<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 Do not cite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">
<h2>Abstract</h2>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">SARS and SARS-CoV-2 coronoviruses enter the cell through interaction with the <a href="https://www.genecards.org/cgi-bin/carddisp.pl?gene=ACE2">ACE2</a> receptor. The first event upon cell entry after uncoating is the primary translation of the <a href="https://www.ncbi.nlm.nih.gov/gene/43740578">ORF1a and ORF1b</a> genomic RNA to produce non-structural proteins (nsps). The nsps structural proteins, and accessory proteins, are encoded by 10 ORFs in the SARS-CoV-2 RNA genome. They may have multiple functions during viral replication as well as in evasion of the host innate immune response, thus augmenting viral replication and spread. The early innate immune system evasion proteins produced in the sub-genomic translation after viral genome replication and transcription within the infected cell suppress the<a href="https://www.wikipathways.org/index.php/Pathway:WP4868"> Interferon-I antiviral response</a> to increase viral load. Beyond potentially contributing to the severity of clinical symptoms and adverse disease outcome in individuals, increase in viral load can lead to proliferation from person-to-person and across species, also increasing the likelihood of mutations that result in more infective or virulant strains. </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Severe accute respiratory syndrome (SARS) and SARS-CoV-2 coronoviruses enter the cell through interaction with the <a href="https://www.genecards.org/cgi-bin/carddisp.pl?gene=ACE2">ACE2</a> receptor. The first event upon cell entry after uncoating is the primary translation of the <a href="https://www.ncbi.nlm.nih.gov/gene/43740578">ORF1a and ORF1b</a> genomic RNA to produce non-structural proteins (nsps). The nsps structural proteins, and accessory proteins, are encoded by 10 ORFs in the SARS-CoV-2 RNA genome. They may have multiple functions during viral replication as well as in evasion of the host innate immune response, thus augmenting viral replication and spread. The early innate immune system evasion proteins produced in the sub-genomic translation after viral genome replication and transcription within the infected cell suppress the<a href="https://www.wikipathways.org/index.php/Pathway:WP4868"> Interferon-I antiviral response</a> to increase viral load. Beyond potentially contributing to the severity of clinical symptoms and adverse disease outcome in individuals, increase in viral load can lead to proliferation from person-to-person and across species, also increasing the likelihood of mutations that result in more infective or virulant strains. </span></span></p>
</div>
<div id="background">
<h3>Background</h3>
<p>This AOP was developed in the context of other COVID-19 AOPs through the work of a larger <span style="font-size:16px">international effort to model the pathogenesis of COVID-19 using the AOP framework (the CIAO project, <a href="https://www.ciao-covid.net/about-us" style="color:#0563c1; text-decoration:underline">https://www.ciao-covid.net/about-us</a>), initiated by the European Commission-Joint Research Centre (EC-JRC), and supported by the Society for the Advancement of Adverse Outcome Pathways (SAAOP). More than 80 scientists from 50 institutions contributed to the fifteen AOPs connected to the molecular initiating event (1739) SARS-CoV-2 binding to ACE2, and other COVID-19-related AOPs. AOP 430 serves as a hub of early key events leading to viral transmission (AO 1939) and the severe disease outcomes described in the networked COVID-19 AOPs.</span></p>
<p>Although COVID-19 has shown to be a more severe illness in older than in young people, there is evidence that viral load was not influenced by age or sex (Challenger et al., 2022), and infection rate and viral load did not differ by sex (Arnold et al., 2022; Qi et al., 2021; Cheemarla et al., 2021). Therefore, this AOP is applicable to all life stages and both sexes.</p>
<p><strong>Taxonomic domain</strong></p>
<p>No non-mammals have been found to be infected by SARS-CoV-2. Mammals listed in the Taxonomic Applicability table were either experimentally or naturally infected, as confirmed by polymerase chain reaction (PCR) or antibody assays, hence evidence is high for these species. Other mammalian species are likely also susceptible, but some mammals experimentally exposed to the virus did not become infected (Bosco-Lauth et al., 2021). The AOP is therefore applicable to humans and other mammals. Infections in non-human mammals is important in the potential for zoonotic spillover and is discussed in more detail in the adverse outcome (AO 1939), with species-specific references.</p>
<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 - ACE2 binding to viral S-protein leading to microvascular disfunction via ACE2 dysregulation</a></td>
<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 acute respiratory distress (via cell death)</a></td>
<td><a href="/aops/468">Aop:468 - Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h3>Evidence for Perturbation by Stressor</h3>
<h4>Overview for Molecular Initiating Event</h4>
<p><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Receptor recognition is an essential determinant of molecular level in this AOP. ACE2 was reported as an entry receptor for SARS-CoV-2. The viral entry process is mediated by the envelope-embedded surface-located spike (S) glycoprotein. Jun Lan and Walls, A.C et al (Nature 581, 215–220; Cell 180, 281–292) demonstrated a critical initial step of infection at the molecular level from the interaction of ACE2 and S protein. ACE2 has shown that receptor binding affinity to S protein is nM range. To elucidate the interaction between the SARS-CoV-2 RBD and ACE2 at a higher resolution, they also determined the structure of the SARS-CoV-2 RBD–ACE2 complex using X-ray crystallography.</span></span> <span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">The expression and distribution of the ACE2 in human body may indicate the potential infection of SARS-CoV-2. Through the developed single-cell RNA sequencing (scRNA-Seq) technique and single-cell transcriptomes based on the public database, researchers analyzed the ACE2 RNA expression profile at single-cell resolution. High ACE2 expression was identified in type II alveolar cells (Zou, X. et al.</span></span> <span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Front. Med.2020)</span></span></p>
<p><span style="font-size:12px"><span style="font-family:"Times New Roman",serif">SARS-CoV-2 belongs to the Coronaviridae family, which includes evolutionary related enveloped (+) strand RNA viruses of vertebrates, such as seasonal common coronaviruses, SARS-CoV and CoV-NL63, SARS-CoV (Kim Young Jun et al)</span></span></p>
<p>The KE is applicable to broad species/life stage/sex. The binding of ACE2 occurs in the cells which express ACE2. </p>
<h4>Key Event Description</h4>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">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). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">A table on ACE2 expression levels according to tissues <em>(Kim et al.)</em></span></span></p>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">ACE2 mean expression</span></strong></span></span></p>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">Standard deviation of expression</span></strong></span></span></p>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.905</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="color:#0070c0">ACE2 receptors in the brain (endothelial, neuronal and glial cells):</span></strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">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. </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">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). </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><strong><em>ACE2 receptors in the intestines</em></strong></span></span></span></p>
<p dir="ltr" style="text-align:justify"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><span style="background-color:transparent">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). </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"><span style="color:black"><strong><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif"><em>In vitro</em> methods supporting interaction between ACE2 and SARS-CoV-2 spike protein</span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"><span style="color:black"><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">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,</span></span></span></span></span></p>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">ACE2 protein </span></strong></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">34.6 nM</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">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.</span></span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Xia, H. and Lazartigues, E. Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010 Rep. 12 (3), 170– 175</span></span></span></p>
<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>MolecularInitiatingEvent</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 acute respiratory distress (via cell death)</a></td>
<td><a href="/aops/468">Aop:468 - Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 vertebrates (Lam et al., 2020)</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">NRP1 in human & rodents (but also present in monkey and other vertebrates </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lu and Meng, 2015)</span></span></p>
<p><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">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 </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Yamamoto et al., 2020, Shang et al., 2020, Shirato et al., 2018)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. 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 </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Murgolo et al., 2021, Kang et al., 2020, Mirabelli et al., 2020, Riva et al., 2020)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. However, even in these cells, heterologous expression of TMPRSS2 abrogates the pharmacological blockade of cathepsin inhibitors </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012, Hoffmann et al., 2020a)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. 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 </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lamers et al., 2020)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, as when both factors are present,d cathepsin inhibitors are less effective than TMPRSS2 inhibitors </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Therefore it is critical to map which cells contain the different types of proteases.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">In summary, TMPRSS2 appears to be expressed in a wide range of healthy adult organs, but in restricted cell types, including:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">AT2 and clara cells of lungs</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">sinusoidal endothelium, and hepatocyte of the liver, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">endocrine cells of the prostate, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">goblet cells , and enterocytes of the small intestine, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">intercalated cells, and the proximal tubular of the kidney.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Ciliated, secretory and suprabasal of nasal</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">spermatogonial stem cells of testes</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">cyto tropoblast and peri vascular cells of placenta</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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.</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">All life stages</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The expression of NRP1 protein in gastric cancer was not related to gender or age (Cao et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">For more information difference of NRP1 expression between male and female see <a href="https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue"><span style="color:blue">https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue</span></a><span style="color:blue">.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">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 </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Moutal et al., 2020)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<h4>Key Event Description</h4>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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) <span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b).</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> 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 </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Lukassen et al., 2020, Lamers et al., 2020, Chen et al., 2020, Jing et al., 2020, Subramanian et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">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 </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Millet and Whittaker, 2018)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">.</span></span></span> 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).<span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> 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 </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">; or (ii) endocytic pathway within the endosomal–lysosomal compartments including processing by lysosomal cathepsin L </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Yang and Shen, 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. 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 </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. When TMPRSS2 (or other serine proteases such as TMPRSS4 </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Zang et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> or human airway trypsin-like protease [HAT]</span></span></span> <span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Bestle et al., 2020a)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">) is expressed, fusion of the virus with the cell surface membrane is preferred </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Shirato et al., 2018)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">, while in their absence, the virus can penetrate the cell by endocytosis </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. 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) </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Cantuti-Castelvetri et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. This key event deals with SARS-CoV-2 entry in host cells and is divided in three categories: TMPRSS2, capthesin L and NRP-1.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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. </span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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). <span style="color:black">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) </span><span style="color:black">(Hasan et al., 2020)</span><span style="color:black">. T</span>he 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. <strong><span style="color:black">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</span></strong> <span style="color:black">(Bestle et al., 2020b, Johnson et al., 2020)</span><span style="color:black">.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Camostat mesylate, an inhibitor of TMPRSS2, blocks SARS-CoV-2 infection of lung cells like <span style="color:black">Calu-3 cells but not Huh7.5 and Vero E6 cells</span>. 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).</span></span></p>
<p> </p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Spike binding to neuropilin-1:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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. </span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">NRP1 is a receptor for <span style="color:black">furin-cleaved SARS-CoV-2 spike peptide </span><span style="color:black">(Cantuti-Castelvetri et al., 2020, Daly et al., 2020, Johnson et al., 2020)</span><span style="color:black">. 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 </span><span style="color:black">(Johnson et al., 2020)</span><span style="color:black">.</span> 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).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID<span style="font-family:"Cambria Math",serif">‑</span>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<span style="font-family:"Cambria Math",serif">‑</span>19 by enhancing the entry of SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 into the brain through the olfactory epithelium. NRP1 is also expressed in the CNS, including olfactory<span style="font-family:"Cambria Math",serif">‑</span>related regions such as the olfactory tubercles and paraolfactory gyri. Supporting the potential role of NRP1 as an additional SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 infection mediator implicated in the neurologic manifestations of COVID<span style="font-family:"Cambria Math",serif">‑</span>19. Accordingly, the neurotropism of SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 via NRP1<span style="font-family:"Cambria Math",serif">‑</span>expressing cells in the CNS merits further investigation (Davies et al., 2020).</span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">More information on tissue distribution and protein expression of NRP1 can be found in https://www.proteinatlas.org/ENSG000000992 50-NRP1</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Spike entry via <span style="color:black">lysosomal cathepsins and endocytosis</span>:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">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.</span> Evidence came from studies<span style="color:black"> 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) </span><span style="color:black">(Rossi et al., 2004, Simmons et al., 2005)</span><span style="color:black">. 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.</span></span></span></p>
<p style="text-align:justify"><strong>Viral entry leads to delivery of virion proteins and translation of viral proteins immediately: </strong></p>
<p style="text-align:justify"><span style="font-size:14px">Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [Kim D,<em> et al., 2020</em>]. The virus contains a <span style="color:#131413">29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively</span><span style="color:black"> </span><span style="color:#131413">that contain cis-acting secondary RNA structures essential for RNA synthesis [</span>Huston N. C.<em> et al., 2021</em>]<span style="color:black">. T</span>he genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [Budzilowicx C.J., <em>et al., 1985</em>]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [Snijder E. J., <em>et al.</em>, 2003]<span style="color:black">.</span> Preceding each ORF there are other TRSs called the body TRS (<span style="color:black">TRS B). </span>The <span style="color:black">5′ two-thirds of the </span>genome contains <span style="color:black">two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [Di H., <em>et al.</em>, 2018].</span> Genome expression starts with the translation of <span style="color:#131413">two large ORFs of the 5’ two-thirds: ORF1a of</span><span style="color:black"> 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a</span><span style="color:#131413"> programmed (- 1) ribosomal frameshifting </span>[Snijder E. J., <em>et al.</em>, 2003]<span style="color:black">, yielding</span><span style="color:#131413"> pp1a and pp1ab</span><span style="color:black">. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a,</span> <span style="color:black">nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain</span> [Sacco M. D. <em>et al., 2020</em>]<span style="color:#131413">. </span><span style="color:black">The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication a</span>nd transcription <span style="color:black">complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [</span>Klein<em> </em>S., <em>et al., 2021</em>, Snijder E. J.<em>, et al., 2020, </em>V'Kovski P. , <em>et al., 2021</em>]<span style="color:black">. 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.</span></span></p>
<h4>How it is Measured or Detected</h4>
<p>SARS-CoV2 entry can be determined by many different ways:</p>
<p>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.</p>
<p>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)].</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 gene expression can be measured by RNAseq and microarray (Baughn et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Expression levels of TMPRSS2 can be measured by RNA in situ hybridization (RNA-ISH) (Qiao et al., 2020)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Several methods have been identified in the literature for measuring and detecting NRP1 receptor binding. Briefly described:</span></span></p>
<ol>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">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 </span><span style="color:black">(Centers for Disease Control and Prevention, 2020, Daly et al., 2020)</span><span style="color:black">.</span></span></span></li>
</ol>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Key findings (Centers for Disease Control and Prevention, 2020, Daly et al., 2020): </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• The S1 fragment of the cleaved SARS-CoV-2 spike protein binds to the cell surface receptor neuropilin-1 (NRP1). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• 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).</span></span></p>
<ol start="2">
<li><span style="font-size:11pt"><span style="color:black"><span style="font-family:"Calibri",sans-serif">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).</span></span></span></li>
</ol>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Key findings (Cantuti-Castelvetri et al., 2020, Centers for Disease Control and Prevention, 2020): </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• 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).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• 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.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">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 </span></span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">(Perez-Miller et al., 2020)</span></span></span></p>
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<h4><a href="/events/1901">Event: 1901: Interferon-I antiviral response, antagonized by SARS-CoV-2</a></h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Broad mammalian host range 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). Some literature found on non-human hosts indicates that NSPs and accessory proteins can interact in a similar manner with bird (chicken) and other mammal proteins in the IFN-I pathway (Moustaqil et al. 2021; Rui et al. 2021).</span></span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">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 <span style="font-size:14px">(</span></span></span><span style="font-size:14px">doi:<a class="article-header__doi__value" href="https://doi.org/10.1016/j.cell.2020.04.011">https://doi.org/10.1016/j.cell.2020.04.01</a></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:14px">). The SAR</span>S-CoV-2 genome contains a 5’-untranslated region (UTR; 265 bp), <a href="https://www.ncbi.nlm.nih.gov/gene/?term=ORF1a+SARS-CoV-2">ORF1ab</a> (21,289 bp) holding two overlapping open reading frames (13,217 bp and 21,289 bp, respectively) that encode two polyproteins (Kim et al. 2020; O’Leary et al. 2020). Viral transcription and replication is explained in depth in <a href="https://aopwiki.org/events/1847" style="color:blue; text-decoration:underline">KE1847</a>. Briefly, the first event upon cell entry is the primary translation of the ORF1a and ORF1b genomic RNA to produce non-structural proteins (NSPs). 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 (duplications of NSP1-11 and five additional proteins, NSP12-16). Viral proteases NSP3 and NSP5 cleave the polypeptides through domains functioning as a papain-like protease and a 3C-like protease, respectively <span style="font-size:14px">(</span></span></span><span style="font-size:14px">doi:<a class="article-header__doi__value" href="https://doi.org/10.1016/j.cell.2020.04.011">https://doi.org/10.1016/j.cell.2020.04.01</a></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:14px">). </span> The NSPs, structural proteins, and accessory proteins are encoded by 10 ORFs in the SARS-CoV-2 RNA genome. They may have multiple functions during viral replication as well as in evasion of the host innate immune response, thus augmenting viral replication and spread<strong> (Amor et al. 2020). </strong>Extensive protein-protein interaction <strong>(Gordon et al. 2020) </strong>and viral protein-host RNA interaction networks have been demonstrated between the viral NSPs and accessory proteins and host molecules. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This key event is focused on the specific viral:host protein interactions within the infected cell that are involved in the <a href="https://www.wikipathways.org/index.php/Pathway:WP4868">IFN-I antiviral response pathways</a>. IFN-I is the main component of the innate immune system that is suppressed by the SARS-CoV-2 coronavirus early in infection. </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The primary form of host intracellular virus surveillance detects viral components to induce an immediate systemic type I interferon (IFN) response. Cellular RNA sensors called pattern recognition receptors (PRRs) such as RIG-I, MDA5 and LGP2 detect the presence of viral RNAs and promote nuclear translocation of the transcription factor IRF3, leading to transcription, translation, and secretion of IFN-α and IFN-β. This in turn leads to interaction with the IFN receptor (IFNAR), phosphorylation of STAT1 and 2, and transcription and translation of hundreds of antiviral genes <strong>(Quarleri and Delpino, 2021).</strong></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Interactions between SARS-CoV-2 proteins and human RNAs thwart the IFN response upon infection: NSP1 binds to 40S ribosomal RNA in the mRNA entry channel of the ribosome to inhibit host mRNA translation; NSP8 and NSP9 displace signal recognition particle proteins (SRP54, 27 and 19) to bind to the 7SL RNA and block protein trafficking to the cell membrane (Banerjee et al. 2020; Gordon et al. 2020). Xia et al. (2020) found that NSP6 and NSP13 antagonize IFN-I production via distinct mechanisms: NSP6 binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, and NSP13 binds and blocks TBK1 phosphorylation. NSP14 induces lysosomal degradation of type 1 IFNAR to prevent STAT activation (Hayn et al. 2021). ORF6 hijacks KPNA2 to block IRF3, and Nup98/RAE1 to block STAT nuclear import, to silence IFN-I gene expression (Xia and Shi, 2020). ORF7a suppresses STAT2 phosphorylation and ORF7b suppresses STAT1 and STAT2 phosphorylation to block ISGF3 complex formation with IRF9 (Xia and Shi, 2020). ORF8 interacts and downregulates MHC-I (Zhang et al 2020), and has been reported to block INF</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> expression, but the mechanism has not been identified (Rashid et al. 2021; Li et al. 2020). ORF9b antagonizes Type I Interferons by targeting multiple components of RIG-I/MDA-5-MAVS, TOMM70, NEMO and cGAS-STING signalling (Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Following is a table of the current state of knowledge of SARS-CoV-2 protein putative functions in relation to IFN-I antiviral response antagonism.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">DNA Polymerase Alpha Complex: Regulates the activation of IFN-I through cytosolic<br />
RNA-DNA synthesis (POLA1/2-PRIM1/2) and primes DNA replication in the nucleus (Gordon et al. 2020; Chaudhuri et al. 2020). Can also inhibit host gene expression by binding to ribosomes and modifying host mRNAs (Shi et al. 2020; Schubert et al. 2020; Thoms et al. 2020). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">While not essential for viral replication, deletion of NSP2 diminishes viral growth and RNA synthesis</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Suppresses IFN-I: Cleaves IRF3 (Moustaqil et al. 2021); binds/cleaves ISG15 (Rui et al. 2021; Shin et al. 2020; Liu et al. 2021; Klemm et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">In complex with NSP8 forms primase as part of multimeric RNA-dependent RNA replicase (RdRp)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds SRP72/54/19 (Gordon et al. 2020) and 7SL RNA to block IFN membrane transport (Banerjee et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds SRP and 7SL RNA with NSP8 to block IFN membrane transport (Banerjee et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Helicase and triphosphatase that initiates the first step in viral mRNA capping.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts with M, S, E and 7a; form viroporins; immune evasion</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds STING (Rui et al 2021)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts with RIG-I and MAVS sensors of viral RNA (Fu et al 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Hijacks the nuclear importin Karyopherin a 2 (KPNA2) to block IRF3 (Xia and Shi, 2020) and Nup98/RAE1 to block STAT nuclear import (Miorin et al. 2020; Kato et al. 2020), leading to the silence of downstream ISGs</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts and downregulates MHC-I (Zhang et al. 2020). May inhibit type I interferon (IFN-β) and interferon-stimulated response element (ISRE) (Rashid et al. 2020; Li et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Attenuates stress granule formation: G3BP1/2 (Chen et al. 2020; Cascarina et al. 2020); G3BP1 also interacts with RIG-I (Kim et al. 2019) and STAT1/2 (Mu et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Membrane protein antagonizes Type I Interferons by targeting multiple components of RIG-I/MDA-5-MAVS, TOMM70, NEMO, and cGAS-STING signaling pathways (Fu et al. 2020; Chen et al. 2020; Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020)</span></span></span></span></p>
</td>
</tr>
</tbody>
</table>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Detection of IFN-I suppression involves measuring gene promoter/transcription activation (luciferase assays), gene up/down regulation (quantitative PCR), protein-protein interaction (immunoprecipitation, immunoblotting) or in-situ co-location of viral and host proteins (immunofluorescent or confocal microscopy) in cell culture. Examples of methods used include the following:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Interferon I decrease (Xia et al. 2020):</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">IFN-I production and signaling luciferase reporter assays</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Co-immunoprecipitation and western blot</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">DNA assembly and RNA transcription of a luciferase replicon for SARS-CoV-2</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Replicon RNA electroporation and luciferase reporter assay</span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2-Human Protein-Protein Interaction Map (Gordon et al. 2020)</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cloning and expression of viral proteins via plasmid transfection into HEK293T cell line</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Protein affinity purification using MagStrep beads with detection by anti-strep western blot of cell lysate</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Global analysis of SARS-CoV-2 host interacting proteins using affinity purification-mass spectrometry</span></span></li>
</ul>
<p> </p>
<h4>References</h4>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Banerjee et al. 2020. SARS-CoV-2 disrupts splicing, translation, and protein trafficking to supress host defenses. Cell 183, 1325–1339. <a href="https://doi.org/10.1016/j.cell.2020.10.004" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.cell.2020.10.004</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cascarina and Ross, 2020. A proposed role for the SARS-CoV-2 nucleocapsid protein in the </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">formation and regulation of biomolecular condensates. The FASEB Journal, 34:9832–9842. DOI: 10.1096/fj.202001351 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chaudhuri, A. 2021. Comparative analysis of non-structural protein 1 of SARS-CoV2 with SARS-CoV1 and MERS-CoV: An in-silico study. Journal of Molecular Structure, Volume 1243, 130854, https://doi.org/10.1016/j.molstruc.2021.130854.</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Jiang et al. 2020. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cellular & Molecular Immunology 17:998–1000; https://doi.org/10.1038/s41423-020-0514-8</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kim et al. 2019. The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-β response. J. Biol. Chem. 294(16): 6430–6438. DOI 10.1074/jbc.RA118.005868</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kim et al. 2020. The Architecture of SARS-CoV-2 Transcriptome. Cell 181, 914–921. <a href="https://doi.org/10.1016/j.cell.2020.04.011" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.cell.2020.04.011</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Li et al. 2020. The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway. Virus Research vol. 286. <a href="https://doi.org/10.1016/j.virusres.2020.198074" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.virusres.2020.198074</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Liu et al. 2021. ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity. Nature Microbiol 6: 467–478. <a href="https://doi.org/10.1038/s41564-021-00884-1" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41564-021-00884-1</a> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">O’Leary et al. 2020 Unpacking Pandora from Its Box: Deciphering the Molecular Basis of the SARS-CoV-2 Coronavirus. Int. J. Mol. Sci. 2021, 22, 386. <a href="https://doi.org/10.3390/ijms22010386" style="color:blue; text-decoration:underline">https://doi.org/10.3390/ijms22010386</a> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rui et al. 2021. Unique and complementary suppression of cGAS-STING and RNA sensing-triggered innate immune responses by SARS-CoV-2 proteins. Sig Transduct Target Ther 6, 123. <a href="https://doi.org/10.1038/s41392-021-00515-5" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41392-021-00515-5</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schubert et al. 2020. SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nature Structural & Molecular Bio. 27:959-966. <a href="https://doi.org/10.1038/s41594-020-0511-8" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41594-020-0511-8</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Thoms et al. 2020. Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2. Science 369(6508): 1249-1255. DOI: 10.1126/science.abc8665</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Wu et al. 2021. SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO. Cell Reports 34, 108761. <a href="https://doi.org/10.1016/j.celrep.2021.108761" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.celrep.2021.108761</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Wu et al. 2020. Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2. Cell Discovery 6:68. <a href="https://doi.org/10.1038/s41421-020-00210-9" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41421-020-00210-9</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xia et al. 2020. Evasion of Type I Interferon by SARS-CoV-2. Cell Reports 33, 108234. <a href="https://doi.org/10.1016/j.celrep.2020.108234" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.celrep.2020.108234</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xia and Shi, 2020. Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2. Journal of Interferon & Cytokine Research v.40, no. 12 DOI:10.1089/jir.2020.0214 </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zhang et al. 2020. The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I. bioRxiv preprint doi: <a href="https://doi.org/10.1101/2020.05.24.111823" style="color:blue; text-decoration:underline">https://doi.org/10.1101/2020.05.24.111823</a></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Broad mammalian host range has been demonstrated based on spike protein tropism for and binding to ACE2 [Conceicao </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020; Wu </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020] and cross-species ACE2 structural analysis [Damas </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 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.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [D. Kim<em> et al.</em>]. The virus contains a <span style="color:#131413">29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively</span><span style="color:black"> </span><span style="color:#131413">[</span>E. J. Snijder<em> et al.</em><span style="color:#131413">] that contain cis-acting secondary RNA structures essential for RNA synthesis [</span>N. C. Huston<em> et al.</em>]<span style="color:black">. T</span>he genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [C.J. Budzilowicx <em>et al.</em>]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [E. J. Snijder <em>et al.</em>]<span style="color:black">.</span> Preceding each ORF there are other TRSs called the body TRS (<span style="color:black">TRS B). </span>The <span style="color:black">5′ two-thirds of the </span>genome contains <span style="color:black">two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [H. Di <em>et al.</em>].</span> Genome expression starts with the translation of <span style="color:#131413">two large ORFs of the 5’ two-thirds: ORF1a of</span><span style="color:black"> 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a</span><span style="color:#131413"> programmed (- 1) ribosomal frameshifting [E. J. Snider <em>et al.</em>]</span><span style="color:black">, yielding</span><span style="color:#131413"> pp1a and pp1ab</span><span style="color:black">. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a,</span> <span style="color:black">nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain</span> [M. D. Sacco <em>et al.</em>]<span style="color:#131413">. </span><span style="color:black">The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication a</span>nd transcription <span style="color:black">complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [</span>S. Klein<em> et al.</em>, E. J. Snijder<em> et al., </em>P. V'Kovski, <em>et al.</em>]<span style="color:black">. Besides replication, which yields the positive-sense gRNA, the replicase also</span> <span style="color:black">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. </span>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]. s<span style="color:black">gRNAs 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 [</span>D. Kim<em> et al.</em>]<span style="color:black">. The gRNA is packaged by the structural proteins to assemble progeny virions.</span></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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<em> et al.</em>]. 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<em> et al.</em>]. Other elements of the genome include are shown below [V. B. O'Leary <em>et al.</em>]. <strong>The first event upon cell entry is the primary translation of the ORF1a and ORF1b gRNA to produce non-structural proteins (nsps).</strong></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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 <em>et al.</em>]. 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<em> et al.</em>]. Two overlapping ORFs, ORF1a and ORF1b, generate continuous polypeptides, which are cleaved into a total of 16 so-called nsps [Y Finkel <em>et al.</em>]. 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 <span style="color:black">pp1a is approximately 1.4–2.2 times more expressed than pp1ab. </span>After translation, the polyproteins are cleaved by viral proteases nsp3 and nsp5. Nsp5 <span style="color:black">protease can be referred to as 3C-like protease (3CL<sup>pro</sup>) or as main protease (M<sup>pro</sup>), as it cleaves the majority of the polyprotein cleavage sites. [H.A. Hussein </span><em>et al.</em><span style="color:black">] Nsp1 cleavage is quick and nsp1 associates with host cell ribosomes and results in host cellular shutdown, </span><span style="color:#231f20">suppressing host gene expression </span><span style="color:#000000">[</span>M. Thoms<em> et al.]</em><span style="color:black">. Fifteen proteins, nsp2–16 constitute the viral RTC. They are targeted to defined subcellular locations and establish a network with host cell factors.</span> N<span style="color:black">sp2–11 remodel host membrane architecture, mediate host immune evasion and provide cofactors for replication, w</span>hilst <span style="color:black">nsp12–16 contain the core enzymatic functions involved in RNA synthesis, modification and proofreading [</span>P. V'Kovski <em>et al.</em>]<span style="color:black">. </span>nsp-7 and nsp-8 form a complex priming the RNA-dependent RNA polymerase (RdRp or RTC) - nsp-12. <span style="color:black">nsp14 provides a 3′–5′ exonuclease activity providing RNA proofreading function.</span> Nsp-10 composes the RNA <span style="color:black">capping machinery</span> nsp-9. <span style="color:black">nsp13 provides the RNA 5′-triphosphatase activity</span>. Nsp-14 is a <em><span style="color:black">N</span></em><span style="color:black">7-methyltransferase and nsp-16 the 2′-<em>O</em>-methyltransferase. </span>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<em> et al.</em>]. Xia et al. [H. Xia<em> et al.</em>] 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.</span></span></p>
<p> </p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Viral transcription and replication:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Viral transcription and replication occur at the viral replication organelle (RO) [E. J. Snijder<em> et al.</em>]. The RO is specifically formed during infection by reshaping ER and other membranes, giving rise to <span style="color:black">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 [</span>S. Klein<em> et al., </em>E. J. Snijder<em> et al.</em>]<span style="color:black">. There is some evidence that DMV accommodate viral replication which is based on radiolabelling viral RNA with nucleoside precursor ([5-<sup>3</sup>[H]uridine) and detection by EM autoradiography</span> <span style="color:#000000">[</span>E. J. Snijder<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">Viral replicative proteins and specific host factors are recruited</span> to ROs [E. J. Snijder<em> et al.</em>]. RNA viral genome is transcribed into messenger RNA by the viral RTC [P. Ahlquist <em>et al.</em>]. 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 <em>et al.</em>]. Positive-sense (messenger-sense) RNA viruses replicate their genomes through negative-strand RNA intermediates [M. Schwartz<em> et al.</em>]. The intermediates comprise <span style="color:black">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. </span>The transcription of coronaviruses <span style="color:black">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 <em>Nidovirales</em> 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</span> <span style="color:black">are unique features of the <em>coronaviruses</em> and <em>arteriviruses</em> [</span>W. A. Miller and G. Koev.]<span style="color:black">. Of note, the produced genomic RNA represents a small fraction of the total vRNA [</span>N. S. Ogando<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">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 [</span>I. Sola, F. Almazan <em>et al., </em>I. Sola, J. L. Moreno, <em>et al.</em>]<span style="color:black">. 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 [</span>H. Di <em>et al.</em>]<span style="color:black">. Therefore, t</span><span style="color:black">he 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 [</span>I. Sola, F. Almazan <em>et al. </em>I. Sola, J. L. Moreno, <em>et al.</em>]<span style="color:black">.</span> <span style="color:black">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 [</span>X. Zhang et al.]<span style="color:black">. 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 [</span>I. Sola <em>et al.</em> 2015, S. G. Sawicki <em>et al.</em>, Y. Yang <em>et al.</em>]<span style="color:black">. 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 [</span>Y. Yang]<span style="color:black">.</span> <span style="color:black">The set of sg mRNAs is then translated to yield </span>29 identified different proteins [F. Wu<em> et al.</em>], although many papers have identified additional ORFs [D. Kim<em> et al.. </em>Y. Finkel<em> et al., </em>A. Vandelli<em> et al.</em>]. 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<em> et al.</em>]. 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 <span style="color:black">5′ last third are found in higher amounts, and the nucleoprotein is the protein found in higher levels [</span>Y. Finkel<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">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 [</span>J. R. Cohen <em>et al.</em><em>, </em>A. Perrier<em> et al.</em>]<span style="color:black"> and v</span>iral 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, <span style="color:black">forming viral ribonucleoprotein (vRNPs) complexes, </span>before being selectively packaged into progeny virions [P. V'Kovski <em>et al.</em>], a step in which vRNPs<span style="color:black"> bud into the lumen of the ER and the ER-Golgi intermediate compartment (ERGIC) [</span>N. S. Ogando<em> et al.</em>]<span style="color:black">. 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.</span> </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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 <em>et al.</em>, D. E. Gordon<em> et al.</em>, 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 <em>et al.</em>], and this way organize viral transcription. Five studies have shown that N protein undergoes liquid-liquid phase separation (LLPS) <em>in vitro</em> [A. Savastano <em>et al.</em>, H. Chen<em> et al.</em>, C. Iserman<em> et al.</em>, T. M. Perdikari<em> et al.</em>, J. Cubuk<em> et al.</em>], dependent on its C-terminal domain (CTD) [H. Chen<em> et al.]</em>. 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<em> et al.</em>], 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 <em>et al.</em>, C. Iserman<em> et al., </em>C. R. Carlson<em> et al.</em>]. </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">The ERGIC constitutes the main assembly site of coronaviruses [</span>S. Klein<em> et al.</em><span style="color:black"><em>, </em></span>E. J. Snijder<em> et al.</em>, L. Mendonca<em> et al.</em>]<span style="color:black"> and budding events have been seen by EM studies.</span> For SARS-CoV-2, v<span style="color:black">irus-budding was mainly clustered in regions with a high vesicle density and close to ER- and Golgi-like membrane arrangements [</span>S. Klein<em> et al.</em><span style="color:black"><em>, </em></span>E. J. Snijder<em> et al.</em>, L. Mendonca<em> et al.</em>]<span style="color:black">. 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</span> <span style="color:black">[</span>S. Klein<em> et al.</em>]<span style="color:black">.</span> </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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<em> et al.</em>]. This process results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation [S. Ghosh<em> et al.</em>].</span></span></p>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">In vivo translation assay</span></span></li>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Transient expression of FLAG-Nsp1 in HeLa cells and puromycin incorporation assay</span></span></li>
</ul>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">SARS-CoV-2 disrupts splicing, translation, and protein trafficking [Banerjee </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020]</span></span></p>
<ul>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">SARS-CoV-2 viral protein binding to RNA</span></span></li>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Membrane SUnSET assay for transport of plasma membrane proteins to the cell surface</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">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 <em>et al.</em>]. 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, <em>et al.</em>]. The viral mRNAs are reverse-transcribed with RT, followed by the amplification by PCR.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">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.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">Plaque assays, infectivity assays, RT-qPCR to detect viral RNA in released virions, sequencing to detect mutations in the genome, electron microscopy.</span></span></p>
<h4>References</h4>
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<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">41. L. Mendonca<em> et al.</em>, SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">42. S. Ghosh<em> et al.</em>, beta-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway. <em>Cell</em> <strong>183</strong>, 1520-1535 e1514 (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">43. Schubert, K., Karousis, E.D., Jomaa, A. <em>et al.</em> SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. <em>Nat Struct Mol Biol</em> <strong>27, </strong>959–966 (2020). </span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">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 <em>In Vitro</em> and with Clinical Specimens. J Clin Microbiol. 2020:58(5)e00310-20. doi:10.1128/JCM.00310-20</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">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</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">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</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">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.</span></span></p>
<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/1939">Event: 1939: Viral infection and host-to-host transmission, proliferated</a></h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Life Stage and Sex.</strong> Viral load was not influenced by age or sex according to Challenger et al. (2022), however more recently Hughes et al. (2023) found wild-type- or Alpha-infected children 5–11-years old had lower viral loads than adults based on PCR cycles, so might transmit less than adults, but smaller differences in viral loads with age were observed in Delta infections. In terms of sex, infection rate and viral load were found not to differ (Arnold et al., 2022; Qi et al., 2021; Cheemarla et al., 2021).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Taxonomic.</strong> No non-mammalian vertebrates have been found to become infected with SARS-CoV-2. Many mammals have tested positive according to PCR tests for viral RNA and antibody test evidence (see compilation by EFSA/Nielson et al, 2023). However, some that have tested positive for RNA or antibodies were determined not to transmit or shed the virus. These include Cattle (<em>Bos taurus</em>; Ulrich et al., 2020), bank vole (<em>Myodes glareolus</em>; Ulrich et al., 2021), and domestic dogs (<em>Canis lupus familiaris</em>; Bosco-Lauth, Hartwig et al., 2021). Several experimentally exposed species did not become infected and hence, did not shed the virus, including coyote (<em>Canis latrans</em>; Porter et al., 2022), pig (<em>Sus scrofa</em>; Schlottau et al., 2020), and in one study by Bosco-Lauth, Root et al. (2021) the house mouse (<em>Mus musculus</em>), Wyoming ground squirrel (<em>Urocitellus elegans</em>), fox squirrel (<em>Sciurus niger</em>), black-tailed prairie dog (<em>Cynomys ludovicianus</em>), raccoon (<em>Procyon lotor</em>), and cottontail rabbits (<em>Sylvilagus</em> sp.).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Studies in which animals experimentally inoculated or naturally infected were tested for viral shedding and found to transmit the original Wuhan virus include Primates and species in Table 1.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:9.0pt"><span style="color:black">Table 1. Species that transmit or shed infectious SARS-CoV-2 virus.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">An example of a study of infection and transmission was conducted among raccoon dogs (Freuling et al., 2020). Nine naive animals received intranasal inoculations with 10<sup>5</sup> 50% tissue culture infectious dose (TCID50) SARS-CoV-2 2019_nCoV Muc-IMB-1, and 3 naive animals were introduced in cages separated from the inoculated animals by meshed wire 24 hours after inoculation. Six inoculated and two contact animals became infected; none showed clinical symptoms. Viral RNA was measured by qPCR in nasal, oropharyngeal, and rectal swab samples collected on days 2, 4, 8, 12, 16, 21, and 28, and the levels of infectious virus was determined by titration on Vero E6 cells. The inoculated animals shed virus in nasal and oropharyngeal swab samples on days 2–4. The mean viral genome load was highest for nasal swab samples at 3.2 (range 1.0–6.45) log10 genome copies/mL, and nasal swab viral titers peaked at 4.125 log10 TCID50/mL on day 2. Viral RNA was first detected in a contact animal 7 days after contact (Freuling et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Early in the pandemic, mink farms were found to be hotspots of non-human COVID-19 spread in both Europe and North America (Fenollar et al., 2021). In the Netherlands, Oude Munnink et al., (2020) showed that the virus was initially introduced from humans working or living at the farms and mutated through widespread circulation among mink. They also documented the first transmission from the mink back to humans (Oude Munnink et al., 2020). Ip et al. (2021) surveyed coronavirus-infected animals in Utah, USA, near mink farms affected by a SARS-CoV-2 outbreak. They suggest that mink farms could be potential hot spots for coronavirus spillover. According to Harrington et al., (2020), wild American minks (<em>Neovison vison</em>) are also a concern for the spread and mutation of SARS-CoV-2, considering their broad native range in North America and introduced range (via escape from farms) across Eurasia and southern South America. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Several researchers have reported wide-spread infection and transmission among wild and captive white-tailed deer: </span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Palmer et al. (2021) conducted intranasal inoculations of deer fawns with SARS-CoV-2, resulting in infection and shedding of infectious virus in nasal secretions. The infected animals were found to transmit the virus to contact deer. </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chandler et al., (2021) conducted SARS-CoV-2 tests on 624 serum samples taken before and during the pandemic from wild deer in the US states of Michigan, Illinois, Pennsylvania, and New York. Antibodies were detected in 152 samples (40%) from 2021, 3 samples from 2020, and one sample from 2019, but all 2011-2018 samples were negative. </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Martins, et al., 2022 found that white-tailed deer fawns shed infectious virus in nasal and oral secretions up to 5 days after intranasal inoculation with SARS-CoV-2 B.1 lineage, with deer-to-deer transmission occurring on day 3 post-inoculation. Contact animals added on days 6 and 9 did not become infected. Multiple sites of virus replication were revealed in adults, as infectious virus was detected up to 6 days after inoculation in nasal secretions, and respiratory-, lymphoid-, and central nervous system tissues.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cool, et al., 2022 investigated transmission in adult white-tailed deer co-infected with both the SARS-CoV-2 ancestral lineage A and the alpha variant of concern (VOC) B.1.1.7. Presence and transmission of each strain was determined using next-generation sequencing, with the finding that the alpha VOC B.1.1.7 isolate outcompeted ancestral lineage A. They found direct contact transmission and also vertical transmission from doe to fetus.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kuchipudi et al., 2022 tested for the presence of SARS-CoV-2 RNA by RT-PCR in 283 retropharyngeal lymph node (RPLN) samples from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021, with positive results in 94 (33.2%) of the 283 samples. Over a 7-wk period during the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples. Whole genome sequencing revealed presence of 12 SARS-CoV-2 lineages with two B lineages accounting for 75% of samples. The results suggest multiple human-to-deer transmission events followed by deer-to-deer spread. </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Pickering et al. (2022) identified a new and highly divergent lineage of SARS-CoV-2 with 76 consensus mutations including 37 previously associated with non-human animal hosts, and evidence of host adaptation under neutral selection. They also provide the first evidence of a SARS-CoV-2 deer-to-human transmission, indicating that a high divergent mutated strain can be generated in deer and transmitted back to humans.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">McBride et al., (2023) found that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA November 2021-March 2022. Transmission within deer populations continued for 2–8 months and over an area covering hundreds of kilometers. They also found SARS-CoV-2 evolution to be three-times faster in white-tailed deer, with different mutational biases and selection pressures compared to humans.</span></span></li>
</ul>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Broad mammalian host range 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). Some literature found on non-human hosts indicates that NSPs and accessory proteins can interact in a similar manner with bird (chicken) and other mammal proteins in the IFN-I pathway (Moustaqil et al. 2021; Rui et al. 2021).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The deer’s susceptibility is in contrast to more resistant species in the Order Artiodactyla including pigs, cattle, and horses. More than 600 race horses in California were tested through 2020 for viral presence in nasal secretions (qPCR) and serum antibodies (ELISA), with 0% positive qPCR tests and 5.9% positive for serum antibodies to SARS-CoV-2 (Lawton et al., 2022). Also note that in the Family Canidae, raccoon dogs and red foxes may transmit the original Wuhan SARS-CoV-2 strain while domestic dogs and coyotes do not, therefore taxonomic relatedness is not necessarily a predictor of infection and transmission. Early in the pandemic, cross-species similarity in the viral entry receptor angiotensin converting enzyme 2 (ACE2) protein sequence to the human ACE2 sequence was studied as a predictor of potential infectability (Damas et al., 2020). However, empirical evidence has shown that some species with low ACE2 similarity, such as the mink, are highly susceptible. While other factors including the type I interferon (IFN-I) pathway proteins are being studied for predictive potential, empirical testing is currently the most reliable method of determining species susceptibility to infection, and more studies are needed to determine which species may be capable of transmitting the virus.</span></span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Much is now understood in terms of human-to-human COVID-19 transmission. Coronaviruses, as with many other respiratory viruses, are transmitted primarily through respiratory droplets, but can also spread through aerosols, fecal-oral transmission, or contact with contaminated surfaces (Harrison et al. 2020). Respiratory droplets and aerosols containing the virus are generated through an infected person coughing, sneezing or talking, and enter the secondary host system through upper and lower respiratory tissues, with the lung being the primary tropism. Barriers to transmission in place worldwide include social distancing, face shields, cloth masks, frequent hand washing, and surface disinfection (Harrison et al. 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Much is now understood in terms of human-to-human COVID-19 transmission. Coronaviruses, as with many other respiratory viruses, are transmitted primarily through respiratory droplets, but can also spread through aerosols, fecal-oral transmission, or contact with contaminated surfaces (Harrison et al. 2020). Respiratory droplets and aerosols containing the virus are generated through an infected person coughing, sneezing or talking, and enter the secondary host system through upper and lower respiratory tissues, with the lung being the primary tropism. Barriers to transmission in place worldwide include social distancing, face shields, cloth masks, frequent hand washing, and surface disinfection (Harrison et al. 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Vaccination is the standard strategy for reducing or eliminating viral disease transmission, symptoms, and mortality in humans, and in some cases domesticated animals. However, the weight of evidence indicates that the reservoir species (bats in the case of betacoronaviruses) and potential intermediate hosts are wildlife, and different control measures will be required to prevent future spillover. Indeed, the intermediate host of the SARS-CoV-2 virus has yet to be identified (Delahay et al. 2021). This key event is therefore focused primarily on the species of potential concern, exposure and transmission routes across species, and the conditions indicative of or conducive toward cross-species spillover of zoonoses or infectious viral diseases of animal origin.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Widespread testing and contact tracing were later instituted, and more effective (medical-grade) masks also became available (Fritz et al., 2023). Fritz et al. (2023) determined that the most effective control measure in reducing COVID-19 spread is a comprehensive testing strategy, until vaccination levels can establish herd immunity.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Vaccination is the standard strategy for reducing or eliminating viral disease transmission, symptoms, and mortality in humans, and in some cases domesticated animals. COVID-19 vaccines were developed using mRNA technology to deliver the viral spike protein sequence against which the host would develop antibodies. The first to gain Emergency Use Authorization from the U.S. Food and Drug Administration (FDA) were the <span style="background-color:white"><span style="color:#0d0d0d">Pfizer-BioNTech (BNT162b2) and Moderna vaccines in December 2020</span></span> (Katella, 2023). The effectiveness of the vaccines is monitored by the U.S. Centers for Disease Control (CDC, 2023) with criteria as follows:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hospitalization for COVID-19 or medically attended COVID-19 (e.g., emergency department visits)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Death due to COVID-19</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Post-COVID Conditions and multisystem inflammatory syndrome (MIS)</span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Prevention of transmission is not part of this monitoring program, however, recent studies have estimated vaccination effect on transmission of the SARS-CoV-2 alpha and delta variants. Vaccines BNT162b2 and ChAdOx1 nCoV-19 (a vaccine developed at Oxford University, England, using an adenoviral vector) were found to be less effective in preventing transmission than preventing serious disease outcomes. Variation in polymerase chain reaction (PCR) cycle-threshold (Ct) values in index patients, which indicate viral load, explained 7 to 23% of vaccine-associated reductions in index-to-secondary patient transmission for the two variants (Eyre et al., 2022). This means viral load was not the only factor in transmission, and other factors associated with positive PCR tests in contacts included the type of exposure between patients and contacts and the age of the index patient. The highest rates of PCR positivity were seen after household exposures of index patients at least 40 years old compared with exposures at the workplace, educational facilities, or events (Eyre et al., 2022). Braeye et al. (2023) in a 2020-21 Belgian contact tracing study showed vaccine effectiveness against transmission (VET) for BNT162b2 for primary vaccination at 96% against Alpha, 87% against Delta and 31% against Omicron. A booster elevated protection against Omicron to 68%, but 150–200 days after booster-vaccination protection waned somewhat for Delta to 71% and for Omicron to 55% (Braeye et al., 2023).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Different control measures will be required to prevent future spillover from the reservoir species (bats in the case of betacoronaviruses) and potential intermediate host species. Indeed, the original intermediate host of the SARS-CoV-2 virus has yet to be identified (Delahay et al. 2021). However, Wuhan, China, was the epicenter of the SARS-CoV-2 pandemic, and Worobey et al. (2022) reported that live animals, many of which proved to be susceptible to the virus, were sold at the Huanan Wholesale Market in Wuhan in late 2019. Worobey et al. (2022) found SARS-CoV-2-positive environmental samples associated with the spaces where the live animals were housed. These animals included raccoon dogs and red foxes (species shown to transmit the virus; Table 1), and other species related to known transmitters like the mink (members of the Mustelidae family including the Asian badger and hog badger).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This key event is therefore focused primarily on the species of potential concern, exposure and transmission routes across species, and the conditions indicative of or conducive toward cross-species spillover of zoonoses or infectious viral diseases of animal origin.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Species of Potential Concern</strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The reservoir host for SARS-CoV-2-like viruses is believed to be the bat.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The reservoir host for SARS-CoV-2-like viruses is believed to be the bat. See Table 1 below for species known to transmit SARS-CoV-2.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Exposure and Transmission Routes</strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2-infected media (respiratory droplets, bodily fluids, tissues, feces): Exposure routes are the pathway into the body of the virus shed from an infected reservoir host animal to the intermediate host, or either type of host animal to humans. These routes may include inhalation, oral, or through broken skin or mucosal membranes (e.g., eyes, nostrils) after touching contaminated media or surfaces and then touching the face (Harrison et al. 2020). Animals may transfer saliva or nasal discharge directly through facial contact, licking or biting. Transmission occurs through these routes when the virus reaches a tissue with cells that allow entry and replication.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Conditions that allow for exposure and transmission across species:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Close proximity of animal communities (bats to potential intermediate hosts; wildlife to domestic animal farms).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Direct human contact with wildlife (Johnson et al. 2015), including:</span></span>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Direct human contact with wildlife (Kreuder Johnson et al. 2015), including:</span></span>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hunting and dressing wild game;</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cleaning of storage buildings, barns, or other structures that may be used by wildlife for shelter, breeding, or feeding, with potential for feces or other contamination (CDC, 2021);</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Wet markets where live animals or bush meat are traded;</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Research facilities that express viruses from wild samples in cell culture, that house potential host species, or that collect and store bodily fluid or tissue samples. </span></span></li>
</ul>
</li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Virus isolated from animal species shows genomic similarity to the human virus, but also high host plasticity to be capable of cross-species viral immune evasion and replication (Johnson et al. 2015).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Virus isolated from animal species shows genomic similarity to the human virus, but also high host plasticity to be capable of cross-species viral immune evasion and replication (Kreuder Johnson et al. 2015).</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Spillover species and new host species share genetic similarity in the components of the cell entry, immune system and replication machinery (Warren et al. 2019). That is, the virus can enter the cell and evade the virus detection and immediate systemic type I interferon (IFN) response to allow replication and generation of viral load in both species. The viral proteins must be capable of interacting with the appropriate cellular proteins in either species. The most studied and considered indicative of infectability is the ACE2 and other cell entry proteins.</span></span></li>
</ul>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Similar host genetics. Spillover species and new host species share genetic similarity in the components of the cell entry, immune system and replication machinery (Warren et al. 2019). That is, the virus can enter the cell and evade the virus detection and immediate systemic type I interferon (IFN) response to allow replication and generation of viral load in both species. The viral proteins must be capable of interacting with the appropriate cellular proteins in either species. The most studied and considered indicative of infectability is the ACE2 and other cell entry proteins.</span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Either the virus or antibodies can be detected with available tests. Active infection can be detected through PCR tests from nasal swab, oropharyngeal swab, rectal swab or saliva samples that indicate the quantity and/or presence of the virus. Antibodies can be detected in blood using various assays including immunofluorescence.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Either the virus or antibodies can be detected with available tests. Active infection can be detected through PCR tests from nasal swab, oropharyngeal swab, rectal swab or saliva samples that indicate the quantity and/or presence of the virus. Antibodies can be detected in blood using various assays including immunofluorescence. Methods used are as follows:</span></span></p>
<p> </p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">ELISA, Indirect immunofluorescence assay (IIFA) for antibodies (Schlottau et al. 2020; Freuling et al. 2020)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Virus neutralization test (VNT) for antibodies (Schlottau et al. 2020; Freuling et al. 2020)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Quantitative reverse transcription PCR (qRT-PCR) for viral load (log10 genome copies) (Freuling et al. 2020)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Titration (Tissue culture infectious dose where 50% of infected cells display cytopathic effect [TCID50 assay]: levels of infectious virus, or viral titre) (Freuling et al. 2020)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Virus-specific immunoglobulin characterization (Freuling et al. 2020)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2 spike protein neutralizing antibodies in saliva from animals that developed serum antibodies (Freuling et al. 2020)</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Serum sample, autopsy, histopathology for tissue lesions (Schlottau et al. 2020; Freuling et al. 2020)</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Viral whole genome sequencing (Kuchipudi et al., 2022)</span></span></li>
</ul>
<h4>Regulatory Significance of the AO</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">There is currently no regulatory guidance for host-to-host transmission of SARS-CoV-2, however mask mandates and institutional controls have been used during the pandemic, and in most countries vaccination is voluntary. The information in this AOP could aid in identification of effective control strategies. With regard to SARS-CoV-2 and other zoonotic disease threats, this AOP points out that more cross-species studies on immune systems are needed to guide which species should be monitored, and need to regulate domestic animal and wildlife trade to avoid future pandemics.</span></span></p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Arnold, C. G., Libby, A., Vest, A., Hopkinson, A., & Monte, A. A. (2022). Immune mechanisms associated with sex-based differences in severe COVID-19 clinical outcomes. <em>Biology of Sex Differences</em>, <em>13</em>(1), 7. <a href="https://doi.org/10.1186/s13293-022-00417-3" style="color:blue; text-decoration:underline">https://doi.org/10.1186/s13293-022-00417-3</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">ELISA, Indirect immunofluorescence assay (IIFA) for antibodies (Schlottau et al. 2020; Freuling et al. 2020)</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bosco-Lauth, A. M., Hartwig, A. E., Porter, S. M., Gordy, P. W., Nehring, M., Byas, A. D., VandeWoude, S., Ragan, I. K., Maison, R. M., & Bowen, R. A. (2020). Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats. <em>Proceedings of the National Academy of Sciences</em>, <em>117</em>(42), 26382–26388. <a href="https://doi.org/10.1073/pnas.2013102117" style="color:blue; text-decoration:underline">https://doi.org/10.1073/pnas.2013102117</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Virus neutralization test (VNT) for antibodies (Schlottau et al. 2020; Freuling et al. 2020)</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bosco-Lauth, A. M., Root, J. J., Porter, S. M., Walker, A. E., Guilbert, L., Hawvermale, D., Pepper, A., Maison, R. M., Hartwig, A. E., Gordy, P., Bielefeldt-Ohmann, H., & Bowen, R. A. (2021). Peridomestic Mammal Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection. <em>Emerging Infectious Diseases</em>, <em>27</em>(8), 2073–2080. <a href="https://doi.org/10.3201/eid2708.210180" style="color:blue; text-decoration:underline">https://doi.org/10.3201/eid2708.210180</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Quantitative reverse transcription PCR (qRT-PCR) for viral load (log10 genome copies) (Freuling et al. 2020)</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Braeye, T., Catteau, L., Brondeel, R., Van Loenhout, J. A. F., Proesmans, K., Cornelissen, L., Van Oyen, H., Stouten, V., Hubin, P., Billuart, M., Djiena, A., Mahieu, R., Hammami, N., Van Cauteren, D., & Wyndham-Thomas, C. (2023). Vaccine effectiveness against transmission of alpha, delta and omicron SARS-COV-2-infection, Belgian contact tracing, 2021–2022. <em>Vaccine</em>, <em>41</em>(20), 3292–3300. <a href="https://doi.org/10.1016/j.vaccine.2023.03.069" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.vaccine.2023.03.069</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Titration (Tissue culture infectious dose where 50% of infected cells display cytopathic effect [TCID50 assay]: levels of infectious virus, or viral titre) (Freuling et al. 2020)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2 spike protein neutralizing antibodies in saliva from animals that developed serum antibodies (Freuling et al. 2020)</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Challenger, J. D., Foo, C. Y., Wu, Y., Yan, A. W. C., Marjaneh, M. M., Liew, F., Thwaites, R. S., Okell, L. C., & Cunnington, A. J. (2022). Modelling upper respiratory viral load dynamics of SARS-CoV-2. <em>BMC Medicine</em>, <em>20</em>(1), 25. <a href="https://doi.org/10.1186/s12916-021-02220-0" style="color:blue; text-decoration:underline">https://doi.org/10.1186/s12916-021-02220-0</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Serum sample, autopsy, histopathology for tissue lesions (Schlottau et al. 2020; Freuling et al. 2020)</span></span></p>
<h4>References</h4>
<p>Under construction</p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cheemarla, N. R., Watkins, T. A., Mihaylova, V. T., Wang, B., Zhao, D., Wang, G., Landry, M. L., & Foxman, E. F. (2021). Dynamic innate immune response determines susceptibility to SARS-CoV-2 infection and early replication kinetics. <em>Journal of Experimental Medicine</em>, <em>218</em>(8), e20210583. <a href="https://doi.org/10.1084/jem.20210583" style="color:blue; text-decoration:underline">https://doi.org/10.1084/jem.20210583</a></span></span></p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Freuling, C. M., Breithaupt, A., Müller, T., Sehl, J., Balkema-Buschmann, A., Rissmann, M., Klein, A., Wylezich, C., Höper, D., Wernike, K., Aebischer, A., Hoffmann, D., Friedrichs, V., Dorhoi, A., Groschup, M. H., Beer, M., & Mettenleiter, T. C. (2020). Susceptibility of Raccoon Dogs for Experimental SARS-CoV-2 Infection. <em>Emerging Infectious Diseases</em>, <em>26</em>(12), 2982–2985. <a href="https://doi.org/10.3201/eid2612.203733" style="color:blue; text-decoration:underline">https://doi.org/10.3201/eid2612.203733</a></span></span></p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Harrison, A. G., Lin, T., & Wang, P. (2020). Mechanisms of SARS-CoV-2 Transmission and Pathogenesis. <em>Trends in Immunology</em>, <em>41</em>(12), 1100–1115. <a href="https://doi.org/10.1016/j.it.2020.10.004" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.it.2020.10.004</a></span></span></p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Jemeršić, L., Lojkić, I., Krešić, N., Keros, T., Zelenika, T. A., Jurinović, L., Skok, D., Bata, I., Boras, J., Habrun, B., & Brnić, D. (2021). Investigating the Presence of SARS CoV-2 in Free-Living and Captive Animals. <em>Pathogens</em>, <em>10</em>(6), 635. <a href="https://doi.org/10.3390/pathogens10060635" style="color:blue; text-decoration:underline">https://doi.org/10.3390/pathogens10060635</a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Katella, K., (2023). Comparing the COVID-19 Vaccines: How Are They Different? Website: YaleMedicine.org, October 5, 2023 (<a href="https://www.yalemedicine.org/news/covid-19-vaccine-comparison" style="color:blue; text-decoration:underline">https://www.yalemedicine.org/news/covid-19-vaccine-comparison</a>)</span></span></p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Ulrich, L., Michelitsch, A., Halwe, N., Wernike, K., Hoffmann, D., & Beer, M. (2021). Experimental SARS-CoV-2 Infection of Bank Voles. <em>Emerging Infectious Diseases</em>, <em>27</em>(4), 1193–1195. <a href="https://doi.org/10.3201/eid2704.204945" style="color:blue; text-decoration:underline">https://doi.org/10.3201/eid2704.204945</a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Ulrich, L., Wernike, K., Hoffmann, D., Mettenleiter, T. C., & Beer, M. (2020). Experimental Infection of Cattle with SARS-CoV-2. <em>Emerging Infectious Diseases</em>, <em>26</em>(12), 2979–2981. <a href="https://doi.org/10.3201/eid2612.203799" style="color:blue; text-decoration:underline">https://doi.org/10.3201/eid2612.203799</a></span></span></p>
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<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>
<p>This KER applies to humans in all life stages and all sexes.</p>
<p> </p>
<p> </p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">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.</span></span></p>
<p> </p>
<p> </p>
<p> </p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:#0070c0">Binding of SARS-CoV-2 S protein to ACE2 receptors present in the brain (endothelial, neuronal and glial cells) :</span></em></strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">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).</span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">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).</span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">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). </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><strong><em>Binding of S protein to ACE2 receptors present in the intestines</em></strong></span></span></span></p>
<strong>Biological Plausibility</strong>
<p>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.</p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">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 <span style="color:#0e101a">SARS-CoV-2</span><strong><span style="color:#0e101a"> </span></strong>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 <sup>680</sup>S<u>PRRA</u>R↓SV<sup>687</sup> forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. In addition, there is a second cleavage site <sup>808</sup>PSKPS<strong>KR</strong>|SFIEDL<sup>822</sup> 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. </span></span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-family:Arial, Helvetica, sans-serif"><span style="font-size:12px">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).</span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">PFAS (PFOS, PFOA, PFNA, PFHxS, and GenX)</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">PFAS (PFOS, PFOA, PFNA, PFHxS, and GenX)</span></span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">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 <em>Ace2</em> and to about 1.5 upregulation of the pulmonary mRNA level of <em>Tmprss2</em> in CD1 mice. [1]</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">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 <em>Ace2</em> and to about 1.5 upregulation of the pulmonary mRNA level of <em>Tmprss2</em> in CD1 mice. [1]</span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">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 <em>Tmprss2. </em>[2]</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">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 <em>Tmprss2. </em>[2]</span></span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">1) DOI: https://doi.org/10.1016/j.toxrep.2021.11.014</span></span></p>
<td><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">female sex (XX chromosomes)</span></span></td>
<td><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">female sex (XX chromosomes)</span></span></td>
<td>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">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]</span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">Estradiol inhibits TMPRSS2, needed to facilitate SARS-CoV-2 entry into the cell. [3]</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">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]</span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">. 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].</span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">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]</span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">Different studies have also illustrated that estradiol increases the expression of ADAM17, leading to high-circulating sACE2 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.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">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.</span></span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">1) doi:10.1177/1933719115597760</span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">2) doi.org/10.1016/j.mce.2015.11.004</span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">Male sex (XY chromosomes)</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Male sex (XY chromosomes)</span></span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">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]</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">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]</span></span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12px">6) doi: 10.1073/pnas.2021450118 </span></span></p>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri","sans-serif"">ACE2 protein expression is increased with aging in several tissues [<strong>1</strong>], including lungs and particularly in patients requiring mechanical ventilation [<strong>2</strong>]. 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 [<strong>3</strong>].</span></span></td>
<td><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">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].</span></span></td>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="background-color:white"><span style="color:#222222">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]. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="background-color:white"><span style="color:#222222"><strong>In COVID-19.</strong> In an<em> in vitro</em> 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]. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="background-color:white"><span style="color:#222222"><strong>In COVID-19.</strong> 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]. </span></span></span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Vitamin D </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="color:black">Vitamin D deficiency</span></span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="color:black">Vitamin D administration enhanced mRNA expression of VDR and ACE2 in a rat model of acute lung injury [</span>1<span style="color:black">]. In particular, vitamin D upregulates the soluble ACE2 form [</span>2<span style="color:black">]. Thus, low vitamin D status may impair the trapping protective mechanism of soluble ACE2 [</span>3<span style="color:black">]. Furthermore, vitamin D deficiency has been shown to reduce the expression of antimicrobial peptides (-defensin, cathelicidin), which act against enveloped viruses [</span>4,5<span style="color:black">]. </span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="color:black"><strong>In COVID-19. </strong>Decreased sACE2 and cellular viral defense might be some mechanisms explaining how low vitamin D modulate SARS-CoV-2 infectibility.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Gut dysbiosis (alteration of gut microbiota)</span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">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]. <em>Coprobacillus</em> enrichment was associated with severe COVID-19 in patients [3] and was shown to upregulate colonic ACE2 in mice [4]. The abundance of<em> Bacteroides </em>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. </span></span></p>
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<td>Genetic factors</td>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">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 [</span>1<span style="color:black">]. Around the twenty natural ACE2 variants, three alleles of 17 variants were found to affect the attachment stability [</span>2<span style="color:black">]. 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 [</span>3<span style="color:black">]. 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 [</span>4<span style="color:black">]. 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 [</span>5<span style="color:black">]. TMPRSS2 single-nucleotide polymorphisms (SNPs) were associated with a frequent “European haplotype” [</span>6<span style="color:black">], 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</span> <span style="color:black">production. Consequently, genomic surveillance is crucial to understand how mutations occurring on SARS-CoV-2 genome influence and drive the pandemic [</span>7<span style="color:black">]. For example, a recent study [</span>8<span style="color:black">] 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 [</span>9<span style="color:black">], 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 [</span>10<span style="color:black">].</span></span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="font-family:URWPalladioL-Roma"><span style="color:black"> </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Are monoclonal antibodies designed to recognize and attach to two</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">different sites of the Receptor-Binding Domain (RBD) of the S protein of SARS-CoV-2,</span></span></p>
<span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">blocking the virus to enter cells [1,2,3].</span></span></td>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">2) EMA Starts Rolling Review of REGN-COV2 Antibody Combination (Casirivimab / Imdevimab). EMA 2021. Available online:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(accessed on 12 May 2022)</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">3) EMA Starts Rolling Review of Sotrovimab (VIR-7831) for COVID-19. EMA 2021. Available online:</span></span></p>
<span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">https://www.ema.europa.eu/en/news/ema-starts-rolling-review-sotrovimab-vir-7831-covid-19 (accessed on 12 May 2022)</span></span></td>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Interacts directly with viral</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">particles and has been shown to bind to the SARS-CoV-2 S1 Spike RBD, causing significant</span></span></p>
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<p>Air pollution induces Increased expression of ACE2 which may result in increased viral entry and coronavirus production. </p>
<p>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). </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">ACE2 mRNA and protein levels, as well as enzymatic activity, were </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">shown to be upregulated in explanted hearts from patients with end-stage HF, as well </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">as in the HF rat model [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">1-3</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">]. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Myocytes, fibroblasts, vascular smooth muscle cells, pericytes [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">4</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] and endothelial cells of the coronaries [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">5</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] express ACE2, while myocytes in patients suffering from heart disease exhibit higher ACE2 expression [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">6</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">]. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Pericytes - the mural cells lining microvasculature, interacting with endothelial cells notably to maintain </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">microvascular stability - exhibited the strongest ACE2 expression in HF patients [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">7</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">], rendering these cells involved in the coronary vasculature of the myocardium, more susceptible to infection. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Furthermore, SARS-CoV-2 infects and replicates in pericytes, and a decrease in their numbers follows [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">8</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">]. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Patients with pre-existing HF showed increased ACE2 levels in myocytes and pericytes, having thereby higher risk of heart injury [</span><span style="color:#0875b8">7, 9</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">].</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">In addition, sACE2 levels are higher in HF patients [</span><span style="color:#0875b8">10, 11</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] and sACE2 activity is increased in HF [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">12</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">].</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">In contrast to a protective role of sACE2, it has been proposed that viral binding to </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">circulating sACE2 forms SARS-CoV-2/sACE2 complexes, which might mediate infection of </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">cells in distal tissues [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">13</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">]; hence, pre-existing HF might disseminate SARS-CoV-2 infection.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">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 [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">10</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] and (ii) the potential of sACE2 activity to be used as a biomarker to distinguish between </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">the two HF types. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Lastly, it is noteworthy that Khoury et al. provided evidence in a different direction, by showing that ADAM17 and TMPRSS2 [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">14</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] expression levels are downregulated in a HF rat model, thus potentially conferring a protective role against infection by SARS-CoV-2 in HF [</span><span style="color:#0875b8">3</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">].</span></span></span></span></span></p>
<td>Chemicals in foods affect ACE2 expression</td>
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<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>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="color:#0070c0">COVID19 References related to CNS:</span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">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</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Xia, H. and Lazartigues, E. Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010 Rep. 12 (3), 170– 175</span></span></span></p>
<p> </p>
<p><strong>References in general:</strong></p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Zhou, P., Yang, XL., Wang, XG. <em>et al.</em> A pneumonia outbreak associated with a new coronavirus of probable bat origin. <em>Nature</em> <strong>579</strong>, 270–273 (2020). doi: 10.1038/s41586-020-2012-7</p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">It has been shown that in human populations males are more likely to suffer severe infections and deaths due to COVID-19 than females. However, in the viral entry and infection phase, one study found that women of working age had higher infection rates than men, but the suggested cause was higher contact rates among women (Doerre and Doblhammer, 2022 <a href="https://doi.org/10.1371/journal.pone.0268119" style="color:blue; text-decoration:underline">https://doi.org/10.1371/journal.pone.0268119</a>). Contact rate increase is an important transmission factor but would not constitute a gender-based biological difference in viral entry or IFN-I pathway antagonism. A biological basis for females having higher levels of Type I IFN has been proposed concerning Toll-like receptor (TLR) 7. TLR7 is expressed in plasmacytoid dendritic cells</span></span> <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(pDCs)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, an immune cell type that </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">on infection with SARS-CoV-2 migrates from peripheral blood into the respiratory tract </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">epithelium. TLR7 stimulates higher IFN-I production in pDCs in women than in men (Van der Sluis et al. 2022 </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><a href="https://doi.org/10.1016/j.celrep.2022.111148" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.celrep.2022.111148</a>). It is proposed that this is due to the TLR7 gene being on the X chromosome, and that X inactivation in males is incomplete regarding the TLR7 gene, creating a double gene-dose effect in females (Spiering and de Vries, 2021 <a href="https://doi.org/10.3389/fimmu.2021.756262" style="color:blue; text-decoration:underline">https://doi.org/10.3389/fimmu.2021.756262</a>). In a mouse SARS-CoV model, XY males had more adverse outcomes than XX females and XXY males (Gadi et al. 2020 <a href="https://doi.org/10.3389%2Ffimmu.2020.02147" style="color:blue; text-decoration:underline">https://doi.org/10.3389%2Ffimmu.2020.02147</a>). Additionally, loss-of-function TLR7 mutations have been identified that are associated with increased COVID-19 severity (Szeto et al. 2021 <a href="https://doi.org/10.1159/000518471" style="color:blue; text-decoration:underline">https://doi.org/10.1159/000518471</a>). However, these results focus on disease outcome as the </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">endpoint, where factors beyond the initial antiviral response could be involved. Also note that the nasal and upper respiratory tract (URT) epithelial cells express ACE2 receptors for SARS-CoV-2 entry while the pDCs do not, relying on viral endocytosis (Van der Sluis et al. 2022). There is not a clear picture in the literature of the timing of pDC arrival in the epithelium after exposure, and the role of TLR7 in sex differences is currently hypothetical (Spiering and de Vries, 2021). </span></span></p>
<p><u>Sex and age applicability</u></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">It has been shown that in human populations males are more likely to suffer severe infections and deaths due to COVID-19 than females. However, in the viral entry and infection phase, one study found that women of working age had higher infection rates than men, but the suggested cause was higher contact rates among women (Doerre and Doblhammer, 2022). Contact rate increase is an important transmission factor but would not constitute a gender-based biological difference in viral entry or IFN-I pathway antagonism. A biological basis for females having higher levels of Type I IFN has been proposed concerning Toll-like receptor (TLR) 7. TLR7 is expressed in plasmacytoid dendritic cells(pDCs), an immune cell type that on infection with SARS-CoV-2 migrates from peripheral blood into the respiratory tract epithelium. TLR7 stimulates higher IFN-I production in pDCs in women than in men (Van der Sluis et al. 2022). It is proposed that this is due to the TLR7 gene being on the X chromosome, and that X inactivation in males is incomplete regarding the TLR7 gene, creating a double gene-dose effect in females (Spiering and de Vries, 2021). In a mouse SARS-CoV model, XY males had more adverse outcomes than XX females and XXY males (Gadi et al. 2020). Additionally, loss-of-function TLR7 mutations have been identified that are associated with increased COVID-19 severity (Szeto et al. 2021). However, these results focus on disease outcome as theendpoint, where factors beyond the initial antiviral response could be involved. Also note that the nasal and upper respiratory tract (URT) epithelial cells express ACE2 receptors for SARS-CoV-2 entry while the pDCs do not, relying on viral endocytosis (Van der Sluis et al. 2022). There is not a clear picture in the literature of the timing of pDC arrival in the epithelium after exposure, and the role of TLR7 in sex differences is currently hypothetical (Spiering and de Vries, 2021). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">In a large study modelling URT viral load dynamics drawn from measurements in 605 human subjects, variations over 5 orders of magnitude in URT viral load from the time of symptom onset was not explained by age, sex, or severity of illness. Additionally, these variables did not explain modelling results concerning control of viral load by immune responses in the early (innate) or late (adaptive) phases (Challenger et al. 2022 <a href="https://doi.org/10.1186/s12916-021-02220-0" style="color:blue; text-decoration:underline">https://doi.org/10.1186/s12916-021-02220-0</a>). Other sources also support that rate of infection and measured viral load does not differ by gender (e.g., Arnold et al. 2022 <a href="https://doi.org/10.1186/s13293-022-00417-3" style="color:blue; text-decoration:underline">https://doi.org/10.1186/s13293-022-00417-3</a>; Qi et al. 2021 <a href="https://doi.org/10.1186/s13293-021-00410-2" style="color:blue; text-decoration:underline">https://doi.org/10.1186/s13293-021-00410-2</a>; Cheemarla et al. 2021 <a href="https://doi.org/10.1084/jem.20210583" style="color:blue; text-decoration:underline">https://doi.org/10.1084/jem.20210583</a>). Therefore, evidence exists that the components of cell entry and the early antiviral response are not influenced by gender specific differences such as sex hormone levels or sex chromosomes to the extent of affecting viral load. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Generally, most mammals are likely susceptible to the SARS-CoV-2 virus based on reports of naturally and experimentally infected animals (See AO 1939). No infections have been reported in other classes of vertebrates. Other than bioinformatic studies on the ACE2 sequence across vertebrates however, there have been few studies on the mechanisms of susceptibility to infection of non-human hosts. Three studies were found on protein targets in the IFN-I innate immune response pathway that included other vertebrates. Rui et al. (2021) showed that SARS-CoV-2 3CLpro and ORF3a inhibit vertebrate (human, mouse, and chicken) STING ability to induce IFNβ promoter activity in a dose-dependent manner in HEK293T cells transfected with IFNβ-luciferase reporter plasmid vectors, together with tagged STING and cGAS vectors and increasing amounts of the SARS-CoV-2 3CLpro or ORF3a expression vectors. This study shows that the vulnerability of the host IFN-I pathway protein components to inhibition by SARS-CoV-2 protein stressors is not limited to humans, however Rui et al. (2021) did not determine the specific amino acids involved in the STING-ORF3a or STING-3CLpro interactions. Mostaquil et al. (2020) studied the cleavage site of IRF3 by PLpro (SARS-CoV-2 NSP3) and compared sequences across mammals. They determined that the IRF3 cleavage site in mammalian species in the taxonomic orders of primates, carnivora, artiodactyla, chiroptera (bats) and a few other mammals was conserved and would generally be susceptible to cleavage, and therefore IFN-I antagonism, but rodentia IRF3 would likely not be susceptible. Hameedi et al. (2022) compared molecular dynamic simulations of 3CLpro cleavage of NEMO in humans and mice showing a decrease in the average number of contacts between mNEMO and 3CLpro compared to hNEMO. Also, hNEMO may be more strongly bound to the catalytic site, and the mNEMO/3CLpro interaction appears more prone to destabilization (Hameedi et al., 2022).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p>Upon entry of a virus into the host cell (KE1738), the virus is unpackaged from the structural nucleocapsid (N), envelope (E), and membrane (M) proteins. <span style="font-size:12.0pt"><span style="font-family:Calibri"><span style="color:black">The viral RNA is detected by Pattern Recognition Receptor (PRR) proteins including RIG-I and MDA5</span></span></span> but the M proteins can interact with these PRRs directly, and block this initial host reaction (https://doi.org/10.1038/s41423-020-00571-x). The viral genomic RNA can then be translated directly at the host ribosomes. The viral proteins may be processed through cleavage by viral protease enzymes. This releases a repertoire of non-structural proteins (NSPs) and accessory open reading frame (ORF) proteins that has evolved, for example in the SARS-CoV-2 virus, to bind and block the proteins in the interferon I (IFN-I) antiviral cascade (KE1901). The timely production of type I IFN by host cells is critical for limiting viral replication and promoting antiviral immunity (10.1126/science.abm8108).</p>
<p><span style="font-size:16px">Upon entry of a virus into the host cell (KE1738), the virus is unpackaged from the structural nucleocapsid (N), envelope (E), and membrane (M) proteins. <span style="font-family:Calibri"><span style="color:black">The viral RNA is detected by Pattern Recognition Receptor (PRR) proteins including RIG-I and MDA5</span></span> but the M proteins can interact with these PRRs directly, and block this initial host reaction (Fu et al., 2021). The viral genomic RNA can then be translated directly at the host ribosomes. The viral proteins are processed through cleavage by viral protease enzymes. This releases a repertoire of non-structural proteins (NSPs) and accessory open reading frame (ORF) proteins that has evolved, for example in the SARS-CoV-2 virus, to bind and block the proteins in the interferon I (IFN-I) antiviral cascade (KE1901). <span style="font-family:"Calibri",sans-serif">The normal function of the host’s IFN-I response to other viruses is the expression of IFN-I which in turn stimulates the expression of many interferon-stimulated gene (ISG) proteins with antiviral functions. The SARS-CoV-2 antagonism of the IFN-I pathway delays or curtails the expression of IFN-I and ISG proteins. </span></span></p>
<h4>Evidence Supporting this KER</h4>
<p>Empirical evidence supporting this relationship is described below.</p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Biological plausibility comes from several studies indicating that if IFN is administered just before or upon exposure, viral production is reduced or eliminated <span style="font-size:14px">(</span></span></span><span style="font-size:14px"><span style="font-family:"Trebuchet MS""><span style="color:#404040">DOI 10.1007/s10517-021-05330-0; <a href="https://doi.org/10.1038/s41467-021-27318-0">https://doi.org/10.1038/s41467-021-27318-0</a>; doi.org/10.1016/j.immuni.2021.01.017</span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:14px">), which supports the linkage that viral entry causes IFN antagonism.</span> </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Also, it has been found that some people have developed autoimmunity toward their own IFN proteins (https://doi.org/10.1084/jem.20210554; </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#404040"><a href="https://doi.org/10.1084/jem.20211211">https://doi.org/10.1084/jem.20211211</a></span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">). They produce autoantibodies that block even exogenously administered IFN, and this has resulted in more severe disease in these patients.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This relationship is concerned with how entry of the virus into the host cell and subsequent release and transcription of viral proteins affects the downstream innate immune response. In particular, literature suggests the main pathway antagonized is the expression of type I interferons (IFN-I), consisting primarily of IFNα and IFNβ, and IFN-I stimulated genes (ISGs) (Banerjee et al., 2020; Blanco-Melo et al., 2020; Cheemarla et al., 2021; Xia et al., 2020; Sharif-Askari et al., 2022). Although there are few studies with evidence for cell entry leading directly to reduced IFN expression (Xia et al., 2020; Hatton et al. 2021), several studies demonstrate individual viral protein interactions with and blocking of host proteins in the IFN-I pathway or ISG proteins (Schubert et al. 2020; Thoms et al. 2020; Rui et al. 2021; Shin et al. 2020; Liu et al. 2021; Mostaqil et al., 2021; Xia et al. 2020; Quarleri and Delpino, 2021; Xia and Shi, 2020; Miorin et al. 2020; Kato et al. 2020; Fu et al. 2020; Chen et al. 2020; Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020; see below and also key event 1901). These studies provide the biological rationale that SARS-CoV-2 entry into the host cell causes interactions between viral proteins and known protein components of the host IFN-I antiviral response, resulting in inhibition of IFN-I and ISG expression.</span></span></p>
<p> </p>
<strong>Empirical Evidence</strong>
<p style="text-align:left"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">There are studies with empirical evidence in support of temporal concordance, showing that interferon expression is delayed by SARS-CoV-2 compared to other viruses like influenza, which is also described as an untuned or imbalanced response between interferons being initially low and inflammatory cytokines elevated in moderate to severe cases (10.1016/j.cell.2020.04.026;</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> </span></span><span style="font-size:14px"><span style="font-family:"Trebuchet MS""><span style="color:#404040"><a href="https://doi.org/10.1038/s41590-020-00840-x">https://doi.org/10.1038/s41590-020-00840-x</a>; <a href="https://www.science.org/doi/10.1126/science.abc6027">https://www.science.org/doi/10.1126/science.abc6027</a>; <a href="https://doi.org/10.1038/s41467-021-27318-0">https://doi.org/10.1038/s41467-021-27318-0</a>; https://doi.org/10.1038/s41598-021-95197-y</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Empirical evidence in support of temporal concordance comes from patient reports, showing that interferon expression is delayed by SARS-CoV-2 compared to other viruses like influenza, which is also described as an untuned or imbalanced response between interferons being initially low in moderate to severe cases (Banco-Melo et al. 2020; Galani et al., 2021; Hadjadj et al., 2020; Hatton et al., 2021; Rouchka et al., 2021). This indicates that SARS-CoV-2 stressors are suppressing the interferon response and highlights an important point regarding the difference between SARS-CoV-2 and other viruses in the stressors produced upon viral entry. Other viruses, as well as non-viral compounds used in research (e.g., polyinosinic:polycytidylic acid or poly[I:C]) enter the cell and stimulate the normal functional operation of the immune response, while SARS-CoV-2 blocks the response at multiple points, acting as a true prototypical stressor. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hatton et al. (2021) used human nasal epithelium differentiated at the air-liquid interface (ALI) cultures (organoids) with several cell types. Secretory cells were the cell type with the highest expression of viral transcripts, with ciliated and deuterosomal cells also showing expression. The SARS-CoV-2-infected secretory and ciliated cells also had many downregulated ISGs. Compared to SARS-CoV-2, influenza A virus induced significantly higher levels of IFN-I (IFNβ) and IFN-III (IFNλ1) at 6 and 24 hours post infection, as well as ISGs Ubiquitin specific peptidase 18 (USP18), radical s-adenosyl methionine domain containing 2 (RSAD2), and ubiquitin-like protein ISG15 at 24 hours post infection (Hatton et al., 2021). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Individual stressors from the virus were investigated by Xia et al. (2020) using an IFN-</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> promoter luciferase assay. HEK293T cells were co-transfected with luciferase reporter plasmids, the specific viral protein expressing plasmid, and stimulator plasmid RIG-I (2CARD). Of the viral proteins tested (NSPs 1, 2, 4-16, S, N, E, M, and ORFs 3a, 3b, 6, 7a, 7b, 8, and 10), four proteins (NSPs 1, 6, and 13 and ORF6) significantly reduced INF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β induction compared to the control (empty vector). A similarly conducted ISRE-promoter luciferase assay showed significant inhibition of the IFN-I signaling pathway (normally resulting in induction of ISGs) by NSPs 1, 6, 7, 13 and 14, ORFs 3a, 6, </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">7a and 7b, and M protein (Xia et al., 2020). See Xia et al. (2020) and Xia and Shi (2020) for schematics depicting the actions of the SARS-CoV-2 proteins on the protein components of the IFN-I antiviral response pathway.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2 stressor proteins and the IFN-I pathway responses were investigated individually in the following studies:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant reductions in IFNβ mRNA induction were seen when SARS-CoV-2 N protein was co-transfected into A549 cells with RIG-I, MAVS, or TBK1, and similar transfections resulted in IFNβ promoter activity reduction in poly(I:C)-stimulated HEK293T cells (Chen et al., 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sun et al. (2022) determined that SARS-CoV-2 and avian coronavirus infectious bronchitis virus (IBV) NSP3 PLpro N-terminal domain directly interacts with MDA5 to inhibit IFNβ expression when co-transfected in HEK293T cells.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fu et al. (2020) found M interaction with MAVS (as determined by coimmunoprecipitation and in vitro pull-down assay) interferes with recruitment of downstream pathway proteins TRAF, TBK1, and IRF3, inhibiting IFNβ1 promoter, IFN-stimulated response element (ISRE), and NFκB promoter activity in a dose-dependent manner. The M protein inhibited the transcription of ISGs (ISG56, CXCL10, and TNF) based on mRNA levels, and inhibited IFNβ and TNFα secretion based on measures of these proteins in HEK293 cell culture.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shin et al. (2020) generated a crystal structure and found that SARS-CoV-2 Plpro preferentially cleaves ISG15. ISG15 functions in antiviral immunity to directly inhibit viral replication (Perng and Lenschow, 2018).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gordon et al. (2020) showed interaction between TOMM70 and ORF9b via affinity purification-mass spectrometry (AP-MS). TOMM70-ORF9b interaction is supported by several studies (Gao et al., 2021; Brandherm et al., 2021; Ayinde et al., 2022). Jiang et al. (2020) used a dual luciferase reporter assay to show human IFN-β promoter activity was significantly reduced in the presence SARS-CoV-2 Orf9b compared to controls.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gordon et al. (2020) showed interaction between ORF6 and the host Nup98-RAE1 protein pair via AP-MS. The interaction was confirmed by Miorin et al., 2020 and Li et al., 2021 (see crystal structures). Miorin et al. (2020) also demonstrate that upon treatment with recombinant IFN-I in HEK293T cells, Nup98 binding to SARS-CoV-2 Orf6 blocks translocation of STAT1 into the nucleus, resulting in suppression of ISRE-dependent gene expression.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Using co-immunoprecipitation, Xia et al. (2020) showed that ORF6 selectively bound with KPNA2. Expression of ORF6 blocked nuclear translocation of IRF3, suggesting that ORF6 inhibited IFN-β production by binding to KPNA2 to block IRF3 nuclear translocation.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Biswal et al. (2022) solved the X-ray crystal structure of the G3BP1 N-terminal nuclear transport factor 2-like domain bound to the first intrinsically disordered region of SARS-CoV-2 N protein.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The interaction of the N-terminus of ORF9b with NEMO upon viral infection interrupts its K63-linked polyubiquitination, thereby inhibiting viral-RNA-induced IFNβ1 activation in HEK293T cells in an ORF9b-dose-dependent manner (Wu et al., 2021)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hameedi et al. (2022) solved the X-ray crystal structure of 3CLpro bound to NEMO and characterized 3CLpro cleavage of NEMO. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kilkenny et al., 2021 demonstrate that components of the host DNA polymerase α (Pol α)–primase complex or primosome directly bind with SARS-CoV-2 NSP1. They also provide a cryo-electron microscopy structure of NSP1 bound to the primosome. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Schubert et al. (2020) provide cryo-EM structures of NSP1 bound to the 40S ribosome subunit, inhibiting translation of host proteins.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sui et al. (2022) show that NSP13 recruits TBK1 to an aggregation of ubiquitinated proteins (p62) for autophagic degradation, resulting in inhibition of IFNβ production, and that NSP13 impaired IRF3 luciferase reporter activity induced by TBK1 in a dose-dependent manner. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xia et al. (2020) co-transfected HEK293T cells with plasmids containing TBK1 and either nsp6 or nsp13. Only NSP13 inhibited TBK1 phosphorylation, and did so in a dose-dependent manner, but both NSP6 and NSP13 suppressed IRF3 phosphorylation. Both NSP6 and NSP13 bind TBK1, as shown by co-immunoprecipitation. NSP6 binds to TBK1 without affecting TBK1 phosphorylation but this decreases IRF3 phosphorylation, while NSP13/TBK1 binding inhibits TBK1 phosphorylation. In both cases, IFN-β production is reduced (Xia et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rui et al. (2021) SARS-CoV-2 ORF3a and 3CLpro inhibited IFNβ promoter activity through cyclic GMP-AMP synthase (cGAS)-STING pathways, specifically through interaction with STING, as indicated by co-immunoprecipitation. 3CLpro also bound to STING and specifically inhibited K63-ubiquitin-mediated modification of STING, which is required for signaling and downstream expression of IFN-I. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mostaquil et al. (2020) showed with a fluorescent-based cleavage assay that NSP3 (Plpro) cleaves IRF-3, and thereby reduces IRF-3 available for induction of IFN-I expression.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>STAT1/STAT2</strong>: Signal transducer and activator of transcription</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mu et al. (2020) used Sendai virus (SeV)-induced ISRE-promoter activation via the luciferase reporter assay to determine that SARS-CoV-2 N protein can inhibit the phosphorylation of STAT1 and STAT2 resulting in decrease in ISG production. They also showed through co-immunoprecipitation that N interacts with both STAT1 and STAT2, and that N inhibits STAT1/2 phosphorylation by blocking interactions with kinases including JAK1.</span></span></p>
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</table>
<strong>Uncertainties and Inconsistencies</strong>
<p>There are uncertainties based on differing disease outcomes, especially associated with timing of administering IFN. Effectiveness of IFN treatment is still uncertain due to some studies evaluating IFN along with other drugs (DOI: 10.1002/jmv.27317). In the small intestine of infected hamsters, a mild antiviral gene signature was observed coinciding with a low-level inflammatory response and low replication similar to some human cases (https://doi.org/10.1016/j.immuni.2021.01.017), in contrast to the robust replication seen in human small intestinal organoids (https://doi.org/10.1126/science.abc1669) and in severely ill patients. See also https://doi.org/10.1186/s12943-021-01363-1.</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">There are uncertainties based on differing disease outcomes, especially associated with timing of IFN increase or suppression under different cell culture circumstances and in different people infected with SARS-CoV-2. Effectiveness of IFN treatment is still uncertain due to some studies evaluating IFN along with other drugs (Sodeifian et al., 2021).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are ISGs that have been implicated in SARS-CoV-2 entry as well as antiviral activity (Prelli Bozzo et al., 2021), in addition to the fact that the SARS-CoV-2 entry receptor ACE2 is an IFN-I stimulated gene (Ziegler et al., 2020). These are some of the paradoxes that confound transcriptomic studies that determine up- or downregulation of IFNs and ISGs in response to infection, and responses are highly dependent on the time points sampled. Efforts to address uncertainties around when and under what circumstances IFNs and ISGs either promote or supress the virus are ongoing. </span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>The current quantitative understanding of this relationship is described below.</p>
<strong>Response-response relationship</strong>
<p>Viral exposure dose threshold to result in IFN antagonism.</p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">A specific titer of virus can be used for infection, but as shown by Hatton et al. (2021), different cell types may express different levels of the actual stressors (viral protein transcripts). Because there are many stressors from each viral particle, which might be differentially expressed and also differentially inhibit each of their targets, a consistent whole viral entry dose leading to IFN-I or ISG response is difficult to measure. However, Chen et al. (2020), Xia et al. (2020), Fu et al. (2021), Wu et al. (2021) and Sui et al., (2022) all showed that individual protein stressor components of SARS-CoV-2 reduced IFN-I expression in a dose-dependent manner. </span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The viral entry MIE and early KEs coincide with the time from exposure to symptoms, within which are the latent period, or time from exposure to infectiousness, and the serial interval, or the time interval between the onset of symptoms in the primary (infector) and secondary case (infectee). V</span></span><span style="font-size:14px">iral entry leading to antagonism of the IFN response occurs d</span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">uring t</span></span><span style="font-size:14px">he latent period of the disease prior to symptom onset. </span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Latent period calculation is based on serial interval and median pre-symptomatic infectious period: Serial interval 5.2 days (Rai et al. 2021) – 2.5 days pre-symptom infectious period (Byrne et al. 2020) ≈ 2.7 days. The latent period was longer in asymptomatic cases (4-9 days); pre-symptomatic transmission occurs from about 3 days after exposure to symptom onset at about day 5-7, viral load peaks from about day 5-7 to day 9-11, and the host can remain infectious to symptom clearance or death (Byrne et al. 2020).</span></span><span style="font-size:14px"> As noted, IFN administered prior to exposure or within the latent period window can stop replication. However, IFN administered too late, in the inflammatory stage (post-symptom onset), led to long-lasting harm and worsened disease outcome (DOI: 10.1002/jmv.27317). </span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In a study using a primary nasal cell model (differentiated at air-liquid interface), the virus did not proliferate beyond the limit of assay detection if treated with IFN beta or lambda 16 hours prior to infection, and virus was significantly reduced in cultures treated 6h post-infection compared to untreated cultures. Treatments 24h post infection were not significantly different from untreated controls for either type of IFN (</span></span><span style="font-size:14px"><span style="font-family:AdvOTea1a7398"><span style="color:black">https://doi.org/10.1038/s41467-021-27318-0</span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">In humans, the viral entry MIE and early KEs coincide with the time from exposure to symptoms, within which are the latent period, or time from exposure to infectiousness, and the serial interval, or the time interval between the onset of symptoms in the primary (infector) and secondary case (infectee). <span style="font-size:14px">V</span></span></span><span style="font-size:14px">iral entry leading to antagonism of the IFN response occurs d</span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">uring t</span></span><span style="font-size:14px">he latent period of the disease prior to symptom onset. </span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Latent <span style="font-size:14px">period</span> calculation is based on serial interval and median pre-symptomatic infectious period: Serial interval 5.2 days (Rai et al. 2021) – 2.5 days pre-symptom infectious period (Byrne et al. 2020) equals approximately 2.7 days. The latent period was longer in asymptomatic cases (4-9 days).</span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<strong>Known modulating factors</strong>
<ul>
<li>Autoimmunity as noted above (patients with autoantibodies that attack IFN).</li>
<li>Factors affecting entry (TMPRSS2 or neuropilin) - therapeutics?</li>
<li>Androgen signaling leads to increased TMPRSS2 production (<a href="https://doi.org/10.1016/j.mce.2009.12.022" rel="noreferrer noopener" target="_blank" title="Persistent link using digital object identifier">https://doi.org/10.1016/j.mce.2009.12.022</a>) in males, and therefore potentially enhanced viral entry.</li>
<li>Weakened IFN antiviral response in the elderly (doi: <a href="https://doi.org/10.3390%2Fcells10030708" rel="noopener noreferrer" target="_blank">10.3390/cells10030708</a>).</li>
<li>Environmental exposures to aryl hydrocarbons and dioxins may block IFN production (doi: <a href="https://doi.org/10.1016%2Fj.cotox.2018.01.004" rel="noopener noreferrer" target="_blank">10.1016/j.cotox.2018.01.004</a>).</li>
</ul>
<div><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Genetic mutations.</strong> Autoimmunity to IFNs has been found in some COVID-19 patients. These individuals produce autoantibodies that attack IFN (Bastard et al., 2021 and 2022), which may be associated with human leukocyte antigen (HLA) gene mutations (Ku et al., 2016; Chi et al., 2013). Zhang et al., 2020 note inborn errors (genetic mutations) in IFN-I immunity that result in severe COVID-19, but some are also genes for proteins involved in the initial response (TBK1, IRF3, NEMO, IFNAR1, IFNAR2, STAT1, and STAT2). Zhang et al. (2022) also found similar mutations (STAT2 and IFNAR1) in children with COVID-19 pneumonia.</span></span></div>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Pollutant exposures.</strong> Most studies have been conducted with the endpoints to determine whether prior or concurrent exposure to chemical or air particulate pollutants exacerbates COVID-19 symptoms resulting in <em>more severe disease or higher mortality rates.</em> This would point to effects downstream of viral replication usually relating to antibody suppression, inflammation and organ/tissue damage. Fewer studies can be found that study pollutant effects on <em>susceptibility to infection</em>, which are relevant to this KER, specifically cell entry or interferon response antagonism. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Marques et al., (2022) reviews associations between COVID-19 and outdoor air pollutants including PM<sub>2.5</sub>, PM<sub>10</sub>, O<sub>3</sub>, NO<sub>2</sub>, SO<sub>2</sub> and CO, reporting that environmental air pollution increases both disease incidence and severity. Physiological mechanism is not investigated for most studies. One relevant study estimated significant odds ratios for increased risk of severe COVID-19 and gene transcriptional analysis showing downregulation of genes associated with the IFN-I pathway in patients with high short-term NO<sub>2</sub> exposure (Feng et al., 2023).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Per- and polyfluoroalkyl substances (PFAS) are a large group of contaminants of current concern, due to their potential for toxicity, ubiquitous presence in the environment and consumer products, as well as their resistance to degradation. Although most community exposure to PFAS is through diet and drinking water, airborne and dermal exposures may also occur, especially in the workplace (CDC/NIOSH 2022). Statistical links between high measured serum or urine concentrations of specific PFAS compounds or mixtures and higher rates of COVID-19-positive cases have been found. One study in Sweden calculated a sex- and age-Standardized Incidence Ratio (SIR) for the town of Ronneby that had highly PFAS-contaminated drinking water compared to a demographically matched town with background PFAS levels (Nielsen et al. 2021). Serum PFAS concentrations were previously measured in 2014-15 for 3507 participants (Xu et al. 2021), after the Ronneby drinking water contamination issue was identified in 2013. Ronneby residents had higher infection risk, with a SIR of 1.19 [95% CI: 1.12-1.27]. Ji et al. (2021) measured urine and serum in a smaller study in China with 160 subjects. They reported statistically significant odds ratios for infection of 1.94 [95% CI: 1.39–2.96] for perfluorooctane sulfonate (PFOS), 2.73 [1.71–4.55] for perfluorooctanoic acid (PFOA), and 2.82 [1.97–3.51] for Σ (12) PFASs, after controlling for age, sex, body mass index (BMI), comorbidities, and urine albumin-to-creatinine ratio (UACR). These odds of infection were clearly higher even though the PFAS-exposed subjects in China had serum concentrations lower than in the Ronneby study participants. Additionally, the risk of infection was similar for residents in a significantly more contaminated section of Ronneby compared with a less contaminated section, so there was no dose-response relationship (Nielsen et al. 2021). However, these associations warrant more study to determine causality. Ji et al. (2021) also found elevated PFAS to be associated with altered mitochondrial metabolism. A potential consideration is that inhibition of mitochondrial oxidative phosphorylation impairs MAVS-mediated induction of IFNs, indicating the coordination between antiviral response and mitochondrial metabolism (Yoshizumi et al., 2017). Another study proposes </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">modulation of ACE2 and TMPRSS2 expression in the lungs of </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">PFAS-exposed mice may play a role in PFAS-associated immune suppression </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Yang et al. 2022). Houck et al., (2022)</span></span> <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">report testing 147 PFAS substances in screening platforms including the BioMAP® Diversity PLUS panel, which is used to model complex tissue adverse effects of pharmaceuticals and environmental chemicals.</span></span> <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Toxicity Signatures within the BioMAP profile indicated the Skin Rash (MEK-Associated) Signature for PFOA, with IFN</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">α/</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> as one of the target mechanisms.</span></span> <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">While not specific to COVID-19, one study found that exposure to aryl hydrocarbons and dioxins may block IFN production (Franchini and Lawrence, 2018).</span></span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>Initial exposure to the virus is exogenous, but disease progression results from viral proliferation and release from the initial site of infection to spread to adjacent cells and eventually distal tissues. </p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">SARS-CoV-2 uses the host ACE2-receptor for entry, upon which the host IFN response could upregulate ACE2 to enhance infection (Ziegler et al., 2020), a positive feedback loop for viral entry, while the IFN response also induces antiviral protein expression to help restore homeostasis as a positive feedback loop to KE 1901.</span></span></p>
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<h4><a href="/relationships/2497">Relationship: 2497: IFN-I response, antagonized leads to SARS-CoV-2 production</a></h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Sex.</strong> In a large study modelling URT viral load dynamics drawn from measurements in 605 human subjects, variations over 5 orders of magnitude in URT viral load from the time of symptom onset was not explained by age, sex, or severity of illness. Additionally, these variables did not explain modelling results concerning control of viral load by immune responses in the early (innate) or late (adaptive) phases (Challenger et al. 2022). Other sources also support that rate of infection and measured viral load does not differ by gender (e.g., Arnold et al. 2022; Qi et al. 2021; Cheemarla et al. 2021). This evidence suggests that the components of the early antiviral response are not influenced by gender specific differences such as sex hormone levels or sex chromosomes to the extent of affecting viral load.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Life Stage. </strong>To apply to this KER, studies would need to show differences in IFN or ISGs correlated with viral load and differing by age. Saheb Sharif-Askari et al. (2022) reported that children had higher expression of IFN-I and associated ISGs than adults, but did not measure viral loads. Euser et al. (2021) found that SARS-CoV-2 viral loads increase with age, but did not measure IFN or ISGs. Literature that connects the two factors for age in humans was not found.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Taxonomic.</strong> No non-mammalian vertebrates have been found to become infected with SARS-CoV-2. Many mammals have tested positive and several are known to shed and transmit the virus, however the prevalent aspects of non-human mammalian infection and transmission found in the literature are ACE2 binding capacity and measures of viral load. For the few species for which IFN is mentioned in the literature (Mostaquil et al., 2020; Rui et al., 2021; Hameedi et al., 2022), the potential IFN antagonism is not linked to resulting increase in viral replication, except in the golden hamster, <em>Mesocricetus auratus</em> (Hoagland et al., 2021). The hamsters were Infected with SARS-CoV-2 resulting in high levels of virus in the upper and lower respiratory tracts and an </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">IFN-I response that was not sufficient to control COVID-19 progression. Direct contact resulted in inoculated hamsters transmitting the virus to naïve hamsters. When intranasal IFN-I was administered to the hamsters, viral replication was reduced and transmission was prevented (Hoagland et al., 2021). For bats, IFN and ISGs are constitutively expressed and therefore may contribute to immune tolerance and lack of replication of SARS-CoV-2 in many bat species (Irving et al., 2021</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">). Differential susceptibility and viral shedding has been found across mammalian species (EFSA/Nielson et al., 2023), and it is likely that differences in IFN-I response may be involved. Therefore, more studies are needed in diverse taxa to assess the tDOA for IFN-I antagonism leading to increase in SARS-CoV-2 replication across the potentially susceptible species.</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The normal function of the host’s innate immune response to viruses is the expression of interferons (IFN) which in turn stimulates the expression of many interferon-stimulated gene (ISG) proteins with antiviral functions (Amor et al., 2020; Harrison et al., 2020). ISGs generally function to inhibit viral replication (Yang and Li, 2020). The SARS-CoV-2 antagonism of the IFN-I pathway delays or curtails the expression of IFN-I and ISG proteins. This results in the downstream event, SARS-CoV-2 production, increased. The increase in SARS-CoV-2 viral production can be measured as viral load, which can contribute to both transmission to new hosts and more severe disease. This KER details the specific ISGs that inhibit viral replication, and demonstrates the difference in how SARS-CoV-2 negates the function of these proteins or delays their expression compared to other viruses to successfully increase its numbers.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<p>See below.</p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The functional relationships between the upstream IFN-I antagonism and downstream increase in SARS-CoV-2 viral replication is biologically plausible via the suppression of IFN through interaction inhibition of the host pathway proteins by viral proteins. This in turn would lead to suppressing the expression of ISGs that have been demonstrated to inhibit replication. The effects of ISGs on viral replication has been demonstrated for several viruses (Schoggins et al., 2011). SARS-CoV-2 replication may be impacted by different ISGs than other families of viruses. A gain-of-function analysis evaluating the impacts of ISGs on SARS-CoV-2 viral replication (Martin-Sancho et al., 2021</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">) showed that a specific subset of ISGs when stably overexpressed in cultured human cells infected with SARS-CoV-2 controlled viral infection, including RNA binding proteins that suppress viral RNA synthesis and ISGs inhibiting viral assembly and egress. Therefore, the lack of these ISGs due to antagonism of the IFN-I pathway leads to increased viral replication.</span></span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Evidence from patients who contracted COVID-19 supports the relationship between IFN antagonism and viral production:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Busnadiego et al. (2020) found that different IFNs upregulate ACE2 to differing degrees, but all IFNs elevated ISGs and inhibited SARS-CoV-2 replication in a dose-dependent manner. Some people have developed autoimmunity toward their own IFN proteins (Bastard et al., 2021; Lopez et al., 2021). They produce autoantibodies that block even exogenously administered IFN, and this has resulted in more severe COVID-19 disease in these patients. Also, Zhang et al., (2020) note inborn errors (genetic mutations) in IFN-I immunity that result in severe COVID-19.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cheemarla et al. (2021) used patient nasopharyngeal samples and airway epithelial organoids. COVID-19 patient samples had upregulated ISG RNAs in the upper respiratory tract. SARS-CoV-2 replicated exponentially when unchecked, doubling in 6 h. ISGs rose with viral replication and peaked as viral load declined. Rhinovirus infection before SARS-CoV-2 exposure caused ISG induction to accelerate and stopped SARS-CoV-2 replication, while blocking ISG induction increased viral replication.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hadjadj et al. (2020) report a phenotype in severe COVID-19 patients with no IFNβ, low IFNα, persistent blood viral load and exacerbated inflammatory response.</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Hatton et al. (2021) use human nasal epithelium differentiated at the air-liquid interface (ALI) cultures (organoids) to show delayed induction of IFN-I and -III in SARS-CoV-2 compared to influenza A virus. They found that exogenous IFNs administered pre-exposure or early in infection controlled SARS-CoV-2 replication.</span></span></li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schuhenn et al. (2022) found that differential immune signatures of IFNα subtypes suppress SARS-CoV-2 infection by treating primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection. The most effective antiviral subtype was IFNα5, against both in vitro and in vivo infected mice, and additive effects with the antiviral drug remdesivir in cell culture.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rouchka et al., (2021) found that there is not only wide variation in nasopharyngeal viral loads in COVID-19 patients early in infection, but also that viral loads were strongly correlated with host gene expression associated with IFNα-inducible cellular antiviral response genes (ISGs). Also, patients with mild symptoms were often found to have a higher viral load than those with severe disease, indicating lack of correlation between susceptibility to severe disease, and susceptibility to viral replication.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In review articles, Yang and Li (2020) and Samuel (2023) discuss the relationship between the IFN antiviral response and viral replication. Yang et al. focus on ISGs with multiple mechanisms that inhibit viral replication by sensing, degrading, or repressing expression of viral RNA. These ISGs may use a variety of co-factors, which indicates the highly complex nature of the type I IFN response. Samuel et al. report that overall genetic variability of both SARS-CoV-2 and the human host affect the IFN response, and viral replication is in turn sensitive to variation in IFN antiviral action.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">These studies point out inconsistencies in quantity and type of IFN expression or administration in patients and COVID-19 disease outcome, but confirming the link between IFN-I response and viral replication. There is uncertainty in the fact that several IFN-I pathway components have been variously implicated. Because many different IFN subtypes and subsequently many different ISGs and cofactors may be involved, not only the specific repertoire of ISGs expressed may differ among individuals, but also the quantity of each ISG may influence viral production.</span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-size:11.0pt"><span style="background-color:white"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">The current quantitative understanding of this relationship is described below.</span></span></span></span></p>
<strong>Response-response relationship</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Busnadiego et al., (2020) found an inverse, linear relationship between IFNβ or IFNλ1 concentration and viral titer, measured as plaque forming units (PFU) in primary human bronchial epithelial cells (BEpCs) differentiated and grown at an air-liquid interface (ALI). However, the upstream event of IFN antagonism is not represented by administered IFN but by antagonism of the IFN response, and does not answer the question of what dose of antagonist results in increased viral replication in a host system, where viral replication is not normal biology. Comparatively, difference in IFN expression between cells infected with influenza A virus vs. SARS-CoV-2 showed significantly higher IFNβ and IFNλ1 for influenza at both 6 and 24 hours post-infection, but this was not tied to relative viral production (Hatton et al., 2021). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The key event of IFN-I response antagonism encompasses a broad range of stressors and targets: 1) viral proteins interacting with pathway proteins leading to IFN expression, 2) the IFN subtypes that induce the expression of ISGs, and 3) the variation in type and amount of ISGs expressed, which also varies with cell/tissue type. Viral replication related to these factors is also dependent on the dose of virus to which the individual host is exposed and the genetic make-up and overall condition of that individual. These factors may explain the variable results in IFN dose-viral production response determination, and why the actual response-response relationship for this KER, between the viral dose resulting in antagonism and viral replication increase, have not been determined. Saheb Sharif-Askari et al., 2022 concluded that more mechanistic studies are needed to quantify the amount of early IFN required to overcome SARS-CoV-2 antagonism and prevent replication. Polyinosinic:polycytidylic acid [poly(I:C)] is a synthetic analog of double-strand RNA (dsRNA) that can stimulate IFN production. The use of poly(I:C) administered before and during SARS-CoV-2 infection in mice increased ISGs and lowered viral loads (Tamir et al., 2022) but was administered at different time points rather than at different dose concentrations. Poly(I:C) dosing may be a potential method to quantify the IFN stimulation needed to overcome SARS-CoV-2 antagonism.</span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The viral entry MIE and early KEs coincide with the time from exposure to symptoms, within which are the latent period, or time from exposure to infectiousness, and the serial interval, or the time interval between the onset of symptoms in the primary (index) and secondary (contact) case. Pre-symptomatic transmission occurs from about 3 days after exposure to symptom onset at about day 5-7, viral load peaks from about day 5-7 to day 9-11, and the host can remain infectious to symptom clearance or death (Byrne et al. 2020). IFN administered prior to exposure or within the latent period window can stop replication (Sodeifian et al., 2021). In a study using a primary nasal cell model (differentiated at air-liquid interface), the virus did not proliferate beyond the limit of assay detection if treated with IFN beta or lambda 16 hours prior to infection, and virus was significantly reduced in cultures treated 6h post-infection compared to untreated cultures. Treatments 24h post infection were not significantly different from untreated controls for either type of IFN (Hatton et al., 2021). This would suggest that viral antagonism of IFN occurring during the first 24h post viral entry allows viral loads to be generated likely concurrently, reaching transmissible levels within 72h post viral entry.</span></span></p>
<strong>Known modulating factors</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">IFN has been the subject of studies for potential therapeutic value to enhance the antiviral response. However, IFN administered too late, in the inflammatory stage (post-symptom onset), led to long-lasting harm and worsened disease outcome (Sodeifian et al., 2021). </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Therapeutics used in COVID-19 patients tend to target either the ACE2 binding, downstream inflammatory response, or viral replication via inhibition of the viral RNA-dependent, RNA polymerase to block viral genome replication (i.e., Remdesivir) (Narayanan and Parimon, 2022). No other therapeutics were found to be relevant to this KER, i.e., specifically targeted to IFN components or ISGs leading to supressed viral replication (see WHO 2021 and Terracciano et al., 2021).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">It is known that per- and poly-fluorinated alkyl substances (PFAS), air pollutants, and other environmental chemicals are implicated in SARS-CoV-2 susceptibility and COVID-19 disease severity (Marques et al., 2022; Nielsen et al., 2021; Xu et al., 2021). However, it is currently unknown whether or how the mechanisms of action are related to blocking IFN components or ISGs, leading to viral replication.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Genetic factors are of importance to this KER: Autoantibodies against IFN, as noted, block even exogenously administered IFN, resulting in more severe disease (Quarleri and Delpino, 2021</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">;</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> Bastard et al., 2021; Busnadiego et al., 2020; Lopez et al., 2021). There are 15 known clinically recessive and inborn errors of type I IFN immunity (Zhang et al., 2022). Four of these including X-linked recessive TLR7 deficiency, and autosomal recessive IFNAR1, STAT2, or TYK2 deficiencies were found in children with moderate to critical pneumonia due to COVID-19. Zhang et al. (2022</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> also reported enhanced SARS-CoV-2 replication measured as expression of viral nucleocapsid (N-protein) in STAT2- and TYK2-deficient patients’ cells.</span></span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">RIG-Like Receptors (RLRs) including MDA5 are Pattern Recognition Receptors (PRRs) that recognize Pathogen-Associated Molecular Patterns (PAMPs) like viral RNA and start signalling cascades to express IFNs. These PRRs and other proteins in the pathway, including STAT1 and STAT2 involved in transcription of the ISGs, are also regulated by IFN, and therefore are themselves ISGs (Yang and Li, 2020). As RNA from most viruses is detected, signalling to express more ISGs increases, and more IFN is expressed (Michalska et al., 2018). However, SARS-CoV-2 inhibits these and other components of the IFN pathway to delay expression of ISGs, and viral production goes unchecked, actually disrupting the normal antiviral positive feedback loop. In fact, SARS-CoV-2 can co-opt another ISG, interferon-induced transmembrane protein 2 (IFITM2), for efficient replication in human lung, heart, and gut cells (Nchioua et al., 2022), which might also be considered a positive feedback loop (i.e., the more IFITM2 is expressed, the more the virus replicates). However, IFITM2 and 3 have also shown antiviral activity toward SARS-CoV-2 (Shi et al., 2021), therefore the conflicting results require more research. </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schuhenn, J., Meister, T. L., Todt, D., Bracht, T., Schork, K., Billaud, J.-N., Elsner, C., Heinen, N., Karakoese, Z., Haid, S., Kumar, S., Brunotte, L., Eisenacher, M., Di, Y., Lew, J., Falzarano, D., Chen, J., Yuan, Z., Pietschmann, T., … Pfaender, S. (2022). Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection. <em>Proceedings of the National Academy of Sciences</em>, <em>119</em>(8), e2111600119. <a href="https://doi.org/10.1073/pnas.2111600119" style="color:blue; text-decoration:underline">https://doi.org/10.1073/pnas.2111600119</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shi, G., Kenney, A. D., Kudryashova, E., Zani, A., Zhang, L., Lai, K. K., Hall‐Stoodley, L., Robinson, R. T., Kudryashov, D. S., Compton, A. A., & Yount, J. S. (2021). Opposing activities of IFITM proteins in SARS‐CoV‐2 infection. <em>The EMBO Journal</em>, <em>40</em>(3), e106501. <a href="https://doi.org/10.15252/embj.2020106501" style="color:blue; text-decoration:underline">https://doi.org/10.15252/embj.2020106501</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sodeifian, F., Nikfarjam, M., Kian, N., Mohamed, K., & Rezaei, N. (2022). The role of type I interferon in the treatment of COVID‐19. <em>Journal of Medical Virology</em>, <em>94</em>(1), 63–81. <a href="https://doi.org/10.1002/jmv.27317" style="color:blue; text-decoration:underline">https://doi.org/10.1002/jmv.27317</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Tamir, H., Melamed, S., Erez, N., Politi, B., Yahalom-Ronen, Y., Achdout, H., Lazar, S., Gutman, H., Avraham, R., Weiss, S., Paran, N., & Israely, T. (2022). Induction of Innate Immune Response by TLR3 Agonist Protects Mice against SARS-CoV-2 Infection. <em>Viruses</em>, <em>14</em>(2), 189. <a href="https://doi.org/10.3390/v14020189" style="color:blue; text-decoration:underline">https://doi.org/10.3390/v14020189</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">WHO Solidarity Trial Consortium. (2021). Repurposed Antiviral Drugs for Covid-19—Interim WHO Solidarity Trial Results. <em>New England Journal of Medicine</em>, <em>384</em>(6), 497–511. <a href="https://doi.org/10.1056/NEJMoa2023184" style="color:blue; text-decoration:underline">https://doi.org/10.1056/NEJMoa2023184</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xu, Y., Nielsen, C., Li, Y., Hammarstrand, S., Andersson, E. M., Li, H., Olsson, D. S., Engström, K., Pineda, D., Lindh, C. H., Fletcher, T., & Jakobsson, K. (2021). Serum perfluoroalkyl substances in residents following long-term drinking water contamination from firefighting foam in Ronneby, Sweden. <em>Environment International</em>, <em>147</em>, 106333. <a href="https://doi.org/10.1016/j.envint.2020.106333" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.envint.2020.106333</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yang, E., & Li, M. M. H. (2020). All About the RNA: Interferon-Stimulated Genes That Interfere With Viral RNA Processes. <em>Frontiers in Immunology</em>, <em>11</em>, 605024. <a href="https://doi.org/10.3389/fimmu.2020.605024" style="color:blue; text-decoration:underline">https://doi.org/10.3389/fimmu.2020.605024</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zhang, Q., Bastard, P., Liu, Z., Le Pen, J., Moncada-Velez, M., Chen, J., Ogishi, M., Sabli, I. K. D., Hodeib, S., Korol, C., Rosain, J., Bilguvar, K., Ye, J., Bolze, A., Bigio, B., Yang, R., Arias, A. A., Zhou, Q., Zhang, Y., … Zhang, X. (2020). Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. <em>Science</em>, <em>370</em>(6515), eabd4570. <a href="https://doi.org/10.1126/science.abd4570" style="color:blue; text-decoration:underline">https://doi.org/10.1126/science.abd4570</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zhang, Q., Matuozzo, D., Le Pen, J., Lee, D., Moens, L., Asano, T., Bohlen, J., Liu, Z., Moncada-Velez, M., Kendir-Demirkol, Y., Jing, H., Bizien, L., Marchal, A., Abolhassani, H., Delafontaine, S., Bucciol, G., COVID Human Genetic Effort, Abel, L., Abolhassani, H., … Casanova, J.-L. (2022). Recessive inborn errors of type I IFN immunity in children with COVID-19 pneumonia. <em>Journal of Experimental Medicine</em>, <em>219</em>(8), e20220131. <a href="https://doi.org/10.1084/jem.20220131" style="color:blue; text-decoration:underline">https://doi.org/10.1084/jem.20220131</a></span></span></p>
</div>
<div>
<h4><a href="/relationships/2498">Relationship: 2498: SARS-CoV-2 production leads to Viral infection, proliferated</a></h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Because this KER describes a relationship within the SARS-CoV-2 infection and transmission process, the domain of susceptible species is the same for AOP 430 as a whole: humans and a broad range of mammals.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Sex and Age</strong></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Age was indicated as a factor in tendency to become infected (Marks et al., 2021); another study found no statistically significant difference in viral loads between age groups or sex (Carrouel et al., <a href="https://doi.org/10.3389/fmicb.2021.786042" style="color:blue; text-decoration:underline">https://doi.org/10.3389/fmicb.2021.786042</a>).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">In the process of SARS-CoV-2 production, the genome is replicated, packaged, and assembled with the structural proteins into virions that are then released from the host cell. The virions can infect nearby cells or be transported to distal organs, or be expelled from the host through coughing, sneezing, or vocalization, or in saliva and bodily waste. The amount of virus expelled from the host is dependent on the viral load produced. The viral load quantity produced in the upstream KE 1847 through the viral hijacking and modifications of host cell resources has been measured or modelled in several studies to determine the downstream terminal KE (1939) response: actual or potential transmission and successful infection of the exposed cell, organ, or new individual host. Transmission at the population level has also been monitored based on contact tracing, or experimental infection and transmission studies, or modelling community spread. Transmission at the ecosystem level has been demonstrated with human-to-animal-to-human transmission.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Empirical evidence supporting this relationship is described below.</span></span></p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In pathogen evolution it is the nature of the virus to replicate in a host (upstream KE 1847) and take advantage of internal and external transport mechanisms to reach another suitable habitat (downstream KE/AO 1939) to replicate again and result in infection. For this AOP and specifically for this KER, it is helpful to be aware of historical development of disease theory, i.e., the germ theory of disease and Koch’s postulates from the 19<sup>th</sup> century. Importantly, the first postulate that the microbe must be found in diseased individuals but not those without symptoms, had to be revised when it was realized that some bacteria like those causing cholera and typhoid could be carried by hosts who were asymptomatic (Fredricks and Relman, 1996). Viruses were discovered and were found to only replicate in cells and cannot be grown in pure culture, confounding the second postulate. Therefore, modifications of these disease principles have been applied to viruses (Rivers, 1937), and are basically an attempt at proving causation. Fredricks and Relman (1996) present a review citing several of these important revisions and their application with current technology like sequence-based identification of pathogens to prove the biological plausibility of the causal agent moving from host to host. Interestingly, Fouchier et al. (2003) carry out a proof that Koch’s postulates, as modified by Rivers (1937), are fulfilled for the (first) SARS virus. Numerous studies on SARS-CoV-2 demonstrate both the presence of the viral sequence by PCR (viral load), and the presence of neutralizing antibodies to the virus in upstream cases as considered by Evans’ (1976) proposed ‘‘Elements of Immunological Proof of Causation.’’ These principles go on to cover the downstream event, transmission to a healthy contact, where the antibody to the agent (SARS-CoV-2) is absent prior to the disease and exposure to the agent, the antibody appears during illness, and a downstream contact with no antibodies to the agent is susceptible to infection and disease produced by the agent (Evans, 1976; Fredricks and Relman, 1996). Literature providing empirical evidence of these principles specific to SARS-CoV-2 is provided below.</span></span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The causal agent of the COVID-19 disease, SARS-CoV-2, the stressor that is increased within a host’s cells in the upstream event, is also the stressor that infects multiple hosts (proliferates) in the downstream event. Evidence is found not only in many human studies, but also in other mammalian studies. Importantly, Worobey et al. (2022) gathered evidence within and around the Huanan Seafood Wholesale Market in Wuhan, China, with several findings on the origins of the human disease caused by the SARS-CoV-2 virus, including: 1) live animals shown to be susceptible to SARS-CoV-2 were sold at the market in late 2019; 2) the SARS-CoV-2 virus was found in environmental samples taken from the live animal vender locations, indicating viral shedding from the animals occurred; 3) earliest known human cases of the COVID-19 disease (December 2019) were geographically centered around the market, and 4) 66% of the 41 people hospitalized with the disease by January 2, 2020 had direct exposure to the market. Pekar et al. (2022) found only two distinct viral lineages of SARS-CoV-2 before February 2020 which epidemic simulations showed were the result of two or more separate zoonotic transmission events to humans, the first lineage introduced between late October and early December, and the second lineage weeks later. Those infected with the first lineage had direct contact with the Huanan market, and those with the second lineage did not, but lived or stayed near the market during that time period (Pekar et al., 2022). This evidence indicates zoonotic transfer to humans, with spread of the disease from human to human in Wuhan as the epicenter.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In humans, as the pandemic spread, household contact studies and tracking were used to determine viral loads and secondary infections in contacts: </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Bhavnani et al. (2022 <a href="https://doi.org/10.1186/s12879-022-07663-1" style="color:blue; text-decoration:underline">https://doi.org/10.1186/s12879-022-07663-1</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">) found an association between SARS-CoV-2 viral load in an individual with a case of COVID-19 and the risk of transmission to contacts.</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> Among the 212 primary (index) cases assessed, median viral load was 5.6 (1.8–10.4) log<sub>10</sub> RNA copies per mL of saliva, with 70 (19%) of their 365 contacts testing positive after exposure. Of those 70, 36 (51%) were exposed to an index case that </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">was asymptomatic or pre-symptomatic on the day of exposure. Contacts infected increased monotonically with index case viral load, resulting in a significant association between viral load and risk of transmission (RR = 1.27, 95% CI 1.22–1.32).</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Marks et al. (2021 <a href="https://doi.org/10.1016/S1473-3099(20)30985-3" style="color:blue; text-decoration:underline">https://doi.org/10.1016/S1473-3099(20)30985-3</a>) conducted a similar study in Catalonia, Spain. Of 282 patients with COVID-19 that had a total of 753 contacts, 17% (125 of 753 contacts) became infected. Infections varied from 12% when the index case viral load was less than 1 × 10<sup>6</sup> copies per mL to 24% when the index case viral load was 1 × 10<sup>10</sup> copies per mL or more. Transmission risk increased for household contacts and by increasing age of the contact. The study results indicated that viral load of index cases was the strongest factor in SARS-CoV-2 transmission.</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">While it has been documented that humans have passively transmitted SARS-CoV-2 back to other mammals (see KE1939), researchers have also conducted controlled exposure experiments in other mammals in which they inoculated test animals with SARS-CoV-2, measured viral shedding, and confirmed infection in contact animals:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Freuling et al. (2020) tested raccoon dogs by administering intranasal inoculations to nine naive animals with SARS-CoV-2 2019_nCoV Muc-IMB-1, and introducing 3 naive animals 24 hours after inoculation. Six inoculated and two contact animals became infected based on viral RNA measured by qPCR in nasal, oropharyngeal, and rectal swab samples. SARS-CoV-2 viral RNA was first detected in a contact animal 7 days after contact.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Palmer et al. (2021) conducted intranasal inoculations of white-tailed deer fawns with SARS-CoV-2, resulting in infection and shedding of infectious virus in nasal secretions. The infected animals were found to transmit the virus to contact deer. </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Martins, et al. (2022) found that white-tailed deer fawns shed infectious virus in nasal and oral secretions up to 5 days after intranasal inoculation with SARS-CoV-2 B.1 lineage, with deer-to-deer transmission occurring on day 3 post-inoculation. </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cool, et al. 2022 investigated transmission in adult white-tailed deer co-infected with both the SARS-CoV-2 ancestral lineage A and the alpha variant of concern (VOC) B.1.1.7. Presence and transmission of each strain was determined using next-generation sequencing, with the finding that the alpha VOC B.1.1.7 isolate outcompeted ancestral lineage A. They found direct contact transmission and also vertical transmission from doe to fetus.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shuai et al. (2020) studied minks and showed that SARS-CoV-2 replicates efficiently in the upper and lower respiratory tracts. To investigate transmission, intranasal inoculations were administered to three animals that were then placed in a separate cages. After 24 hours, three naïve minks were placed in cages adjacent to the virus-inoculated mink without direct contact. Viral RNA was detected in the nasal washes of all three introduced animals after 3-9 days showing that SARS-CoV-2 was transmitted via respiratory droplets.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kim et al. (2021) found that SARS-CoV-2 replicated in 6 inoculated ferrets, and was transmitted to all 6 direct contact ferrets and two of 6 indirect contact ferrets, indicating direct contact was more efficient, but airborne transmission also occurred. Airborne transmission likely results in a lower dosage than direct contact.</span></span></li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The main area of uncertainty is in quantifying viral load either by measuring viral RNA by PCR or by isolating the virus in cell culture and determining numbers of plaque forming units (PFU).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">A review by Puhach et al. (2022 <a href="https://doi.org/10.1038/s41579-022-00822-w" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41579-022-00822-w</a>) points out several issues affecting quantification: </span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Quantitative real-time polymerase chain reaction (RT-PCR) cannot differentiate between replication-competent (infectious) virus and residual (non-infectious) viral RNA.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">RT-PCR cannot determine whether the RNA viral load is increasing or decreasing; peak viral load may have passed or has not yet been reached.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Analytical sensitivity of measured RT-PCR cycles (Ct values) used to determine RNA copy numbers and limits of detection may vary between the tests and laboratories.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Site of specimen collection (e.g., nasal or nasopharyngeal swabs or throat samples) can affect viral load measurement. </span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">For cell culture, only live (infectious) viruses are counted, however:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Quality of the sample affects success of viral culture.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Loss of infectiousness can occur due to unsuitable storage conditions like high temperatures (requirement for −80 °C) or repeated freeze–thaw cycles.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cell lines used for isolation can show a high variability between laboratories.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Culture medium or additives such as fetal bovine serum and antibiotics may affect success of viral culture. </span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Infectious virus determined using Vero E6 cells might overestimate transmission risks in vivo.</span></span></li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Data is available for a quantitative understanding between the upstream SARS-CoV-2 production increased and the downstream host-to-host transmission proliferation key events in this KER, epitomized by the computation of the basic reproduction number (<em>R</em><sub>0</sub>). The R<sub>0</sub> is the number of secondary cases from each index case in a population, assuming no immunization, and is a fundamental epidemiological metric used as an indicator of the contagiousness or transmissibility of infectious agents in populations (Delamater et al., 2019 <a href="https://doi.org/10.3201/eid2501.171901" style="color:blue; text-decoration:underline">https://doi.org/10.3201/eid2501.171901</a>). Interpretation of an <em>R</em><sub>0</sub> > 1 is that the virus is spreading exponentially, and if <em>R</em><sub>0</sub> < 1, the number becoming infected is decreasing. Time interval is important in the calculation; the R<sub>0</sub> at a given time during an epidemic is called R<sub>t</sub> or R<sub>e</sub> (effective reproduction number). The R<sub>0</sub> varies due to viral load, regional social factors, and differences in underlying health and numbers of contacts of individuals (Karimizadeh et al., 2023). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">A review and comparison of the original Wuhan strain of SARS-CoV-2 and the first SARS virus indicated that SARS-CoV-2 had higher R<sub>0</sub> values than SARS, with SARS-CoV-2 R<sub>0</sub> averaging 3.28 (median 2.79), exceeding WHO estimates of 1.4-2.5 (Liu et al., 2020 <a href="https://doi.org/10.1093%2Fjtm%2Ftaaa021" style="color:blue; text-decoration:underline" target="_blank">10.1093/jtm/taaa021</a>). A comparison of R<sub>0</sub> values for several variants worldwide found the highest values for Alpha (1.22), Beta (1.19), Gamma (1.21), Delta (1.38) and Omicron (1.90) from Japan, Belgium, the United States, France and South Africa, respectively (Manathunga et al., 2023 <a href="https://doi.org/10.1186/s12985-023-02018-x" style="color:blue; text-decoration:underline">https://doi.org/10.1186/s12985-023-02018-x</a>). A recent review cites several studies in which R<sub>0</sub> has been calculated by various means, for the original and SARS-CoV-2 variants, in several countries (Karimizadeh et al., 2023 <a href="https://doi.org/10.1186%2Fs40001-023-01047-0" style="color:blue; text-decoration:underline" target="_blank">10.1186/s40001-023-01047-0</a>). As an example, an Iranian study used four different models to calculate R<sub>0</sub> for two COVID-19 variants (Sheikhi et al. 2022 <a href="https://doi.org/10.1371/journal.pone.0265489" style="color:blue; text-decoration:underline">https://doi.org/10.1371/journal.pone.0265489</a>). Results for the Exponential Growth Rate (EGR), Maximum Likelihood (ML), Sequential Bayesian (SB), and time-dependent susceptible, infectious, and recovered or removed (SIR) models for the Alpha variant were 0.9999 (95% Confidence Interval [CI]: 0.9994-1), 1.046 (95% CI: 1.044-1.049), 1.06 (95% CI: 1.03-1.08), and 2.79 (95% CI: 2.77-2.81), respectively, for March10-June 10, 2021. However, during the exponential growth period for Alpha in Iran of April 3-9, the <em>R</em><sub>0</sub> of the respective models were 2.26 (95% CI: 2.04-2.49), 2.64 (95% CI: 2.58-2.7), 11.38 (95% CI: 11.28-11.48), and 12.13 (95% CI: 12.12-12.14). For the Delta variant exponential growth period from July 3-8, 2021 <em>R</em><sub>0</sub> calculated for the respective models were 3 (95% CI: 2.34-3.66), 3.1 (95% CI: 3.02-3.17), 12 (95% CI: 11.89-12.12), and 23.3 (95% CI: 23.19-23.41), with the interval of June 22-September 22, 2021 R<sub>0</sub> close to 1, similar to the longer interval for the Alpha variant (Sheikhi et al. 2022).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">To determine the specific dose in the index case required to infect a secondary host, viral loads have been measured by using qRT-PCR to enumerate viral RNA genomes (Bhavnani et al., 2022; Marks et al., 2021) and by determining the number of infectious units in tissue culture. Such empirically determined dose-response parameters have been used in models that incorporate dynamics of the exposure pathways, such as airborne transmission (the link between viral production in the index case and infection in the contact case), as discussed below.</span></span></p>
<strong>Known modulating factors</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Vaccination causes the production of antibodies in the host that can quickly mobilize to attack the invading virus, ultimately inhibiting viral replication and transmission. Pfizer-BioNTech (BNT162b2) vaccine using mRNA technology to deliver the viral spike protein sequence and other vaccines reduced index-to-secondary patient transmission (Eyre et al., 2022). Braeye et al. (2023) in a 2020-21 Belgian contact tracing study showed vaccine effectiveness against transmission (VET) for BNT162b2 for primary vaccination at 96% against Alpha, 87% against Delta and 31% against Omicron. Mentzer et al. (2023) found that certain human leukocyte antigen (HLA) gene alleles are associated with COVID-19 breakthrough infection in vaccinated individuals.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">It is known that per- and poly-fluorinated alkyl substances (PFAS), air pollutants, and other environmental chemicals are implicated in SARS-CoV-2 susceptibility and COVID-19 disease severity (Marques et al., 2022; Nielsen et al., 2021; Xu et al., 2021). However, it is currently unknown whether or how the mechanisms of action are related to transmission risk.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The drug Remdesivir (GS-5734) is a small molecule adenosine analogue that</span></span><span style="font-size:1rem"> </span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">binds to the viral RNA-dependent RNA polymerase and inhibits viral replication by incorporating into the nascent viral RNA chain causing pre-mature termination (Warren et al., 2016 <a href="https://doi.org/10.1038%2Fnature17180" style="color: blue; text-decoration-line: underline;" target="_blank">10.1038/nature17180</a>). Wang et al. (2020 <a href="https://doi.org/10.1038%2Fs41422-020-0282-0" style="color: blue; text-decoration-line: underline;" target="_blank">10.1038/s41422-020-0282-0</a>) found remdesivir to inhibit viral yield in cell culture by more than 90%. However, Williamson et al. (2020 <a href="https://doi.org/10.1038/s41586-020-2423-5" style="color: blue; text-decoration-line: underline;">https://doi.org/10.1038/s41586-020-2423-5</a></span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">) report that in </span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">rhesus macaques, </span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">remdesivir treatment did not reduce virus shedding from the upper respiratory tract but prevented disease progression to pneumonia.</span></span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multilevel feedback mechanisms have been suggested regarding behaviors such as social distancing and how they influence SARS-CoV-2 mutational adaptations geospatially (Barrett et al., 2022 <a href="https://doi.org/10.1089/cmb.2020.0343" style="color:blue; text-decoration:underline">https://doi.org/10.1089/cmb.2020.0343</a>). A transfer entropy (TE) framework was used to illustrate the feedback between macrolevel dynamics of socio-behavioral measures and microlevel mutational composition of the viral population. For example, A23404G leading to the D614G mutation in the viral spike protein significantly increases the viral load in patients, in turn increasing transmission rates. Differences in culture, policy, and the severity of infections resulted in distinct selective pressures on the virus in different geospatial blocks, producing different mutational signals for human populations in California versus New York versus Washington, suggesting a feedback loop connecting socio-behavioral patterns with mutational signatures. Additionally, the framework shows adaptability of the virus based on a noncoding mutation G29540A, highly localized in NY (> 95%), and extended incubation periods in China possibly due to the pressure imposed by drastic social distancing measures (Barrett et al., 2022). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bradley et al. (2020 <a href="https://doi.org/10.1016/j.eclinm.2020.100325" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.eclinm.2020.100325</a>) use a systems approach with causal loop diagrams as tools to also illustrate the dynamics of a societal response to the threat of COVID-19. They propose CLDs that include numbers of infectious and susceptible people in feedback loops with policies that influence risk of transmission and transmission events. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Wanelik et al. (2023 <a href="https://doi.org/10.1016/j.isci.2023.106618" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.isci.2023.106618</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">) focus on “superspreaders”, i.e., individuals who are capable of infecting more than the average number of secondary contacts, who, evidence suggests, may be more likely to become superspreaders themselves. They used a generic model with hypothetical parameters to show that this positive feedback loop had effects on the herd immunity threshold, basic reproduction number (R<sub>0</sub>), the peak prevalence of superspreaders, and the final epidemic size.</span></span></p>