
<td>
Air pollution induces increased expression of ACE2 which may result in increased viral entry and coronavirus production.
Increased ACE2 expression has been reported in the respiratory system in response to air pollution exposure (1-4). Increased expression may affect susceptibility to SARS-CoV-2 infection. Similarly, some constituents of air pollution (PM, ozone) have been reported to increase the expression of TMPRSS2. (3, 5-6)
</td>
<td>
1) https://doi.org/10.1186/s12989-015-0094-4
2) 10.1016/j.burns.2015.04.010
3) 10.1016/j.envres.2021.110722
4) 10.3390/ijerph17155573
5) 10.1186/s12989-021-00404-3
6) https://doi.org/10.1038/s41598-022-04906-8
</td>
</tr>
<tr>
<td colspan="1">Diet</td>
<td>Chemicals found in foods impact viral replication</td>
<td>
<ul>
<li>Pelargonidin, found in red and black berries, was shown to dose-dependently block SARS-CoV-2 binding to ACE2, reduce SARS-CoV-2 replication in vitro and reduce ACE2 expression [213].</li>
<li>
Further evidence comes from SARS-CoV. Many plant-derived terpenoids and curcumin inhibited viral replication in vitro [218].
</li>
</ul>
</td>
<td>
213: <a href="http://doi.org/10.1016/j.bcp.2021.114564">http://doi.org/10.1016/j.bcp.2021.114564</a>
218:<a href="http://%C2%A0http://doi.org/10.1021/jm070295s"> </a><a href="http://doi.org/10.1021/jm070295s">http://doi.org/10.1021/jm070295s</a>
</td>
</tr>
</tbody>
</table>
</div>
<div>
<h4><a href="/relationships/2545">Relationship: 2545: SARS-CoV-2 production leads to Sustentacular cells, decrease </a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
<h4><a href="/relationships/2362">Relationship: 2362: Sustentacular cells, decrease leads to olfacotry neurons, decrease</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
<h4><a href="/relationships/2363">Relationship: 2363: olfacotry neurons, decrease leads to Olfactory epithelium degeneration</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
<h4><a href="/relationships/2364">Relationship: 2364: Olfactory epithelium degeneration leads to anosmia</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2365">Relationship: 2365: Sustentacular cells, decrease leads to anosmia</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>non-adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
<h4><a href="/relationships/2366">Relationship: 2366: olfacotry neurons, decrease leads to anosmia</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/394">SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>non-adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
</div>
</div>
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