AOP ID and Title:

Short Title: SARS-CoV-2 leads to infection proliferation

Authors

Sally Mayasich, University of Wisconsin-Madison Aquatic Sciences Center at US Environmental Protection Agency, Duluth, MN, USA

Maria João Amorim, Instituto Gulbenkian de Ciência, Oeiras, and Universidade Católica Portuguesa, Católica Medical School, Católica Biomedical Research Centre, Portugal

Laure-Alix Clerbaux, European Commission-Joint Research Centre (EC-JRC), Ispra, Italy

Alicia Paini, EC-JRC/EsqLab

Nikolaos Parissis, EC-JRC

Young Jun Kim, KIST Europe, Germany

Penny Nymark, Institute of Environmental Medicine, Karolinska Institute, Sweden

Status

Abstract

Severe accute respiratory syndrome (SARS) and SARS-CoV-2 coronoviruses enter the cell through interaction with the ACE2 receptor. The first event upon cell entry after uncoating is the primary translation of the ORF1a and ORF1b 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 Interferon-I antiviral response 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.

Background

This AOP was developed in the context of other COVID-19 AOPs through the work of a larger international effort to model the pathogenesis of COVID-19 using the AOP framework (the CIAO project, https://www.ciao-covid.net/about-us), 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.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

See details below.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Life stage and sex

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.

Taxonomic domain

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.

References

Appendix 1

List of MIEs in this AOP

Event: 1739: Binding to ACE2

Short Name: Binding to ACE2

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The KE is applicable to broad species/life stage/sex. The binding of ACE2 occure in the cells which express ACE2. 

Key Event Description

Angiotensin-converting enzyme 2 (ACE2) is an enzyme that can be found either attached to the membrane of the cells (mACE2) in many tissues and in a soluble form form (sACE2).

A table on ACE2 expression levels according to tissues (Kim et al.)

	Sample size	ACE2 mean expression	Standard deviation of expression
Intestine	51	9.50	1.183
Kidney	129	9.20	2.410
Stomach	35	8.25	3.715
Bile duct	9	7.23	1.163
Liver	50	6.86	1.351
Oral cavity	32	6.23	1.271
Lung	110	5.83	0.710
Thyroid	59	5.65	0.646
Esophagus	11	5.31	1.552
Bladder	19	5.10	1.809
Breast	113	4.61	0.961
Uterus	25	4.37	1.125
Protaste	52	4.35	1.905

ACE2 receptors in the brain (endothelial, neuronal and glial cells):

The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010). The neurons and glial cells, like astrocytes and microglia also express ACE-2.

In the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020). Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018).

ACE2 receptors in the intestines

The highest levels of ACE2 are found at the luminal surface of the enterocytes, the differentiated epithelial cells in the small intestine, lower levels in the crypt cells and in the colon (Liang et al, 2020; Hashimoto et al., 2012, Fairweather et al. 2012; Kowalczuk et al. 2008).

 

 

How it is Measured or Detected

In vitro methods supporting interaction between ACE2 and SARS-CoV-2 spike protein

Several reports using surface plasmon resonance (SPR) or biolayer interferometry binding (BLI) approaches. to study the interaction between recombinant ACE2 and S proteins have determined a dissociation constant (Kd) for SARS-CoV S and SARS-CoV-2 S as follow,

Reference	ACE2 protein	SARS-CoV S	SARS-CoV2 S	Method	Measured Kd
doi:10.1126/science.abb2507	1–615 aa	306–577 aa		SPR	325.8 nM
			1–1208 aa		14.7 nM
doi:10.1001/jama.2020.3786	19–615 aa	306–527 aa		SPR	408.7 nM
			319–541 aa		133.3 nM
Lan et al., 2020	19–615 aa	306–527 aa		SPR	31.6 nM
			319–541 aa		4.7 nM
doi:10.1016/j.cell.2020.02.058	1–614 aa	306–575 aa		BLI	1.2 nM
			328–533 aa		5 nM
doi:10.1126/science.abb2507	1–615 aa	306–577 aa		BLI	13.7 nM
			319–591 aa		34.6 nM

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.

References

de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.

Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.

Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.

Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.

Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.

Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.

Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.

Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468

Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.

Xia, H. and Lazartigues, E.  Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010  Rep. 12 (3), 170– 175

List of Key Events in the AOP

Event: 1738: SARS-CoV-2 cell entry

Short Name: SARS-CoV-2 cell entry

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

TMPRSS2 vertebrates (Lam et al., 2020)

NRP1 in human & rodents (but also present in monkey and other vertebrates (Lu and Meng, 2015)

The ability for SARS-CoV-2 to use multiple host pathways for viral entry, means that it is critical to map which viral entry pathway is prevalent in specific cell types. This is key for understanding coronavirus biology, but also use informed decisions to select cells for cell-based genetic and small-molecule screens and to interpret data. In fact, a combination of protease inhibitors that block both TRMPSS2 and cathepsin L is the most efficient combination to block coronavirus infection (Yamamoto et al., 2020, Shang et al., 2020, Shirato et al., 2018). In accordance, SARS-CoV-2 entry processes are highly dependent on endocytosis and endocytic maturation in cells that do not express TMPRSS2, such as VeroE6 or 293T cells (Murgolo et al., 2021, Kang et al., 2020, Mirabelli et al., 2020, Riva et al., 2020). However, even in these cells, heterologous expression of TMPRSS2 abrogates the pharmacological blockade of cathepsin inhibitors (Kawase et al., 2012, Hoffmann et al., 2020a). Treatment of SARS-CoV-2 with trypsin enables viral cell surface entry, even when TMPRSS2 is absent. Moreover, TMPRSS2 is more efficient to promote viral entry than cathepsins (Lamers et al., 2020), as when both factors are present,d cathepsin inhibitors are less effective than TMPRSS2 inhibitors (Hoffmann et al., 2020b). Therefore it is critical to map which cells contain the different types of proteases.

In summary, TMPRSS2 appears to be expressed in a wide range of healthy adult organs, but in restricted cell types, including:

• AT2 and clara cells of lungs
• sinusoidal endothelium, and hepatocyte of the liver,
• endocrine cells of the prostate,
• goblet cells , and enterocytes of the small intestine,
• intercalated cells, and the proximal tubular of the kidney.
• Ciliated, secretory and suprabasal of nasal
• spermatogonial stem cells of testes
• cyto tropoblast and peri vascular cells of placenta
• 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.

There is a shift in TMPRSS2 regulation during nasal epithelium differentiation in young individuals that is not occurring in old individuals (Lin et al., 1999, Lucas et al., 2008, Singh et al., 2020).

Only a small minority of human respiratory and intestinal cells have genes that express both ACE2 and TMPRSS2. Amongst the ones that do, three main cell types were identified: A) lung cells called type II pneumocytes (which help maintain air sacs, known as alveoli); B) intestinal cells called enterocytes, which help the body absorb nutrients; and C) goblet cells in the nasal passage, which secrete mucus (Ziegler et al., 2020).

The clinical manifestations of COVID‐19 include not only complications from acute myocardial injury, elevated liver enzymes, and acute kidney injury in patients presenting to hospitals, but also gastrointestinal symptoms in community patients experiencing milder forms of the disease (Madjid et al., 2020, Pan et al., 2020).

 

NRP-1:

All life stages

The expression of isoforms 1 (NRP1) and 2 (NRP2) does not seem to overlap. Isoform 1 is expressed by the blood vessels of different tissues. In the developing embryo it is found predominantly in the nervous system. In adult tissues, it is highly expressed in heart and placenta; moderately in lung, liver, skeletal muscle, kidney and pancreas; and low in adult brain. Isoform 2 is found in liver hepatocytes, kidney distal and proximal tubules. Expressed in colon and 234 other tissues with Low tissue specificity (UniProtKB).

The expression of NRP1 protein in gastric cancer was not related to gender or age (Cao et al., 2020).

 

Sex Applicability:

TMPRSS2:

Androgen receptors (ARs) play a key role in the transcription of TMPRSS2 (Fig. 1). This may explain the predominance of males to COVID-19 infection, fatality, and severity because males tend to have a higher expression and activation of ARs than females, which is due to the presence of dihydrotestosterone (DHT).

Regulation of COVID-19 severity and fatality by sex hormones. Females have aromatase, the enzyme that converts androgen substrates into estrogen. On the other hand, males have steroid 5α reductase, the enzyme that is responsible for the conversion of testosterone into dihydrotestosterone (DHT). In case of males, DHT activates androgen receptor (AR) that binds to the androgen response element (ARE) present in the promoter of TMPRSS2 gene, leading to its transcription. This ultimately results into enhanced processing of viral spike protein for greater entry and spread of SARS-CoV-2 into host cells. On the other hand,in females, estrogen activates estrogen receptor (ER), which binds to the estrogen response element (ERE) present in the promoter of eNOS gene to drive its transcription and catalyze the formation of nitric oxide (NO) from L-arginine. This NO is involved in vasodilation as well as inhibition of viral replication.

NRP-1:

For more information difference of NRP1 expression between male and female see https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue.

The expression of NRP1 protein in gastric cancer was not related to gender, age. The expression of NRP1 protein in gastric cancer is closely correlated to clinical stage, tumor size, TNM stage, differentiation, and lymph node metastasis (Cao et al., 2020).

SARS-CoV-2 Spike protein co-opts VEGF-A/Neuropilin-1 receptor signalling to induce analgesia had same results on both male and female rodents (Moutal et al., 2020).

Key Event Description

Coronavirus is recognized by the binding of S protein on the viral surface and angiotensin-converting enzyme 2 (ACE2) receptor on the cellular membrane, followed by viral entry via processing of S protein by transmembrane serine protease 2 (TMPRSS2) (Hoffmann et al., 2020b). ACE2 is expressed on epithelial cells of the lung and intestine, and also can be found in the heart, kidney, adipose, and male and female reproductive tissues (Lukassen et al., 2020, Lamers et al., 2020, Chen et al., 2020, Jing et al., 2020, Subramanian et al., 2020).

SARS-CoV-2 is an enveloped virus characterized by displaying spike proteins at the viral surface (Juraszek et al., 2021). Spike is critical for viral entry (Hoffmann et al., 2020b) and is the primary target of vaccines and therapeutic strategies, as this protein is the immunodominant target for antibodies (Yuan et al., 2020, Ju et al., 2020, Robbiani et al., 2020, Premkumar et al., 2020, Liu et al., 2020). Spike is composed of S1 and S2 subdomains. S1 contains the N-terminal (NTD) and receptor-binding (RBD) domains, and the S2 contains the fusion peptide (FP), heptad repeat 1 (HR1) and HR2, the transmembrane (TM) and cytoplasmic domains (CD) (Lan et al., 2020). S1 leads to the recognition of the angiotensin-converting enzyme 2 (ACE2) receptor and S2 is involved in membrane fusion (Hoffmann et al., 2020b, Letko et al., 2020, Shang et al., 2020).

Upon binding to ACE2, the spike protein needs to be activated (or primed) through proteolytic cleavage (by a host protease) to allow membrane fusion. Fusion is a key step in viral entry as it is the way to release SARS-CoV-2 genetic material inside the cell. Cleavage happens between its spike’s S1 and S2 domains, liberating S2 that inserts its N-terminal domain into a host cell membrane and mediates membrane fusion (Millet and Whittaker, 2018). Many proteases were identified to activate coronaviruses including furin, cathepsin L, trypsin-like serine proteases TMPRSS2, TMPRSS4, TMPRSS11, and human airway trypsin-like protease (HATs). These may operate at four different stages of the virus infection cycle: (a) pro-protein convertases (e.g., furin) during virus packaging in virus-producing cells, (b) extracellular proteases (e.g., elastase) after virus release into extracellular space, (c) cell surface proteases [e.g., type II transmembrane serine protease (TMPRSS2)] after virus attachment to virus-targeting cells, and (d ) lysosomal proteases (e.g., cathepsin L) after virus endocytosis in virus-targeting cells (Li, 2016). SARS-CoV-2 lipidic envelope may fuse with two distinct membrane types, depending on the host protease(s) responsible for cleaving the spike protein: (i) cell surface following activation by serine proteases such as TMPRSS2 and furin (Hoffmann et al., 2020b); or (ii) endocytic pathway within the endosomal–lysosomal compartments including processing by lysosomal cathepsin L (Yang and Shen, 2020). These flexibility for host cell factors mediating viral entry, highlights that the availability of factors existing in a cell type dictates the mechanism of viral entry (Kawase et al., 2012). When TMPRSS2 (or other serine proteases such as TMPRSS4 (Zang et al., 2020) or human airway trypsin-like protease [HAT] (Bestle et al., 2020a)) is expressed, fusion of the virus with the cell surface membrane is preferred (Shirato et al., 2018), while in their absence, the virus can penetrate the cell by endocytosis (Kawase et al., 2012). A third factor has also been shown to facilitate SARS-CoV-2 entry in cells that have ACE2 and even promote, although to very low levels, SARS-CoV-2 entry in cells that lack ACE2 and TMPRSS2 which is the neuropilin-1 (NRP-1) (Cantuti-Castelvetri et al., 2020). This key event deals with SARS-CoV-2 entry in host cells and is divided in three categories: TMPRSS2, capthesin L and NRP-1.

TMPRSS2 Spike cleavage:

TMPRSS2 (transmembrane serine protease 2, (https://www.ncbi.nlm.nih.gov/gene/7113) is a cell-surface protease (Hartenian et al., 2020) that facilitates entry of viruses into host cells by proteolytically cleaving and activating viral envelope glycoproteins. Viruses found to use this protein for cell entry include Influenza virus and the human coronaviruses HCoV-229E, MERS-CoV, SARS-CoV and SARS-CoV-2 (COVID-19 virus).

TMPRSS2 is a membrane bound serine protease also known as epitheliasin. TMPRSS2 belongs to the S1A class of serine proteases alongside proteins such as factor Xa and trypsin. Whilst there is evidence that TMPRSS2 autoclaves to generate a secreted protease, its physiological function has not been clearly identified. However, it is known to play a crucial role in facilitating entry of coronavirus particles into cells by cleaving the spike protein (Huggins, 2020).

After ACE2 receptor binding, SARS-CoV-2 S proteins can be subsequently cleaved and activated by host cell-surface protease at the S1/S2 and S2’ sites, generating the subunits S1 and S2 that remain non-covalently linked. Cleavage leads to activation of the S2 domain that drives fusion of the viral and host membranes (Hartenian et al., 2020, Walls et al., 2016). For other coronaviruses, processing of spike was proposed to be sequential with S1/S2 cleavage preceding that of S2. Cleavage at S1/S2 may be crucial for inducing conformational changes required for receptor binding or exposure of the S2 site to host proteases.

The S1/S2 site of SARS-CoV-2 S protein contains an insertion of four amino acids providing a minimal furin cleavage site (RRAR685↓) (that is absent in SARS-CoV). Interestingly, the furin cleavage site has been implicated in increased viral pathogensis (Bestle et al., 2020b, Huggins, 2020). Processing of the spike protein by furin at the S1/S2 cleavage site is thought to occur following viral replication in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) (Hasan et al., 2020). The spike S2’ cleavage site of SARS-CoV-2 possesses a paired dibasic motif with a single KR segment (KR815↓) (as SARS-CoV) that is recognized by trypsin-like serine proteases such as TMPRSS2. The current data support a model for SARS-CoV-2 entry in which furin-mediated cleavage at the S1/S2 site pre-primes spike during biogenesis, facilitating the activation for membrane fusion by a second cleavage event at S2’ by TMPRSS2 following ACE2 binding (Bestle et al., 2020b, Johnson et al., 2020).

Virus	S1/S2 site	S2’ site
SARS-CoV-2	TNSPRRAR|SVA	PSKPSKR|SFIEDL
SARS-CoV	S----LLR|STS	PLKPTKR|SFIEDL

Camostat mesylate, an inhibitor of TMPRSS2, blocks SARS-CoV-2 infection of lung cells like Calu-3 cells but not Huh7.5 and Vero E6 cells. Cell entry was assessed using a viral isolate and viral pseudotypes (artificial viruses) expressing the COVID-19 spike (S) protein. The ability of the viral pseudotypes (expressing S protein from SARS-CoV and SARS-CoV-2) to enter human and animal cell lines was demonstrated, showing that SARS-CoV-2 can enter similar cell lines as SARS-CoV. Amino acid analysis and cell culture experiments showed that, like SARS-CoV, SARS-CoV-2 spike protein binds to human and bat angiotensin-converting enzyme 2 (ACE2) and uses a cellular protease TMPRSS2 for priming. Priming activates the spike protein to facilitate viral fusion and entry into cells. Cell culture experiments were performed using immortalized cell lines and primary human lung cells (Hoffmann et al., 2020b, Rahman et al., 2020).

 

Spike binding to neuropilin-1:

Neuropilin-1 (NRP1) is a transmembrane glycoprotein that serves as a cell surface receptor for semaphorins and various ligands involved in angiogenesis in vertebrates. NRP1 is expressed in neurons, blood vessels (endothelial cells), immune cells and many other cell types in the mammalian body (maternal fetal interface) and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function (Raimondi et al., 2016).  NRP1 is well described as a co-receptor for members of the class 3 semaphorins (SEMA3) or vascular endothelial growth factors (VEGFs) (Gelfand et al., 2014). Structurally, NRP1 comprises seven sub-domains, of which the first five are extracellular; two CUB domains (a1 and a2), two coagulation factor V/VIII domains (FV/VIII; b1 and b2) and a meprin, A5 μ-phosphatase domain (MAM; c). NRP1 contains only a short cytosolic tail with a PDZ-binding domain lacking internal signaling activity. The different ligand families bind to different sites of NRP1; SEMA3A binding requires the first three sub-domains of NRP1 (a1, a2, and b1), whereas binding of VEGF-A requires the b1 and b2 domains (Muhl et al., 2017). Additional studies conducted by means of in silico computational technology to identify and validate inhibitors of the interaction between NRP1 and SARS-CoV-2 Spike protein are reported in (Perez-Miller et al., 2020).  Represents a schematic picture of VEGF-A triggered phosphorylation of VEGF-R2. Screening of NRP-1/VEGF-A165 inhibitors by in-cell Western (Perez-Miller et al., 2020).v NRP1 acts as a co-receptor for SARS-CoV-2.

NRP1 is a receptor for furin-cleaved SARS-CoV-2 spike peptide (Cantuti-Castelvetri et al., 2020, Daly et al., 2020, Johnson et al., 2020). Blockade of NRP1 reduces infectivity and entry, and alteration of the furin site leads to loss of NRP1 dependence, reduced replication in Calu3, augmented replication in Vero E6, and attenuated disease in a hamster pathogenesis disease model (Johnson et al., 2020). In fact, a small sequence of amino acids was found that appeared to mimic a protein sequence found in human proteins that interact with NRP1. The spike protein of SARS-CoV-2 binding with NRP1 aids viral infection of human cells. This was confirmed by applying a range of structural and biochemical approaches to establish that the spike protein of SARS-CoV-2 does indeed bind to NRP1. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic RRAR C-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors. It was reported that the S1 CendR motif directly bound NRP1 by X-ray crystallography and biochemical approaches. Blocking this interaction using RNAi or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture (Daly et al., 2020).

NRP1, known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was revealed by a monoclonal blocking antibody against NRP1. It was found that a SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells faced the nasal cavity (Cantuti-Castelvetri et al., 2020). Furthermore, it has been found that NRP1 is a new potential SARS‑CoV‑2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID‑19.  Preclinical studies have suggested that NRP1, a transmembrane receptor that lacks a cytosolic protein kinase domain and exhibits high expression in the respiratory and olfactory epithelium, may also be implicated in COVID‑19 by enhancing the entry of SARS‑CoV‑2 into the brain through the olfactory epithelium. NRP1 is also expressed in the CNS, including olfactory‑related regions such as the olfactory tubercles and paraolfactory gyri. Supporting the potential role of NRP1 as an additional SARS‑CoV‑2 infection mediator implicated in the neurologic manifestations of COVID‑19. Accordingly, the neurotropism of SARS‑CoV‑2 via NRP1‑expressing cells in the CNS merits further investigation (Davies et al., 2020).

 

Up-regulation of NRP1 protein in diabetic kidney cells hints at its importance in a population at risk of severe COVID-19. Involvement of NRP-1 in immune function is compelling, given the role of an exaggerated immune response in disease severity and deaths due to COVID-19. NRP-1 has been suggested to be an immune checkpoint of T cell memory. It is unknown whether involvement and up-regulation of NRP-1 in COVID-19 may translate into disease outcome and long-term consequences, including possible immune dysfunction (Mayi et al., 2021).

The main feature of NRP1 co-receptor is to form complexes with multiple other receptors. Hence, there is a competition between receptors to complex with NRP-1, which may determine their abilities both quantitatively and qualitatively to transduce signals. It is tempting to hypothesize that the occupancy of NRP-1 with one receptor may thus decrease its availability for virus entry. Recent proteomics work has shown that NRP-1 can form a complex with the α7 nicotinic receptor in mice. Both receptors are expressed in the human nasal and pulmonary epithelium (Mayi et al., 2021).

NRP1, is highly expressed in the respiratory and olfactory epithelium; it is also expressed in the CNS, including olfactory related regions such as the olfactory tubercles and paraolfactory gyri (Davies et al., 2020).

More information on tissue distribution and protein expression of NRP1 can be found in https://www.proteinatlas.org/ENSG000000992 50-NRP1

Spike entry via lysosomal cathepsins and endocytosis:

Evidence shows the role of TMPRSS2 and other serine proteases in activating the coronavirus spike protein for plasma membrane fusion. However, studies using various cell culture systems showed that SARS-CoV2 could enter cells via an alternative endosomal–lysosomal pathway. Evidence came from studies demonstrating that lysosomotropic agents reduced SARS-CoV replication in cells lacking TMPRSS2 and other studies, using highly potent and specific small-molecule cathepsin inhibitors, to understand the role of cathepsins in processing and activating the spike for membrane fusion, mainly of cathepsin L (one of the 11 cathepsins) (Rossi et al., 2004, Simmons et al., 2005). SARS-CoV-2 and other coronaviruses can establish infection through endosomal entry in commonly used in vitro cell culture systems. Of relevance, inhibitors of the endosomal pathway, as the cathepsin inhibitor Z-FA-FMK and PIKfyve inhibitor apilimod, blocked viral entry in Huh7.5 and Vero E6 cells but not Calu-3 cells.

Viral entry leads to delivery of virion proteins and translation of viral proteins immediately:

Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [Kim D, et al., 2020]. The virus contains a 29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively that contain cis-acting secondary RNA structures essential for RNA synthesis [Huston N. C. et al., 2021]. The genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [Budzilowicx C.J., et al., 1985]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [Snijder E. J., et al., 2003]. Preceding each ORF there are other TRSs called the body TRS (TRS B). The 5′ two-thirds of the genome contains two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [Di H., et al., 2018]. Genome expression starts with the translation of two large ORFs of the 5’ two-thirds: ORF1a of 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a programmed (- 1) ribosomal frameshifting [Snijder E. J., et al., 2003], yielding pp1a and pp1ab. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a, nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain [Sacco M. D. et al., 2020]. The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication and transcription complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [Klein S., et al., 2021, Snijder E. J., et al., 2020, V'Kovski P. , et al., 2021]. This association leads to replication factories or organelles, that are originate new membranous structures that are observed by electron mciroscopy . They are a feature of all coronaviridae and the site of viral replication and transcription hidden from innate immune molecules.

How it is Measured or Detected

SARS-CoV2 entry can be determined by many different ways:

1) quantitative RT-PCR specific to the subgenomic mRNA of the N transcript, in cells manipulated with host factors that express of not TMPRSS2, cathepsinL, neuropilin-1, hACE2 [Glowacka I, et al. (2011)], or exogenous addition of HAT or furin.

2) using spike-pseudotyped viral particles expressing GFP/luciferase/bgalactosidase and comparing with vesicular stomatitis virus G seudotyped particles expressing the same reporter analysed in manipulated cultured with cell lines, followed by determining fluorescence, biolumincescence, luciferase activity in cell lysates  [Hoffmann M, et al. (2020)].

TMPRSS2:

TMPRSS2 gene expression can be measured by RNAseq and microarray (Baughn et al., 2020).

Expression levels of TMPRSS2 can be measured by RNA in situ hybridization (RNA-ISH) (Qiao et al., 2020)

NRP-1:

Several methods have been identified in the literature for measuring and detecting NRP1 receptor binding. Briefly described:

1. X-ray crystallography  and biochemical approaches help to show that the S1 CendR motif directly bound NRP1 (1).  Binding of the S1 fragment to NRP1 was assessed and ability of SARS-CoV-2 to use NRP1 to infect cells was measured in angiotensin-converting enzyme-2 (ACE-2)-expressing cell lines by knocking out NRP1 expression, blocking NRP1 with 3 different anti-NRP1 monoclonal antibodies, or using NRP1 small molecule antagonists (Centers for Disease Control and Prevention, 2020, Daly et al., 2020).

Key findings (Centers for Disease Control and Prevention, 2020, Daly et al., 2020):

• The S1 fragment of the cleaved SARS-CoV-2 spike protein binds to the cell surface receptor neuropilin-1 (NRP1).

• SARS-CoV-2 utilizes NRP1 for cell entry as evidenced by decreased infectivity of cells in the presence of: NRP1 deletion (p <0.01). Three different anti-NRP1 monoclonal antibodies (p <0.001). Selective NRP1 antagonist, EG00229 (p <0.01).

2. 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).

Key findings (Cantuti-Castelvetri et al., 2020, Centers for Disease Control and Prevention, 2020):

• Infectivity of cells expressing angiotensin converting enzyme-2 (ACE2, receptor for SARS-CoV-2), transmembrane protease serine-2 (TSS2, primes the Spike [S] protein), and neuropilin-1 (NRP1) with pseudovirus expressing the SARS-CoV-2 S1 protein was approximately 3-fold higher than in cells expressing either ACE2 or TSS2 alone (p<0.05).

• Analysis of autopsy tissue from COVID-19 patients showed co-localization of the SARS-CoV-2 spike (S) protein and NRP1 in olfactory and respiratory epithelium.

Virtual screen of nearly 0.5 million compounds against the NRP-1 CendR site, resulting in nearly 1,000 hits. A pharmacophore model was derived from the identified ligands, considering both steric and electronic requirements. Preparation of receptor protein and grid for virtual screening, docking of known NRP-1 targeting compounds, ELISA based NRP1-VEGF-A165 protein binding assay; more details on methodology in the referenced paper (Perez-Miller et al., 2020)

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UNIPROTKB - O14786 (NRP1_HUMAN)

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Event: 1901: Interferon-I antiviral response, antagonized by SARS-CoV-2

Short Name: IFN-I response, antagonized

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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).

Key Event Description

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 (doi:https://doi.org/10.1016/j.cell.2020.04.01). 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 (Kim et al. 2020; O’Leary et al. 2020). Viral transcription and replication is explained in depth in KE1847. 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 (doi:https://doi.org/10.1016/j.cell.2020.04.01). 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 (Amor et al. 2020). Extensive protein-protein interaction (Gordon et al. 2020) and viral protein-host RNA interaction networks have been demonstrated between the viral NSPs and accessory proteins and host molecules. 

This key event is focused on the specific viral:host protein interactions within the infected cell that are involved in the IFN-I antiviral response pathways. IFN-I is the main component of the innate immune system that is suppressed by the SARS-CoV-2 coronavirus early in infection. 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 (Quarleri and Delpino, 2021).

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 INFβ 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).

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.

 

Gene	Protein	Function	Role in early innate immune evasion
ORF1a	NSP1	NSP1 antagonizes interferon induction to suppress host antiviral response.	DNA Polymerase Alpha Complex: Regulates the activation of IFN-I through cytosolic 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).
	NSP2	While not essential for viral replication, deletion of NSP2 diminishes viral growth and RNA synthesis	Translation repression through binding GIGYF2and EIF4E2 (4EHP) (Gupta et al. 2021)
	NSP3	Papain-like protease (Plpro); Cleaves the ORF1a and 1ab polypeptides	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)
	NSP5	3C-like protease (3CLpro); Cleaves the ORF1a and 1ab polypeptides	Binds STING (Rui et al. 2021)
	NSP6	Limits autophagosome expansion	Suppresses IFN-I expression: Binds TBK-1 to supress IRF3 phosphorylation (Xia et al. 2020; Quarleri and Delpino, 2021)
	NSP7	In complex with NSP8 forms primase as part of multimeric RNA-dependent RNA replicase (RdRp)
	NSP8	Replication complex with NSP7, NSP9 and NSP12	Binds SRP72/54/19 (Gordon et al. 2020) and 7SL RNA to block IFN membrane transport (Banerjee et al. 2020)
	NSP9	Replication complex with NSP7, NSP8 and NSP12	Binds SRP and 7SL RNA with NSP8 to block IFN membrane transport (Banerjee et al. 2020)
ORF1b	NSP13	Helicase and triphosphatase that initiates the first step in viral mRNA capping.	Binds TBK1 (Xia et al. 2020)
	NSP14		Induces lysosomal degradation of IFNAR1 (Hayn et al. 2021)
ORF2	Spike (S)	ACE2 interaction, cell entry
ORF3a	ORF3a	Interacts with M, S, E and 7a; form viroporins; immune evasion	Binds STING (Rui et al 2021)
ORF4	Envelope (E)	Viral assembly and budding
ORF5	Membrane (M)	Viral assembly	Interacts with RIG-I and MAVS sensors of viral RNA (Fu et al 2020)
ORF6	ORF6	Viral pathogenesis and virulence; interacts with ORF8; promotes RNA polymerase activity	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
ORF7a	ORF7a	Interacts with S, ORF3a; immune evasion	Suppresses STAT2 phosphorylation to block IFN-I response (Xia and Shi, 2020).
ORF7b	ORF7b	Structural component of virion	Suppresses STAT1 and STAT2 phosphorylation to block IFN-I response (Xia and Shi, 2020)
ORF8	ORF8	Immune evasion	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)
ORF9	Nucleocapsid (N)	Stabilizes viral RNA	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)
ORF9b	ORF9b	Immune evasion	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)

How it is Measured or Detected

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:

Interferon I decrease (Xia et al. 2020):

• IFN-I production and signaling luciferase reporter assays
• Co-immunoprecipitation and western blot
• Indirect immunofluorescence assays
• DNA assembly and RNA transcription of a luciferase replicon for SARS-CoV-2
• Replicon RNA electroporation and luciferase reporter assay

SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling (Wu et al. 2021)

• Viral- and host-protein-specific antibodies
• Immunoprecipitation
• Immunofluorescent microscopy
• Dual-luciferase reporter assays
• Fluorescence quantification immunoblotting

SARS-CoV-2-Human Protein-Protein Interaction Map (Gordon et al. 2020)

• Cloning and expression of viral proteins via plasmid transfection into HEK293T cell line
• Protein affinity purification using MagStrep beads with detection by anti-strep western blot of cell lysate
• Global analysis of SARS-CoV-2 host interacting proteins using affinity purification-mass spectrometry

 

References

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Andres et al. 2020. SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.18.256776

Banerjee et al. 2020. SARS-CoV-2 disrupts splicing, translation, and protein trafficking to supress host defenses. Cell 183, 1325–1339. https://doi.org/10.1016/j.cell.2020.10.004

Cascarina and Ross, 2020. A proposed role for the SARS-CoV-2 nucleocapsid protein in the formation and regulation of biomolecular condensates. The FASEB Journal, 34:9832–9842. DOI: 10.1096/fj.202001351

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.

Chen et al. 2021. SARS-CoV-2 Nucleocapsid Protein Interacts with RIG-I and Represses RIG-Mediated IFN-β Production. Viruses. 13(1):47. https://doi.org/10.3390/v13010047

Conceicao 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

Damas et al. 2020. Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates. PNAS vol. 117 no. 36:22311–22322 www.pnas.org/cgi/doi/10.1073/pnas.2010146117 

Fu et al. 2021. SARS-CoV-2 membrane glycoprotein M antagonizes the MAVS-mediated innate antiviral response. Cell Mol Immunol 18: 613–620. https://doi.org/10.1038/s41423-020-00571-x

Gordon et al. 2020. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 483:459-473. https://doi.org/10.1038/s41586-020-2286-9

Gupta et al. 2021. CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes. bioRxiv 2021.05.10.443524; doi: https://doi.org/10.1101/2021.05.10.443524

Han et al. 2020. SARS-CoV-2 ORF9b Antagonizes Type I and III Interferons by Targeting Multiple Components of RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING Signaling Pathways. bioRX https://doi.org/10.1101/2020.08.16.252973

Hayn et al. 2021. Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities. Cell Reports 35, 109126. https://doi.org/10.1016/j.celrep.2021.109126

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

Kato et al. 2021. Overexpression of SARS-CoV-2 protein ORF6 dislocates RAE1 and NUP98 from the nuclear pore complex. Biochemical and Biophysical Research Communications 536:59-66 https://doi.org/10.1016/j.bbrc.2020.11.115

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

Kim et al. 2020. The Architecture of SARS-CoV-2 Transcriptome. Cell 181, 914–921. https://doi.org/10.1016/j.cell.2020.04.011

Li et al. 2020. The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway. Virus Research vol. 286. https://doi.org/10.1016/j.virusres.2020.198074

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. https://doi.org/10.1038/s41564-021-00884-1

Moustaqil et al. 2021. SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species, Emerging Microbes & Infections, 10:1, 178-195. https://doi.org/10.1080/22221751.2020.1870414

Mu et al. 2020. SARS-CoV-2 N protein antagonizes type I interferon signaling by suppressing phosphorylation and nuclear translocation of STAT1 and STAT2. Cell Discov 6, 65. https://doi.org/10.1038/s41421-020-00208-3

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. https://doi.org/10.3390/ijms22010386

Quarleri and Delpino, 2020. Type I and III IFN-mediated antiviral actions counteracted by SARS-CoV-2 proteins and host inherited factors. Cytokine & Growth Factor Reviews, 58: 55-65. https://doi.org/10.1016/j.cytogfr.2021.01.003

Rashid et al. The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon beta. Virus Research 296, 198350. https://doi.org/10.1016/j.virusres.2021.198350

Ren et al. 2020. The ORF3a protein of SARS-CoV-2 induces apoptosis in cells. Cellular & Molecular Immunology 17:881–883; https://doi.org/10.1038/s41423-020-0485-9

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. https://doi.org/10.1038/s41392-021-00515-5

Schubert et al. 2020. SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nature Structural & Molecular Bio. 27:959-966. https://doi.org/10.1038/s41594-020-0511-8

Shin et al. 2020. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature 587: 657–662. https://doi.org/10.1038/s41586-020-2601-5

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

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Event: 1847: Increased SARS-CoV-2 production

Short Name: SARS-CoV-2 production

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Broad mammalian host range has been demonstrated based on spike protein tropism for and binding to ACE2 [Conceicao et al. 2020; Wu et al. 2020] and cross-species ACE2 structural analysis [Damas et al. 2020]. No literature has been found on primary translation and molecular interactions of nsps in non-human host cells, but it is assumed to occur if the virus replicates in other species.

Very broad mammalian tropism: human, bat, cat, dog, civet, ferret, horse, pig, sheep, goat, water buffalo, cattle, rabbit, hamster, mouse

Key Event Description

This KE1847 "Increase coronavirus production" deals with how the genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is translated, replicated, and transcribed in detail, and how the genomic RNA (gRNA) is packaged, and the virions are assembled and released from the cell. 

Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [D. Kim et al.]. The virus contains a 29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively [E. J. Snijder et al.] that contain cis-acting secondary RNA structures essential for RNA synthesis [N. C. Huston et al.]. The genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [C.J. Budzilowicx et al.]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [E. J. Snijder et al.]. Preceding each ORF there are other TRSs called the body TRS (TRS B). The 5′ two-thirds of the genome contains two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [H. Di et al.]. Genome expression starts with the translation of two large ORFs of the 5’ two-thirds: ORF1a of 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a programmed (- 1) ribosomal frameshifting [E. J. Snider et al.], yielding pp1a and pp1ab. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a, nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain [M. D. Sacco et al.]. The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication and transcription complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [S. Klein et al., E. J. Snijder et al., P. V'Kovski, et al.]. Besides replication, which yields the positive-sense gRNA, the replicase also mediates transcription leading to the synthesis of a nested set of subgenomic (sg) mRNAs to express all ORFs downstream of ORF1b that encode structural and accessory viral proteins. These localize to the 3′ one-third of the genome, as stated above, and result in a 3′ coterminal nested set of 7–9 mRNAs that share ~70–90 nucleotide (nt) in the 5′ leader and that is identical to the 5′ end of the genome [P. Liu, and J. Leibowitz]. sgRNAs encode conserved structural proteins (spike protein [S], envelope protein [E], membrane protein [M], and nucleocapsid protein [N]), and several accessory proteins. SARS-CoV-2 is known to have at least six accessory proteins (3a, 6, 7a, 7b, 8, and 10). Overall the virus is predicted to express 29 proteins [D. Kim et al.]. The gRNA is packaged by the structural proteins to assemble progeny virions.

Viral translation:

SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of ~30 kb, sequence orientation in a 5’ to 3’ direction typical of positive sense and reflective of the resulting mRNA [D. Kim et al.]. The SARS-CoV-2 genome contains a 5’-untranslated region (UTR; 265 bp), ORF1ab (21,289 bp) holding two overlapping open reading frames (13,217 bp and 21,289 bp, respectively) that encode two polyproteins [D. Kim et al.]. Other elements of the genome include are shown below [V. B. O'Leary et al.]. The first event upon cell entry is the primary translation of the ORF1a and ORF1b gRNA to produce non-structural proteins (nsps).

This is completely dependent on the translation machinery of the host cell. Due to fewer rare “slow-codons”, SARS-CoV-2 may have a higher protein translational rate, and therefore higher infectivity compared to other coronavirus groups [V. B. O'Leary et al.]. The ORF1a produces polypeptide 1a (pp1a, 440–500 kDa) that is cleaved into nsp-1 through nsp-11. A -1 ribosome frameshift occurs immediately upstream of the ORF1a stop codon, to allow translation through ORF1b, yielding 740–810 kDa polypeptide pp1ab, which is cleaved into 15 nsps [D. Kim et al.]. Two overlapping ORFs, ORF1a and ORF1b, generate continuous polypeptides, which are cleaved into a total of 16 so-called nsps [Y Finkel et al.]. Functionally, there are five proteins from pp1ab (nsp-12 through nsp-16) as nsp-1-11 are duplications of the proteins in pp1a due to the ORF overlap. The pp1a is approximately 1.4–2.2 times more expressed than pp1ab. After translation, the polyproteins are cleaved by viral proteases nsp3 and nsp5. Nsp5 protease can be referred to as 3C-like protease (3CL^pro) or as main protease (M^pro), as it cleaves the majority of the polyprotein cleavage sites. [H.A. Hussein et al.] Nsp1 cleavage is quick and nsp1 associates with host cell ribosomes and results in host cellular shutdown, suppressing host gene expression [M. Thoms et al.]. Fifteen proteins, nsp2–16 constitute the viral RTC. They are targeted to defined subcellular locations and establish a network with host cell factors. Nsp2–11 remodel host membrane architecture, mediate host immune evasion and provide cofactors for replication, whilst nsp12–16 contain the core enzymatic functions involved in RNA synthesis, modification and proofreading [P. V'Kovski et al.].  nsp-7 and nsp-8 form a complex priming the RNA-dependent RNA polymerase (RdRp or RTC) - nsp-12. nsp14 provides a 3′–5′ exonuclease activity providing RNA proofreading function. Nsp-10 composes the RNA capping machinery nsp-9. nsp13 provides the RNA 5′-triphosphatase activity. Nsp-14 is a N7-methyltransferase and nsp-16 the 2′-O-methyltransferase. Many of the nsps have multiple functions and many viral proteins are involved in innate immunity inhibition. Nsp-3 is involved in vesicle formation along with nsp-4 and nsp-6 where viral replication occurs. Interactions between SARS-CoV-2 proteins and human RNAs thwart the IFN response upon infection: nsp-16 binds to U1 and U2 splicing RNAs to suppress global mRNA splicing; nsp-1 binds to 40S ribosomal RNA in the mRNA entry channel of the ribosome to inhibit host mRNA translation; nsp-8 and nsp-9 bind to the 7SL RNA to block protein trafficking to the cell membrane [A. K. Banerjee et al.]. Xia et al. [H. Xia et al.] found that nsp-6 and nsp-13 antagonize IFN-I production via distinct mechanisms: nsp-6 binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, and nsp-13 binds and blocks TBK1 phosphorylation.

 

Viral transcription and replication:

Viral transcription and replication occur at the viral replication organelle (RO) [E. J. Snijder et al.]. The RO is specifically formed during infection by reshaping ER and other membranes, giving rise to small spherular invaginations, and large vesiculotubular clusters, consisting of single- and/or double-membrane vesicles (DMV), convoluted membranes (CM) and double-membrane spherules invaginating from the ER  [S. Klein et al., E. J. Snijder et al.]. There is some evidence that DMV accommodate viral replication which is based on radiolabelling viral RNA with nucleoside precursor ([5-³[H]uridine) and detection by EM autoradiography [E. J. Snijder et al.].

Viral replicative proteins and specific host factors are recruited to ROs [E. J. Snijder et al.]. RNA viral genome is transcribed into messenger RNA by the viral RTC [P. Ahlquist et al.]. Viral RTC act in combination with other viral and host factors involved in selecting template RNAs, elongating RNA synthesis, differentiating genomic RNA replication from mRNA transcription, modifying product RNAs with 5’ caps or 3’ polyadenylate [P. Ahlquist et al.]. Positive-sense (messenger-sense) RNA viruses replicate their genomes through negative-strand RNA intermediates [M. Schwartz et al.]. The intermediates comprise full-length negative-sense complementary copies of the genome, which functions as templates for the generation of new positive-sense gRNA, and a nested set of sg mRNAs that lead to the expression of proteins encoded in all ORFs downstream of ORF1b. The transcription of coronaviruses is a discontinuous process that produces nested 3′ and 5′ co-terminal sgRNAs. Of note, the synthesis of sg mRNAs is not exclusive to the order Nidovirales but a discontinuous minus-strand synthesis strategy to produce a nested set of 3′ co-terminal sg mRNAs with a common 5′ leader in infected cells are unique features of the coronaviruses and arteriviruses [W. A. Miller and G. Koev.]. Of note, the produced genomic RNA represents a small fraction of the total vRNA [N. S. Ogando et al.].

The discontinuous minus-strand synthesis of a set of nested sg mRNAs happens during the synthesis of the negative-strand RNA, by an interruption mechanism of the RTC as it reads the TRS-B preceding each gene in the 3′ one-third of the viral genome [I. Sola, F. Almazan et al., I. Sola, J. L. Moreno, et al.]. The synthesis of the negative-strand RNA stops and is re-initiated at the TRS-L of the genome sequence close from the 5′ end of the genome [H. Di et al.]. Therefore, the mechanism by which the leader sequence is added to the 5' end requires that the RTC switches template by a jumping mechanism. This interruption process involves the interaction between complementary TRSs of the nascent negative-strand RNA TRS-B and the positive-strand gRNA at the positive-sense TRS-L. The TRS-B site has a 7-8 bp conserved core sequence (CS) that facilitates RTC template switching as it hybridizes with a near complementary CS in the TRS-L [I. Sola, F. Almazan et al. I. Sola, J. L. Moreno, et al.]. Upon re-initiation of RNA synthesis at the TRS-L region, a negative-strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs. This means that all sg mRNAs as well as the genomic RNA share a common 5' sequence, named leader sequence [X. Zhang et al.]. This programmed template switching leads to the generation of sg mRNAs with identical 5' and 3' sequences, but alternative central regions corresponding to the beginning of each structural ORF [I. Sola et al. 2015, S. G. Sawicki et al., Y. Yang et al.]. Of note, the existence of TRSs also raises the possibility that these sites are at the highest risk of recombining through TRS-B mediated template switching [Y. Yang]. The set of sg mRNAs is then translated to yield 29 identified different proteins [F. Wu et al.], although many papers have identified additional ORFs [D. Kim et al.. Y. Finkel et al., A. Vandelli et al.]. The translation of the linear single-stranded RNA conducts to the generation of the following proteome: 4 are structural proteins, S, N, M, and E; 16 proteins nsp: the first 11 are encoded in ORF1a whereas the last 5 are encoded in ORF1ab. In addition, 9 accessory proteins named ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10 have been identified [F. Wu et al.]. At the beginning of infection, there is the predominant expression of the nsp that result from ORF1a and ORF1ab, however, at 5 hpi, the proteins encoded by the 5′ last third are found in higher amounts, and the nucleoprotein is the protein found in higher levels [Y. Finkel et al.].

 

Viral assembly:

The final step of viral production requires virion assembly and this process is not well explored for SARS-CoV-2. For example, the role of the structural proteins of SARS-CoV-2 in virus assembly and budding in not known. In general, all beta-coronavirus structural proteins assemble at the endoplasmic reticulum (ER)-to-Golgi compartment [J. R. Cohen et al., A. Perrier et al.] and viral assembly requires two steps: Genome packaging which is a process in which the SARS-CoV-2 gRNA must be coated by the viral protein nucleoprotein (N) protein, forming viral ribonucleoprotein (vRNPs) complexes, before being selectively packaged into progeny virions [P. V'Kovski et al.], a step in which vRNPs bud into the lumen of the ER and the ER-Golgi intermediate compartment (ERGIC) [N. S. Ogando et al.]. This results in viral particles enveloped with host membranes containing viral M, E, and S transmembrane structural proteins that need to be released from the cell.

SARS-CoV-2 gRNA packaging involves the N protein. The N protein of human coronaviruses is highly expressed in infected cells. It is considered a multifunctional protein, promoting efficient sub-genomic viral RNA transcription, viral replication, virion assembly, and interacting with multiple host proteins [P. V'Kovski et al., D. E. Gordon et al., R. McBride, and M. van Zyl, B. C.]. In relation to transcription and replication, the N protein could provide a cooperative mechanism to increase protein and RNA concentrations at specific localizations S. Alberti, and S. Carra, S. F. Banani et al.], and this way organize viral transcription. Five studies have shown that N protein undergoes liquid-liquid phase separation (LLPS) in vitro [A. Savastano et al., H. Chen et al., C. Iserman et al., T. M. Perdikari et al., J. Cubuk et al.], dependent on its C-terminal domain (CTD) [H. Chen et al.]. It has been hypothesised that N could be involved in replication close to the ER and in packaging of gRNA into vRNPs near the ERGIC where genome assembly is thought to take place [A. Savastano et al.], but so far this is still speculative. Phosphorylation of N could adjust the physical properties of condensates differentially in ways that could accommodate the two different functions of N: transcription and progeny genome assembly [A. Savastano et al., C. Iserman et al., C. R. Carlson et al.].

The ERGIC constitutes the main assembly site of coronaviruses [S. Klein et al., E. J. Snijder et al., L. Mendonca et al.] and budding events have been seen by EM studies. For SARS-CoV-2, virus-budding was mainly clustered in regions with a high vesicle density and close to ER- and Golgi-like membrane arrangements [S. Klein et al., E. J. Snijder et al., L. Mendonca et al.]. The ectodomain of S trimers were found facing the ERGIC lumen and not induce membrane curvature on its own, therefore proposing that vRNPs and spike trimers [S. Klein et al.].

Finally, it has been shown that SARS-CoV-2 virions de novo formed traffic to lysosomes for unconventional egress by Arl8b-dependent lysosomal exocytosis [S. Ghosh et al.]. This process results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation [S. Ghosh et al.].

How it is Measured or Detected

Viral translation:

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation [Schubert et al. 2020]

• Sucrose pelleting binding assay to verify Nsp1–40S complex formation
• In vivo translation assay
• Transient expression of FLAG-Nsp1 in HeLa cells and puromycin incorporation assay

SARS-CoV-2 disrupts splicing, translation, and protein trafficking [Banerjee et al. 2020]

• SARS-CoV-2 viral protein binding to RNA
• Interferon stimulation experiments
• Splicing assessment experiments
• IRF7-GFP splicing reporter, 5EU RNA labeling, capture of biotinylated 5EU labeled RNA

Membrane SUnSET assay for transport of plasma membrane proteins to the cell surface

Viral transcription:

The mRNA transcripts are detected by the real-time reverse transcription-PCR (RT-PCR) assay. Several methods targeting the mRNA transcripts have been developed, which includes the RT-PCR assays targeting RdRp/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 [Chan et al.]. RT-PCR assays detecting SARS-CoV-2 RNA in saliva include quantitative RT-PCR (RT-qPCR), direct RT-qPCR, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) [Nagura-Ikeda M, et al.]. The viral mRNAs are reverse-transcribed with RT, followed by the amplification by PCR.

Viral replication:

viral replication is measured by RT-qPCR in infected cells, formation of liquid organelles is assessed in vitro reconstitution systems and in infected cells. Labelling by radioactive nucleosides.

Viral production:

Plaque assays, infectivity assays, RT-qPCR to detect viral RNA in released virions, sequencing to detect mutations in the genome, electron microscopy.

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List of Adverse Outcomes in this AOP

Event: 1939: Viral infection and host-to-host transmission, proliferated

Short Name: Viral infection, proliferated

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Life Stage and Sex. 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).

Taxonomic. 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 (Bos taurus; Ulrich et al., 2020), bank vole (Myodes glareolus; Ulrich et al., 2021), and domestic dogs (Canis lupus familiaris; Bosco-Lauth, Hartwig et al., 2021). Several experimentally exposed species did not become infected and hence, did not shed the virus, including coyote (Canis latrans; Porter et al., 2022), pig (Sus scrofa; Schlottau et al., 2020), and in one study by Bosco-Lauth, Root et al. (2021) the house mouse (Mus musculus), Wyoming ground squirrel (Urocitellus elegans), fox squirrel (Sciurus niger), black-tailed prairie dog (Cynomys ludovicianus), raccoon (Procyon lotor), and cottontail rabbits (Sylvilagus sp.).

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.

Table 1. Species that transmit or shed infectious SARS-CoV-2 virus.
Common name	Species	References
White-tailed deer	Odocoileus virginianus texanus	Cool, 2022; Palmer, 2021; Martins, 2022; Chandler, 2021; Kuchipudi, 2022; Pickering, 2022; McBride, 2023
Cat	Felis catus	Bosco-Lauth, Hartwig, et al. 2021
European (NZ white) rabbit	Oryctolagus cuniculus	Myktykyn, 2021
Golden (Syrian) hamster	Mesocricetus auratus	Sia, 2020; Hoagland, 2021
Raccoon dog	Nyctereutes procyonoides	Freuling 2020
European mink	Mustela lutreola	Oude Munnink, 2020; Mastutik, 2022; Fenollar, 2021; Molenaar 2022
American mink	Neovison vison	Ip, 2021; Harrington, 2021
Striped skunk	Mephitis mephitis	Bosco-Lauth, Root, et al. 2021
Deer mouse	Peromyscus maniculatus bairdii	Bosco-Lauth, Root, et al. 2021
Bushy-tailed wood rat	Neotoma cinerea	Bosco-Lauth, Root, et al. 2021
Tree shrew	Tupaia belangeris	Zhao 2020
Ferret	Mustela putorius furo	Schlottau 2020; Kim, 2020
Red fox	Vulpes vulpes	Porter, 2022, Yes/Jemersic, 2021, No

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 10⁵ 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).

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 (Neovison vison) 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.

Several researchers have reported wide-spread infection and transmission among wild and captive white-tailed deer:

• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.

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.

Key Event Description

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). 

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.

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 Pfizer-BioNTech (BNT162b2) and Moderna vaccines in December 2020 (Katella, 2023). The effectiveness of the vaccines is monitored by the U.S. Centers for Disease Control (CDC, 2023) with criteria as follows:

• Hospitalization for COVID-19 or medically attended COVID-19 (e.g., emergency department visits)
• Death due to COVID-19
• Post-COVID Conditions and multisystem inflammatory syndrome (MIS)
• Symptomatic SARS-CoV-2 infection

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).

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).

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.

Species of Potential Concern

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.

Exposure and Transmission Routes

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.

Spillover Conditions

Conditions that allow for exposure and transmission across species:

• Close proximity of animal communities (bats to potential intermediate hosts; wildlife to domestic animal farms).
• Direct human contact with wildlife (Kreuder Johnson et al. 2015), including:
  • Zoos, wildlife farms, domesticated animal farms, feeding and animal care;
  • Hunting and dressing wild game;
  • 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);
  • Wet markets where live animals or bush meat are traded;
  • 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.
• 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).
• 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.

How it is Measured or Detected

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:

• ELISA, Indirect immunofluorescence assay (IIFA) for antibodies (Schlottau et al. 2020; Freuling et al. 2020)
• Virus neutralization test (VNT) for antibodies (Schlottau et al. 2020; Freuling et al. 2020)
• Quantitative reverse transcription PCR (qRT-PCR) for viral load (log10 genome copies) (Freuling et al. 2020)
• 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)
• Virus-specific immunoglobulin characterization (Freuling et al. 2020)
• SARS-CoV-2 spike protein neutralizing antibodies in saliva from animals that developed serum antibodies (Freuling et al. 2020)
• Serum sample, autopsy, histopathology for tissue lesions (Schlottau et al. 2020; Freuling et al. 2020)
• Viral whole genome sequencing (Kuchipudi et al., 2022)

Regulatory Significance of the AO

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.

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Appendix 2

List of Key Event Relationships in the AOP