key: cord-299093-zp07aqpm
authors: Harrison, Andrew G.; Lin, Tao; Wang, Penghua
title: Mechanisms of SARS-CoV-2 transmission and pathogenesis
date: 2020-10-14
journal: Trends Immunol
DOI: 10.1016/j.it.2020.10.004
sha: 
doc_id: 299093
cord_uid: zp07aqpm

The emergence of SARS-coronavirus 2 (SARS-CoV-2) marks the third highly pathogenic coronavirus to spill over into the human population. SARS-CoV-2 is highly transmissible with a broad tissue tropism that is likely perpetuating the pandemic. However, important questions remain regarding its transmissibility and pathogenesis. In this review, we summarize current SARS-CoV-2 research, with an emphasis on transmission, tissue tropism, viral pathogenesis, and immune antagonism. We further present advances in animal models that are important for understanding the pathogenesis of SARS-CoV-2, vaccine development, and therapeutic testing. When necessary, comparisons are made from studies with SARS to provide further perspectives on COVID-19, as well as draw inferences for future investigations.

infection. First, viral entry may heavily depend on the expression of TMPRSS2, as nearly undetectable amounts of ACE2 still support SARS-CoV entry, so long as TMPRSS2 is present [27] . Second, the mRNA expression of cellular genes such as ESCRT (endosomal sorting complex required for transport) machinery gene members (including CHMP3, CHMP5, CHMP1A, and VPS37B) related to pro-SARS-CoV-2 lifecycle is higher in a small population of human type II alveolar cells with abundant ACE2, relative to ACE2-deficient cells [28] . This suggests that SARS-CoV-2 hijacks a small population of type II alveolar cells with high expression of ACE2 and other pro-viral genes for its productive replication. Third, the lungas the main tropism of SARS-CoVs-may be contingent on the regulation of ACE2 at the transcriptional and protein levels [24, 29, 30] [25, 31] . For example, in human airway epithelial cells, ACE2 gene expression is upregulated by type I and II interferons [25, 31] during viral infection. Lastly, compared to other SARS-CoVs, SARS-CoV-2 Spike contains a unique insertion of RRAR at the S1/S2 cleavage site [17] [18] . This site can be pre-cleaved by furin, thus reducing the dependence of SARS-CoV-2 on target cell proteases (TMPRSS2/cathepsin L) for entry [17] [18] and potentially extending its cellular tropism, given that proteolytically active furin is abundantly expressed in human bronchial epithelial cells [32, 33] .

One of the distinctions between SARS-CoV and SARS-CoV-2 is the latter's ability to efficiently infect the upper respiratory tract (URT), such as nasopharyngeal (NP) and/or oropharyngeal (OP) tissues, possibly due to its higher affinity for ACE2, which is expressed in human nasal and oral tissues [34] [35] [23, 25, 36] . The readily detectable titers of SARS-CoV-2 in the URT mucus of COVID-19 patients during prodromal periods might contribute to explaining the more rapid and effective transmissibility of SARS-CoV-2 relative to SARS-CoV [37] .

Human CoVs often cause enteric infections, with variable degrees of pathogenicity [38] .

Indeed, ACE2 and TMPRSS2 are abundantly expressed within the human and many other mammalian intestinal tracts, specifically the brush border of intestinal enterocytes [23, 25, 26, 39] . Accordingly, gastrointestinal illness has been frequently reported in COVID-19 patients [40, 41] , consistent with the recovery of SARS-CoV from SARS patients' stool samples [42] , suggesting a potential fecal-oral route of transmission for these two CoVs. Of note, ~20% of COVID-19 patients examined have had detectable SARS-CoV-2 RNA in feces, even after respiratory symptoms subsided, suggesting that SARS-CoV-2 titers might be prolonged in the intestinal tract [41] . Although further testing is warranted, these data suggest the possibility that fecal-oral transmission of SARS-CoV-2 might occur. Evidently, robust epidemiological studies are needed to conclusively demonstrate if COVID-19 patients recovering from respiratory illness are able to spread SARS-CoV-2.

Human CoVs are transmitted primarily through respiratory droplets, but aerosol, direct contact with contaminated surfaces, and fecal-oral transmission were also reported during the SARS epidemic [43] [44] [45] . Early reports of patients with cough, lung ground glass opacities, and symptom progression to severe pneumonia, suggested communicability of SARS-CoV-2 via the respiratory route (Figure 2 ) [1] [2] [3] . Direct transmission by respiratory droplets is reinforced by productive SARS-CoV-2 replication in both the URT and LRT, and the increasing number of reports indicating human-to-human spread among close contacts exhibiting active coughing ( Figure 2 ) [35, [46] [47] [48] . So far, the basic reproduction number (R 0 ) is ~2.2, based on early case J o u r n a l P r e -p r o o f Journal Pre-proof tracking in the beginning of the pandemic, with a doubling time of 5 days [47] [49] . Furthermore, there is now evidence for non-symptomatic/pre-symptomatic spread of SARS-CoV-2, which is in contrast to the transmission dynamics of SARS-CoV [50] . This finding underscores the ability of SARS-CoV-2 to colonize and replicate in the throat during early infection [37, 51, 52] . Based on these apparent disparities in virus transmission, one study modeled the transmission dynamics of SARS-CoV-2 in pre-symptomatic individuals, and indicated that the pre-symptomatic R 0 has approached the threshold for sustaining an outbreak on its own (R 0 >1); by contrast, the corresponding estimates for SARS-CoV were approximately zero [49] . Similarly, asymptomatic spread of SARS-CoV-2 has been documented throughout the course of the pandemic [48] [51, [53] [54] [55] [56] . Understanding the relative importance of cryptic transmission to the current COVID-19 pandemic is essential for public health authorities to make the most comprehensive and effective disease control measures that include mask-wearing, contact tracing, and physical isolation.

For SARS-CoV-2, various modes of transmission have been proposed, including aerosol, surface contamination, fecal-oral route, representing confounding factors in the current COVID-19 pandemic; thus, their relative importance is still being investigated (Figure 2 ) [57] . Aerosol transmission (spread >1m) was implicated in the Amoy Gardens outbreak during the SARS epidemic, but the inconsistency of these findings in other settings suggested that SARS-CoV was likely an opportunistic airborne infection [43, 58] . Similarly, no infectious SARS-CoV-2 virions have been isolated, though viral RNA was detectable in the air of COVID-19 hospital wards [59] . Generation of experimental aerosols carrying SARS-CoV-2 (comparable to those that might be generated by humans) have offered the plausibility of airborne transmission, but the aerodynamic characteristics of SARS-CoV-2 during a natural course of infection is still an area of intense inquiry [60] . Nonetheless, deposition of virus-laden aerosols might contaminate J o u r n a l P r e -p r o o f objects (e.g. fomites) and contribute to human transmission events [59, 61] . Finally, fecal-oral transmission has also been considered as a potential route of human spread, but this route remains an enigma despite evidence of RNA-laden aerosols being found nearby toilet bowls, along with detectable SARS-CoV-2 RNA in rectal swabs during the precursor epidemic of COVID-19 in China [41, 59, 62] .

In general, common cold CoVs tend to cause mild URT symptoms and occasional gastrointestinal involvement (Figure 3) . On the contrary, infection with the highly pathogenic CoVs, including SARS-CoV-2, causes severe 'flu'-like symptoms that can progress to acute respiratory distress (ARDS), pneumonia, renal failure, and death [46, 48, 63, 64] . The most common symptoms are fever, cough and dyspnea, accounting for 83%, 82% and 31% of COVID-19 patients (n=99), respectively in one epidemiological study [65] . The incubation period in COVID-19 is rapid, ~5-6 days, versus 2-11 days in SARS-CoV infections [38, 47, 48] .

As the pandemic is progressing, it has become increasingly clear that COVID-19 encompasses not only rapid respiratory/gastrointestinal illnesses, but can have long-term ramifications such as myocardial inflammation [66] . Furthermore, severe COVID-19 is not restricted to the aged population as initially reported; children and young adults are also at risk [67] . From a diagnostic perspective, COVID-19 presents with certain 'hallmark' laboratory and radiological indices, which can be helpful in assessing disease progression ( Table 1) 

It is widely accepted that the aging process predisposes individuals to certain infectious diseases [68] . In the case of COVID-19, older age is associated with greater COVID-19 morbidity, admittance to the ICU, progressing to ARDS, higher fevers and greater mortality rates [69] [70, 71] . Moreover, lymphocytopenia, neutrophilia, elevated inflammation-related indices, and coagulation-related indicators have been consistently reported in older (≥65 years old) relative to young and middle-aged COVID-19 patients [ Table 1 ;( [72, 73] ) [46, 65, 71, 74, 75] . At the cellular level, a lower capacity of CD4 + and CD8 + T-cells to produce IFN-γ and IL-2, as well as an impairment in T-cell activation from dendritic cells (DCs) in acute COVID-19 patients (≥55 years old), might potentially compromise an optimal adaptive immune response [76] . Based on examples from mice, a productive CD4 + T-cell response relies heavily on lung resident DCs (rDCs) and abates SARS-CoV infection [77, 78] . However, whether a reduction in the DC population in the lungs of older, more severe patients causes sub-optimal T-cell activation during SARS-CoV-2 infection remains to be robustly investigated.

Higher proportions of proinflammatory macrophages and neutrophils have also been observed in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients with severe symptoms compared with those exhibiting mild symptoms (Key Figure, Figure 4 ) [79] .

Accordingly, proinflammatory cytokines (e.g. IL-6, IL-8) are elevated in the BALF of severe J o u r n a l P r e -p r o o f Journal Pre-proof COVID-19 patients, along with higher expression of inflammatory chemokines (e.g. CCL2) in macrophages relative to non-severe COVID-19 patients [79] [80] [81] [82] . Indeed, similar inflammatory milieux have been associated with severe lung pathology in SARS patients, along with the notable 'cytokine storm' that can present in critically ill COVID-19 patients [83, 84] [71, [85] [86] [87] . These proinflammatory mediators can, in turn, perpetuate lung disease by elevating Creactive protein (CRP) from the liver ( Table 1) products [93] . Specifically, CoVs can avoid immune sensing via i) the formation of DMVs that sequester viral nucleic acid from being recognized by PRRs and ii) direct ablation of the functionality of immune signaling molecules by viral proteins [11, 94] . The structural and functional conservation of these proteins across the Betacoronavirus genus and in nsps between SARS-CoV and SARS-CoV-2, suggests that some of these suppressive mechanisms might be employed by SARS-CoV-2 (see below) [1] . Indeed, patients with severe COVID-19 have reported an imbalanced immune response with high concentrations of inflammatory cytokines/chemokines, but little circulating IFN-β or IFN-λ, resulting in persistent viremia [95] .

Of note, among several respiratory viruses tested, SARS-CoV-2 has demonstrated to most potently suppress type I and type III IFN expression in both human bronchial epithelial cells and ferrets [81] . Thus, evasion of IFN signaling by SARS-CoV-2 and impaired IFN production in J o u r n a l P r e -p r o o f human peripheral blood immune cells might contribute to the productive viral replication, transmission, and severe pathogenesis during COVID-19, although further testing is warranted to fully dissect these putative evasion pathways [95] .

With regard to functional conservation of viral proteins, SARS-CoV and MERS-CoV nsps and accessory proteins circumvent viral RNA-sensing pathways at multiple stages (e.g.

RIG-I, MDA-5) through proteasomal degradation and/or prevention of protein activation ( Figure   5 ) [94] . Functional conservation between SARS-CoV and MERS-CoV PL pro (encoded by nsp3) proteins has been reported, where these proteins target the initial PRR signaling cascade at multiple levels of the pathway includingbut not limited to-RIG-I, MAVS, TBK1, IRF3 and NF-K B ( Figure 5 ) [96] [97] [98] . The SARS-CoV PL pro also targets the DNA-sensing pathway at STING ( Figure 5) ; antagonizing this pathway might be important as mitochondrial stress during dengue virus infection triggers IFN-β production that is dependent on STING activation [99, 100] . Recent evidence suggests the SARS-CoV-2 PL pro might also inhibits IFN-I expression in human kidney epithelial cells, yet the mechanisms remain to be defined [101] . Moreover, nsp1 of highly pathogenic HCoVs, including SARS-CoV and MERS-CoV displays a pleiotropic effect, targeting several components of IFN-I signaling ( Figure 5 ) [102, 103] . This potent suppressive function of nsp1 also appears to be maintained in SARS-CoV-2, primarily through shutdown of translational machinery and prevention of immune gene expression [101, 104, 105] .

Furthermore, because there are only five accessory genes in the MERS-CoV genome compared to eight and seven in the SARS-CoV and SARS-CoV-2 genomes, respectively, similar immunosuppressive mechanisms may exist but appear to be mediated via different proteins [106, 107] . For example, SARS-CoVs ORF6 can inhibit IRF3 activation and STAT1 nuclear translocation, whereas this same effect is obtained by ORF4a/b and ORF5 of MERS-CoV J o u r n a l P r e -p r o o f ( Figure 5 ) [118, 119] . Coincidently, the apparent loss of these proteins may provide evidence for why MERS-CoV is more sensitive to IFN treatment than SARS-CoVs in primary and continuous cells of the human airways [108] . The SARS-CoV-2 proteins appear to have stronger inhibitory effects than their counterparts of highly pathogenic SARS-and MERS-CoV [105] . In light of these findings, SARS-CoV-2 has replicated more efficiently than SARS-CoV in ex vivo human lung explants, possibly through the greater suppression of IFN-I/III cytokines [109] ; further work 

Given that SARS-CoV-2 uses the same ACE2 entry receptor as SARS-CoV, rapidly deploying mouse models for pathogenesis studies were well underway within weeks of the pandemic's inception. However, various impediments remain for SARS-CoV-2 in productively infecting mice in these models, as it is unable to bind mouse ACE2 (mACE2) [111] . To overcome these prerequisites, several mouse models have been developed that recapitulate certain components of J o u r n a l P r e -p r o o f human COVID-19. One of these strategies is to genetically modify mice to express human ACE2 (hACE2) (humanized mice) under the epithelial cell-specific cytokeratin-18 (Krt 18 ) promoter [112] , a universal chicken beta-actin promoter [113] , or the endogenous mACE2 promoter [111] .

All these mice are susceptible to SARS-CoV-2 infection, but phenotypic disease varies because of differential hACE2 tissue expression [112] [113] [111] . For instance, Krt18-hACE2 and betaactin-hACE2-transgenic mice rapidly succumb to SARS-CoV-2 infection with lung infiltration of inflammatory immune cells inducing severe pulmonary disease, accompanied by evident thrombosis and anosmia, which partially recapitulate human COVID-19 [114] [115] . As the onset of severe histopathological changes occurs days after peak virus infection, these models recapture the delayed morbidity seen in COVID-19 patients as a result of inflammatory cell infiltration [115] . Therefore, employing humanized mouse models of severe SARS-CoV-2 infection might be useful for testing the efficacy of antiviral drugs, vaccines, and immune therapeutics that ablate hyperinflammation [114] . However, the broad expression of hACE2 in these models significantly expands SARS-CoV-2 tissue tropisms and might alter its pathogenic mechanisms [114] [115] . For example, both SARS-CoV and SARS-CoV-2 infection lead to encephalitis in these mouse models, which is not common in COVID-19 patients [113, 115, 116] . Considering the fact that the majority of human SARS-CoV-2 infections are asymptomatic or mild, mice originally bearing mACE2 that is replaced by hACE2 may be more appropriate for assessing pathogenesis and tissue tropism [111] . This model develops mild lung pathology, with SARS-CoV-2 infection being restricted to the lung and intestine [111] . In addition to the transgenic modification, mice can also be sensitized to SARS-CoV-2 infection via transient transduction of adenovirus (Ad5)-or adeno-associated virus (AAV)-expressing hACE2 in respiratory tissues, akin to the approach previously used for MERS-CoV infection [117] [118] [119] .

These mice develop viral pneumonia, weight loss, severe pulmonary pathology, and a high viral load in the lung, consistent with human COVID-19 [119] . This approach might be quickly adapted to many genetically modified mouse strains that might provide mechanisms of SARS-CoV-2 pathogenesis and protective immune responses. This model is limited, however, by the transient ectopic expression of hACE2 from the Ad5/AAV vector that can induce mild bronchial inflammation and expand cell tropism of SARS-CoV-2 and thus, presumably alter disease pathogenesis [120] .

Rather than genetic modification in host animals, viruses can also be genetically modified and be used in model animals [121, 122] . For instance, in one study, serial passaging of SARS-CoV-2 in mice led to enrichment of a N501Y viral mutant that elicited interstitial pneumonia and inflammatory responses in both aged and young wild-type BALB/c mice [123] . Another mouseadapted SARS-CoV-2 strain (MA10) carrying three mutations in the RBD of Spike protein caused severe lung pathology and ARDS in mice, characteristic of severe COVID-19 [124] .

Despite the three mutations in the RBD of the mouse-adapted Spike, vaccination with full length SARS-CoV-2 Spike elicited robust neutralizing antibody titers and complete protection against a secondary challenge with MA10 [124] ; these findings suggest that this strain may be applicable to pathogenesis studies, as well as antiviral drug and vaccine testing in rodents.

The role of non-human primates (NHP) in evaluating coronavirus pathogenesis cannot be understated. Depending on the NHP model utilized, clinical signs/symptoms may be mild or absent entirely [125] [126] [127] . In rhesus macaques, several studies have noted reduced appetite, J o u r n a l P r e -p r o o f transient fevers (1 day post infection: dpi) and mild weight loss without overt signs of respiratory distress or mortality [125] [126] [127] . By contrast, cynomolgus macaques did not display any observational signs of disease in another study [126] . Although certain NHPs appear to only mimic mild disease (if any), rhesus macaques have exhibited high viral loads in nasal swabs, throat samples, and BALF early post inoculation, and viral RNA was still measurable by qPCR in the trachea and lung on day 21 p.i., highlighting the apparent tropism of SARS-CoV-2 for the URT and lingering viral nucleic acid in respiratory tissues after resolution of disease [51, 125] .

SARS-CoV-2 has also been detected in nasal swabs at 10 dpi in NHPs, consistent with the prolonged URT shedding of virus in COVID-19 patients at ~9 dpi [51, 125, 126, 128] . The tropism of SARS-CoV-2 for the LRT in NHPs has also been recapitulated by the development of multifocal lesions and interstitial pneumonia, supporting the hypothesis that lung injury is driven by increased infiltration of neutrophils and macrophages into the lung following viral infection such as thickened alveolar septum and diffuse severe interstitial pneumonia when compared to young macaques (3-5 years old) [129] . Therefore, these studies highlight the importance of also considering the age factor, as an additional variable, when selecting animal models that might closely, or accurately, recapitulate human disease.

Evaluating efficacious vaccine candidates in NHPs will also be important for understanding correlates of protection against SARS-CoV-2. Accordingly, reports of antibodydependent enhancement, as well as of non-neutralizing humoral responses to the conserved regions of SARS-CoV-2, raise concerns on our future ability to effectively administer an immunogen without inducing immunopathology [130, 131] . Furthermore, upon viral challenge, lymphocytes have expanded in rhesus macaque models around 5 dpi with complementary B-cell responses against SARS-CoV-2 Spike appearing 10-15 dpi in blood samples [125] ; expansion of these adaptive immune compartments was analogous to those observed in COVID-19 patients [37, 125, [132] [133] [134] . Subsequent re-challenged rhesus macaque have presented a rapid anamnestic immune response characterized by significantly higher neutralizing antibody (NAb) titers than the primary infection macaques [127] . Thus, protective efficacy seems to depend primarily on NAb titers, at least in NHPs, and so far, T-cell numbers have not substantially increased following re-challenge in the serum of these animals, and in a secondary study, CD4 + and CD8 + cytokine (e.g. IFN-γ) responses did not correlate with immune protection from DNA vaccines with different components of the SARS-CoV-2 Spike protein [135] [127] .

Although these animals have failed to manifest overt signs of infection and respiratory compromise, NHPs still represent the 'gold standard' for evaluating the protective efficacy of human-bound SARS-CoV-2 vaccines based on parallels to humans in terms of viral tropism, immunopathology, and correlates of protection [127] . Further research is urgently needed to J o u r n a l P r e -p r o o f explore the durability of immune responses to SARS-CoV-2, considering reports of waning immunity to other CoVs and the detection of pre-existing cross-reactive 'common-cold' CoV Tcells with SARS-CoV-2 in naïve humans (see Outstanding Questions) [136, 137] .

The emergence of SARS-CoV-2 as the most recent example of zoonotic virus spillovers into In the last 24 hours leading up to death, all 13 patients which were included for this metric had a prothrombin time of >12.1s.

Aerosol: suspension of fine solid or liquid droplets in the air (or a gas medium), such as dusts, mists, or fumes. Anamnestic immune response: memory immune response to a previously encountered antigen. Angiotensin-converting enzyme 2 (ACE2): cell surface enzyme of endothelial, epithelial, and other cells, with a well-defined function in maintaining normal blood pressure. Anosmia: partial or complete loss of the sense of smell. Antibody-dependent enhancement: phenomenon by which antibodies against a virus are suboptimal to the virus and enhance its entry into host cells. Convalescence period: the time of gradual recovery after an illness or injury. Correlates of protection: quantifiable parameters such as antibodies, indicating that a host is protected against microbial infection. Cytokine storm: severe immune reaction in which the body releases too many cytokines into the blood too quickly. D-dimer: fibrin degradation product in the blood after a clot is degraded by fibrinolysis. Disseminated intravascular coagulation (DIC): condition in which blood clots form throughout the body and block small blood vessels, leading to multiorgan failure. Fecal-oral transmission: route of disease transmission by which an infectious agent in fecal materials is passed to the mouth of another. Fomite: inanimate object (clothes, utensil, and furniture etc.) that, when contaminated with an infectious agent, can transfer the infectious agent to a new host. Furin: proprotein convertase that cleaves a precursor protein into a biologically active state. Incubation period: timeframe elapsed between when a host is first exposed to an infectious agent and when signs or symptoms begin to appear. Lung ground glass opacity: nonspecific radiological description of an area of increased opacity in the lung through which vessels and bronchial structures are still visible. Neutralizing antibody (NAb): an antibody that binds a pathogen with high affinity and prevents the latter from exerting its biological effect.

Neutrophil extracellular traps (NETs): networks of extracellular fibers, primarily composed of DNA from neutrophils due to chromatin decondensation, which can 'trap' extracellular pathogens. Pattern recognition receptor: germline-encoded host sensor that recognizes a signature pattern in microbial molecules. Prodromal period: the time immediately following the incubation period of a microbial infection in which a host begins to experience symptoms or changes in behavior/functioning. Prothrombin time: measurement of the extrinsic pathway of coagulation. R 0 (reproductive number): the expected number of new disease cases generated by one case. An R 0 > 1 indicates the outbreak will expand; R 0 <1 the outbreak will die out.

Respiratory droplet: small aqueous droplet produced by exhalation, consisting of saliva or mucus and other matter derived from respiratory tract surfaces. Zoonotic disease: infectious disease caused by a pathogen that has crossed a species barrier from animals to humans. The ongoing COVID-19 pandemic has resulted in numerous accounts of different transmission routes between humans. Droplet transmission (>5μM) is the most pronounced and heavily implicated mode of transmission reported during the pandemic. Direct contact spread from one infected individual to a second, naïve person has also been considered a driver of human-to-human transmission, especially in households with close interactions between family members. The contagiousness of SARS-CoV-2 after disposition on fomites (e.g. door handle) is still under investigation, but is likely a compounding factor for transmission events, albeit less frequently than droplet or contact-driven transmission. Both airborne and fecal-oral human-to-human transmission events were reported in J o u r n a l P r e -p r o o f the precursor SARS-CoV epidemic but have yet to be observed in the current crises. Solid arrows show confirmed viral transfer from one infected person to another with a declining gradient in arrow width denoting the relative contributions of each transmission route. Dashed lines show the plausibility of that transmission type but have yet to be confirmed. SARS-CoV-2 symbol in "infected patient" indicate where RNA/infectious virus has been detected [43, 44, 47-49, 57, 59] 

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Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Interleukin-1β Induces mtDNA Release to Activate Innate Immune Signaling via cGAS-STING

Coronavirus Papain-like Proteases Negatively Regulate Antiviral Innate Immune Response through Disruption of STING-Mediated Signaling

Activation and evasion of type I interferon responses by SARS-CoV-2

Severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation

Middle East Respiratory Syndrome Coronavirus nsp1 Inhibits Host Gene Expression by Selectively Targeting mRNAs Transcribed in the Nucleus while Sparing mRNAs of Cytoplasmic Origin

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

Evasion of type-I interferon by SARS-CoV-2

The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists

Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame (ORF) 3b, ORF 6, and Nucleocapsid Proteins Function as Interferon Antagonists

Human Cell Tropism and Innate Immune System Interactions of Human Respiratory Coronavirus EMC Compared to Those of Severe Acute Respiratory Syndrome Coronavirus

Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19

Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

Lethal Infection of K18-hACE2 Mice Infected with Severe Acute Respiratory Syndrome Coronavirus

Severe Acute Respiratory Syndrome Coronavirus Infection of Mice Transgenic for the Human Angiotensin-Converting Enzyme 2 Virus Receptor

K18-hACE2 Mice for Studies of COVID-19 Treatments and Pathogenesis Including Anosmia

SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function

Severe Acute Respiratory Syndrome Coronavirus Infection Causes Neuronal Death in the Absence of Encephalitis in Mice Transgenic for Human ACE2

A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by Neutralizing Antibodies

Rapid generation of a mouse model for Middle East respiratory syndrome

Generation of a Broadly Useful Model for COVID-19 Pathogenesis Vaccination, and Treatment

Adenovirus vectors for gene therapy, vaccination and cancer gene therapy

A Mouse-Adapted SARS-Coronavirus Causes Disease and Mortality in BALB/c Mice

A mouse-adapted SARS-CoV-2 model for the evaluation of COVID-19 medical countermeasures

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

A Mouse-adapted SARS-CoV-2 induces Acute Lung Injury (ALI) and mortality in Standard Laboratory Mice

Respiratory disease in rhesus macaques inoculated with SARS-CoV-2

Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model

SARS-CoV-2 infection protects against rechallenge in rhesus macaques

Cynomolgus Macaque as an Animal Model for Severe Acute Respiratory Syndrome

Age-related rhesus macaque models of COVID-19

Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus

Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection

Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease

Antibody responses to SARS-CoV-2 in patients with COVID-19

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Seasonal coronavirus protective immunity is short-lasting

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

Type-I, type-III IFNs then signal in an autocrine or paracrine manner through the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 1 and 2 (STAT1/2) pathway, culminating in antiviral interferon-stimulated gene (ISG) transcription. Listed here are SARS-CoV (abbreviated CoV), SARS-CoV-2 (abbreviated CoV-2) and MERS-CoV (abbreviated M-CoV) IFN-I antagonists, which make these viruses resistant to interferon responses. IFN-III is also implicated in exhibiting potent antiviral effects in lung/intestinal tissues, but the underlying evasion strategies of this pathway for these viruses are currently unknown. SARS-CoV proteins are highlighted in blue, while functions of SARS-CoV-2 and MERS-CoV proteins are highlighted in red and green, respectively. Question mark symbol (?) denotes SARS-CoV-2 protein bound a member of that signaling pathway in [123], but further work is necessary to confirm its immunological mechanism. SARS-CoV-2 proteins with * denotes functional conservation with SARS-CoV

Which animal(s) serves as the natural reservoir of SARS-CoV-2?  Does active replication of SARS-CoV-2 in the upper respiratory tract contribute to enhanced transmissibility in humans?  Is intestinal SARS-CoV-2 infection a source of virus transmission?  Which SARS-CoV-2 proteins antagonize innate and adaptive immune responses? Do the SARS-CoV-2 proteins with more potent antagonistic immune functions increase virulence in humans compared to other HCoVs?  Why do some recovered patients fail to develop neutralizing antibodies?  What are the host and/or viral factors driving

 What are the underlying mechanisms contributing to an inadequate IFN response to SARS-CoV-2?

 What are the correlates of immune protection for SARS-CoV2 and will they provide sterilizing immunity?

 Will candidate vaccines against SARS-CoV2 also be effective in elderly subpopulations

This work was supported by a National Institutes of Health grant R01AI132526 to P.W.The authors declare no competing financial/non-financial interest.J o u r n a l P r e -p r o o f Journal Pre-proof

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