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SARS-CoV-2/COVID-19: Evolving Reality, Global Response, Knowledge Gaps, and Opportunities

ABSTRACT Approximately 3 billion people around the world have gone into some form of social separation to mitigate the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The uncontrolled influx of patients in need of emergency care has rapidly brought several national health systems to near-collapse with deadly consequences to those afflicted by Coronavirus Disease 2019 (COVID-19) and other critical diseases associated with COVID-19. Solid scientific evidence regarding SARS-CoV-2/COVID-19 remains scarce; there is an urgent need to expand our understanding of the SARS-CoV-2 pathophysiology to facilitate precise and targeted treatments. The capacity for rapid information dissemination has emerged as a double-edged sword; the existing gap of high-quality data is frequently filled by anecdotal reports, contradictory statements, and misinformation. This review addresses several important aspects unique to the SARS-CoV-2/COVID-19 pandemic highlighting the most relevant knowledge gaps and existing windows-of-opportunity. Specifically, focus is given on SARS-CoV-2 immunopathogenesis in the context of experimental therapies and preclinical evidence and their applicability in supporting efficacious clinical trial planning. The review discusses the existing challenges of SARS-CoV-2 diagnostics and the potential application of translational technology for epidemiological predictions, patient monitoring, and treatment decision-making in COVID-19. Furthermore, solutions for enhancing international strategies in translational research, cooperative networks, and regulatory partnerships are contemplated.

The Interferons. The precise role of IFNs in SARS-CoV-2 infection is yet unclear and requires further investigation. It is unclear whether patients by default produce amounts of interferon like certain mammals (84) or low IFN concentrations may reflect the coronaviruses' capacity to prevent/reduce its production and action (85-92). The latter may be plausible given that CoV are well capable of preventing NF-B activation (93) and protein translation (94). It is also important to define the specific role played by IFN-ε in the mucosa; this may explain differences between humans versus bats/pangolins regarding their response to a CoV infection (95-97). Better understanding of the crosstalk between SARS-CoV-2 and IFNs will offer insights into the COVID-19 immunopathogenesis. Some data suggest that the strategy of a very early administration of type I IFN could be considered (98-100). However, the negative effect of a late exposure to IFNs must also be considered; a study combining single-cell RNA-sequencing data and in vitro analysis showed that ACE2 is upregulated by type I and II IFNs in human and primate airway epithelial cells (101). Coagulopathy & endotheliopathy. Coagulopathy appears to be a critical element in the context of severe COVID-19 courses. Elevation of D-dimers (fibrin degradation products) has been frequently observed in severe cases and identified as a significant risk factor (104).
One study revealed presence of a procoagulant state even during the early COVID-19 stage (19) and disseminated intravascular coagulation (DIC) has been diagnosed in most critically ill patients (105). Moreover, the incidence of thromboembolic events is high (comparable to sepsis) and most likely underdiagnosed (106). It can be hypothesized that there is dominant local pulmonary vessel microthrombosis which corelates with the severity of hypoxemia and high compliance (107) and stays in accordance with fibrin cumulation found in the lungs (107, 108). Presumably, there are several pathways which may contribute to the clinical coagulopathy observed. Direct infection of the endothelium, thereby triggering endothelial injury, inflammation and cell death (20), can direcly activate the coagulation cascade.
Pyroptosis and inflammasome-released mediators are other potent coagulation cascade activators (109, 110). Histopathological examination revealed deposition of activated complement complexes that may propel microvascular injury and subsequent activation of the clotting pathway (111). Neutrophilic infiltrates can also activate coagulation through the generation of neutrophil extracellular traps (NETs) (112) and a recent anecdotal study confirmed the presence of antiphospholipid antibodies in three COVID-19 patients with severe coagulopathy (113). An analysis of over 3,500 genes (differentially expressed in the lungs) following murine SARS-CoV infection identified various pro-coagulatory factors (especially urokinase) to be strongly associated with mortality (114). Urokinase activity results in the generation of plasmin and in turn in fibrinolysis with elevated D-dimers manifested by alveolar coagulopathy and pulmonary hemorrhage. Furthermore, serpin1 knockout mice confirmed an enhanced pulmonary expression of procoagulatory and profibrinolytic proteins and clinical susceptibility to SARS-CoV (114). Apart from cytokine effects on the pulmonary endothelium, it has also been proposed that dysruptions of the kallikrein-bradykine axis can increase microvascular permeablity and cause angioedema (115).

Experimental therapies and pre-clinical evidence
The number of proposed therapeutic strategies against COVID-19 grows weekly; presently, there are over fifty substances considered as potential remedies including brand new (siRNA; 119) as well as well-known/repurposed chemicals (chloroquin, interferons, remdesivir). Yet, the currently available pre-and clinical evidence supportive of the experimental therapies is suggestive at best. Several substances with different targets have been proposed based on sparse peer-reviewed publications ( Table 3); some based on nonpeer reviewed (medRxiv and bioRxiv) pre-prints and/or anecdotal evidence only. As of April 30, 2020, no peer-reviewed study has ever tested any anti-COVID-19 candidate drug in a relevant SARS-CoV-2 animal model; many drugs have not yet been directly tested against SARS-CoV-2 in vitro. The only available animal model-based evidence stems from MERS/SARS-CoV studies ( Table 3) but these diseases are not identical with COVID-19 (Table 2).
Mechanistically, we are only at the very beginning to understand how SARS-CoV-2 infects, targets the lungs (and other organs) and causes severe vascular and tissue damage. However, small laboratory animals remain the most accessible and cost-effective model to study. Transgenic human ACE-2 mice (both sexes) infected with the HB-01 strain developed COVID-19-like interstitial pneumonia, high viral load, and produced high specific IgG titer but the disease was generally mild (125). Syrian hamsters constitute another potential option; two groups recently recapitulated a mild but widely symptomatic COVID-19 phenotype (126,127). In contrast, species such as pigs, chickens and ducks were virus-free when either inoculated or exposed to the virus, cats were asymptomatic despite post-exposure infection whereas dogs were minimally susceptible to SARS-CoV-2 exposure (128). Experimental drugs will soon be tested in the newly emerging COVID-19 models. While modeling of SARS-CoV-2 infection in healthy and young animals will be informative, it is important to consider that the most severe COVID-19 phenotypes appear in aged patients with comorbidies -the animal models should reflect this to maximize their translational capability for pathophysiology studies and drug testing. The most recent macaque experiment attests to that; 15-year-old animals developed an exacerbated COVID-19 phenotype while the young ones did not (129). Another important element in pre-clinical modeling is to promote study designs that allow drug testing in divergent, precisely defined and relatively homogeneous COVID-19 phenotypes (provided they can be recapitulated in animals). It is likely that a given therapeutic may be either beneficial or detrimental contingent upon the timing of its administration and/or specific COVID-19 pathophysiological characteristic. Compared to patients, animal studies present a relatively cheap and safe platform to establish such relationships.
The yet limited evidence derived from pre-clinical studies coupled with the emerging clinical data as discussed previously indicate that SARS-CoV-2-induced coagulopathy may be one of the key interests for experimental studies (114). In this context, another less apparent but interesting target for potential COVID-19 therapy is the complement activation pathway. Genetic absence of the complement C3 component was associated with reduced pulmonary/systemic inflammation and improved lung function (130). MERS-CoV-infected mice displayed elevated complement component C5a in the lungs and blood and blockade of the C5aR receptor attenuated inflammation in the lungs and spleen and reduced pulmonary viral replication (131 and clinical studies will gradually reveal the cons and pros of the experimental therapeutics.

Clinical management and trials
With the exception of remdesivir, which has received an emergency use authorization (EUA) 83%) (158). This French study was further confirmed by the same team on 80 patients (159). These studies were heavily criticized by the MRC-NIHR Trials Methodology Research Partnership for numerous shortcomings (158). The two subsequent studies with hydroxychloroquine indicated no improvement (160,161). The most recent reports emphasize risks associated with chloroquine-based trials; due to adverse cardiac reactions, chloroquine treatment (with and without azithromycin) was stopped in Brazilian (162) and French (163) trials; and several hospitals in Sweden stopped administering hydroxychloroquine to COVID-19 patients based on similar findings (164). A recent preprint article that retrospectively analyzed hydroxychloroquine use in hospitalized US veterans found an association of increased overall mortality with its use (165).   (175). An additional major concern of many ongoing COVID-19 trials is their relatively low power to detect significant differences in meaningful outcomes. Low-powered trials rarely provide unequivocal evidence justifying the use of tested therapeutics. In this respect, pandemics provide a unique window of opportunity for large-scale collaborative initiatives, thus, enabling networks to jointly generate and address common goals as well as to standardise data collection in large patient year.

SARS-CoV-2 diagnosis and optimization of testing
The differentiation of the SARS-CoV-2-positive cases from the healthy is among the main clinical challenges. Many asymptomatic persons are a source of infection despite being considered healthy prior to a positive test result (180). The prevalence of asymptomatic (SARS-CoV-2 positive) patients ranges between 1.5 and 30% (180)(181)(182). Non-test based COVID-19 diagnosis is difficult given that the most common clinical symptoms, e.g., fever, fatigue, dry cough, myalgia, and dyspnea (48,183) are unspecific and overlap with other viral diseases (183). Several hematologic and immuno-inflammatory abnormalities observed in SARS-CoV-2-positive patients resemble MERS/SARS-CoV infection symptoms (183). To reduce the transmission risk, aggressive containment, mitigation and treatment strategies must be combined and rapid and accurate testing is key. A few countries/territories such as Taiwan, Hong Kong, Singapore and South Korea have rapidly implemented aggressive testing (184); South Korea has performed >300,000 tests (5829 tests/million population) within the 9 weeks after the first SARS-CoV-2 case was identified, effectively containing the SARS-CoV-2 spread (185,186). The majority of other countries have been struggling with tests approval, availability and/or operationalization of high-throughput testing, which was accompanied by substantial differences in reported numbers.
The current recommendation of many professional radiological associations and societies is that imaging should not be employed as a screening nor diagnostic tool for COVID-19 but reserved for the evaluation of complications. Although used frequently in early reports with some characteristic features described (25, Figure 2) (189,190). The WHO testing recommendations differ depending on the SARS-CoV-2 prevalence. In the virus-free areas, a positive NAAT for at least two target SARS-CoV-2 genes is considered reliable; in areas with SARS-CoV-2 presence, a confirmation of a single discriminatory target is sufficient (183,187 (192). Therefore, a negative RT-PCR result does not necessarily rule out the possibility of SARS-CoV-2 infection (192). The WHO quotes several culprit factors including a poor specimen quality, timing of specimen collection, specimen handling and/or shipping and various technical problems (187). A small Chinese study demonstrated that some COVID-19 patients who met criteria for hospital discharge and/or quarantine discontinuation (free of clinical symptoms, radiological abnormalities and with two negative RT-PCR tests) were tested positive (by RT-PCR) 5 to 13 days later (193). A South Korean study reported that two out of ten negative COVID-19 cases by RT-PCR were later confirmed as positive (194). In a large study, the second RT-PCR test was positive in 12.5% of initially negative 384 patients (195). Thus, the detection of SARS-CoV-2 is highly specific (no false positives) but the sensitivity is not ideal (196). These findings reveal limitations of RT-PCR testing and indicate that some recovered patients may continue to be virus carriers (193). This phenomenon, if confirmed on a larger scale, may force changes in the current criteria for hospital discharge and quarantine discontinuation.
Furthermore, the SARS-CoV-2 diagnostics should account for clinical findings; preliminary data from China showed that isolated patients (with presumed COVID-19) with initially negative RT-PCR but with typical clinical and radiological COVID-19 symptoms were confirmed as SARS-CoV-2-positive after repeated swab testing (192). Thus, a combination of repeated RT-PCR testing and radiological imaging may be helpful in determining suspected false-negative cases.
Although the RT-PCR constitutes the current diagnostic standard, the test kits suffer considerable limitations. In 610 patients, in the first all-patient test, 27.5% cases were positive, 0.2% weakly positive, 9.3% dubious and 63% were negative (195). Among the patients with initial negative results, the second test was positive in 12.5%, dubious in 7% patients, negative in 73% patients, and results were not available for 7.6% patients (195). The false negative readouts are also contingent on the workflow for molecular detection (e.g. and isolation method with/without commercial kit) (192,193,197  Despite its epidemiological and diagnostic potential, it is premature to expect AI to be a game-changer in real-time patient monitoring (214). It is unclear whether AI can precisely prognosticate, and its usefulness has been modest in the context of therapy design. Even though AI algorithms have been used to predict the efficacy of existing drugs repurposed to treat COVID-19 (215,216), the candidate drugs (and pharmacological targets) still require experimental validation before translating them to bedside and into clinical practice.
Interestingly, promising results were obtained from the analysis of high-throughput data on the interaction between SARS-CoV-2 and ACE2 receptors encouraging research on interventions preventing the internalization of SARS-CoV-2 (144,217).
The contribution of data-driven models in the improvement of patient monitoring and in the design of new therapeutic approaches critically depends on the quality of the available data; it is key to ensure the best possible consistency and homogeneity across databases.  (229) and neurological manifestations (230).
Retrospective studies will be necessary to build and validate physiological multiscale models. The success of this effort will rely on the availability of well-annotated, cohesive and consistent databases and corresponding biobanks. The integration of statistical (data-driven) modeling and physiological (knowledge-based) modeling should be pursued to enhance the translational potential of computational analytics for: i) diagnostics and monitoring, ii) the design of novel, more effective treatments, iii) the guidance of therapy by decision support systems.

Dissemination of data and large-scale translational research networks
The SARS-CoV-2 outbreak has triggered an unprecedented cooperative activity worldwide, continents (https://esicm-tv.org/covid19/). However, perception of the rapid data dissemination by the pre-print servers (e.g. bioRxiv.org; medRxiv.org) is less favorable.
There has been a growing concern that such a steep growth of unscrutinized scientific evidence (2492 bio/medRxiv.org articles on SARS-CoV-2/COVID-19; accessed April 30, 2020) rather provokes more confusion than meritorious guidance and indiscriminately selected pieces of the pre-printed data are recurrently sensationalized by the lay press.   Despite relatively clear genetic relationships among sampled viruses, an uncertainty for specific transmission dates and the reconstruction of the geographic spread remains. Note, that the specific inferred transmission patterns (connecting lines) are only hypothetical (25). Thousands of complete genomes are available and increase on a daily basis. The visualization is based upon sub-sampled available genome data (see more under: https://nextstrain.org/ncov). As the pathogen replicates and spreads, its genome is replicated and random mutations/errors accumulate in the genome. Such random mutations allow tracking of the SARS-CoV-2 spread inferrences regarding its transmission routes and dynamics. The colors indicate the origin/source of the various viral strains, while circle diameters reflect the size of the transmission clusters.      anti-type I IFN strategies preventing its production and its effects anti-type I IFN strategies preventing its production and its effects unclear; under investigation *Due to the similar origin and genome, the immune response to human coronaviruses shares several confirmed and assumed aspects but numerous differences have already been reported. The characterized immune responses are of relevance to their pathophysiology and treatment (116)(117)(118).