FAIR Sharing of Reproducible Models of Epidemic and Pandemic Forecast (preprint)

A major challenge for the dissemination, replication, and reuse of epidemiological forecasting studies during COVID-19 pandemics is the lack of clear guidelines and platforms to exchange models in a Findable, Accessible, Interoperable, and Reusable (FAIR) manner, facilitating reproducibility of research outcomes. During the beginning of pandemics, models were developed in diverse tools that were not interoperable, opaque without traceability and semantics, and scattered across various platforms - making them hard to locate, infer and reuse. In this work, we demonstrate that implementing the standards developed by the systems biology community to encode and share COVID-19 epidemiological models can serve as a roadmap to implement models as a tool in medical informatics, in general. As a proof-of-concept, we encoded and shared 24 epidemiological models using the standard format for model exchange in systems biology, annotated them with cross-references to data resources, packed up all associated files in COMBINE archives for easy sharing, and finally, disseminated the models through BioModels repository to significantly enhance their reproducibility and repurposing potential. We recommend the use of systems biology standards to encode and share models of epidemic and pandemic forecasts to improve their findability, accessibility, interoperability, reusability, and reproducibility.

COVID-19 Disease Map, a computational knowledge repository of SARS-CoV-2 virus-host interaction mechanisms (preprint)

We hereby describe a large-scale community effort to build an open-access, interoperable, and computable repository of COVID-19 molecular mechanisms - the COVID-19 Disease Map. We discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. We highlight the role of relevant databases and text mining approaches in enrichment and validation of the curated mechanisms. We describe the contents of the map and their relevance to the molecular pathophysiology of COVID-19 and the analytical and computational modelling approaches that can be applied to the contents of the COVID-19 Disease Map for mechanistic data interpretation and predictions. We conclude by demonstrating concrete applications of our work through several use cases.

Induced pulmonary comorbidities render CD-1 mice sensitive to SARS-CoV-2 (preprint)

Severe manifestations of COVID-19 are mostly restricted to persons with comorbidities, and they form a significantly high proportion of those which develop life-endangering lung injury. Nevertheless, COVID-19 animal models established to date are not based on preexistence of comorbidities. Here we report that mild pulmonary injury induced by administration of acute-lung-injury stimulants, renders outbred CD-1 mice to be sensitive to SARS-CoV-2. Following intranasal pretreatment of mice with low doses of ricin or bleomycin, SARS-CoV-2 infection caused a severe disease manifested by sustained body loss and mortality rates of >50%. Low-dose-ricin pretreated mice displayed markedly higher levels of viral RNA than mice not pretreated with ricin, not only in the nasal turbinate, trachea and lungs but also in the serum and heart. The deleterious effects of SARS-CoV-2 infection in ricin-pretreated mice were effectively alleviated by passive transfer of polyclonal and monoclonal antibodies generated against SARS-CoV-2 or SARS-CoV-2 RBD. Notably, viral cell entry in the sensitized mice model seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel animal model in mice that express native murine ACE2 yet are susceptible to genetically unaltered SARS-CoV-2, for the study of comorbidity-dependent COVID-19 pathology and treatment.

A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor (preprint)

Genome-wide CRISPR/Cas9 knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. With the aim of identifying new cellular inhibitors of HIV-1, we have developed a strategy in which we took advantage of the ability of type 1 interferon (IFN) to potently inhibit HIV-1 infection, in order to create a cellular environment hostile to viral replication. This approach led to the identification of the DEAD-box RNA helicase DDX42 as an intrinsic inhibitor of HIV-1. Depletion of endogenous DDX42 using siRNA or CRISPR/Cas9 knock-out increased HIV-1 infection, both in model cell lines and in physiological targets of HIV-1, primary CD4+ T cells and monocyte-derived macrophages (MDMs), and irrespectively of the IFN treatment. Similarly, the overexpression of a dominant-negative mutant of DDX42 positively impacted HIV-1 infection, whereas wild-type DDX42 overexpression potently inhibited HIV-1 infection. The positive impact of endogenous DDX42 depletion on HIV-1 infection was directly correlated to an increase in viral DNA accumulation. Interestingly, proximity ligation assays showed that DDX42, which can be mainly found in the nucleus but is also present in the cytoplasm, was in the close vicinity of HIV-1 Capsid during infection of primary monocyte-derived macrophages. Moreover, we show that DDX42 is also able to substantially decrease infection with other retroviruses and retrotransposition of long interspersed elements-1 (LINE-1). Finally, we reveal that DDX42 potently inhibits other pathogenic viruses, including Chikungunya virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Aglycone polyether ionophores as broad-spectrum agents inhibit multiple enveloped viruses including SARS-CoV-2 in vitro and successfully cure JEV infected mice (preprint)

Infections with zoonotic viruses, such as flaviviruses, influenza virus, and the SARS-CoV-2 pandemic coronavirus constitute an increasing global risk. Hence, an urgent need exists for the development of broad-spectrum antivirals to prevent such outbreaks. Here, we show that the maduramycin and CP-80,219 aglycone polyether ionophores exhibit effective broad-spectrum antiviral activity, against various viruses, including Japanese encephalitis virus (JEV), Dengue virus (DENV), Zika virus (ZIKV), and Chikungunya virus (CHIKV), while also exhibiting promising activity against PR8 influenza virus and SARS-CoV-2. Moreover, liposome-encapsulated maduramycin and CP-80,219 provide full protection for mice from infection with JEV in vivo. Mechanistic studies suggest that aglycone polyether ionophores primarily inhibit the viral replication step without blocking endosome acidification to promote the fusion between viral and cellular membranes. The successful application of liposomes containing aglycone polyether ionophores in JEV-infected mice offers hope to the development of broad-spectrum antiviral drugs like penicillin back to 1940s.

Extracellular vesicle-based vaccine platform displaying native viral envelope proteins elicits a robust anti-SARS-CoV-2 response in mice. (preprint)

Extracellular vesicles (EVs) emerge as essential mediators of intercellular communication. DNA vaccines encoding antigens presented on EVs efficiently induce T-cell responses and EV-based vaccines containing the Spike (S) proteins of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) are highly immunogenic in mice. Thus, EVs may serve as vaccine platforms against emerging diseases, going beyond traditional strategies, with the antigen displayed identically to the original protein embedded in the viral membrane and presented as such to the immune system. Compared to their viral and pseudotyped counterparts, EV-based vaccines overcome many safety issues including pre-existing immunity against these vectors. Here, we applied our technology in natural EV's engineering, to express the S proteins of SARS-CoV-2 embedded in the EVs, which mimic the virus with its fully native spikes. Immunizations with a two component CoVEVax vaccine, comprising DNA vector (DNAS-EV) primes, allowing in situ production of Spike harbouring EVs, and a boost using S-EVs produced in mammalian cells, trigger potent neutralizing and cellular responses in mice, in the absence of any adjuvants. CoVEVax would be the prototype of vaccines, where the sole exchange of the envelope proteins on EVs leads to the generation of new vaccine candidates against emerging viruses.

SARS-CoV-2 desensitizes host cells to interferon through inhibition of the JAK-STAT pathway (preprint)

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.

In vitro assessment of the virucidal activity of four mouthwashes containing Cetylpyridinium Chloride, ethanol, zinc and a mix of enzyme and proteins against a human coronavirus (preprint)

Background: saliva is established to contain high counts SARS-CoV-2 virus and contact with saliva droplets, contaminated surfaces or airborne particles are sources of viral transmission. The generation of infective aerosols during clinical procedures is of particular concern. Therefore, a fuller understanding of the potential of mouthwash to reduce viral counts and modulate the risk of transmission in medical professional and public context is an important research topic. Method: we determined the virucidal activity of four anti-bacterial mouthwashes against a surrogate for SARS-CoV-2, Human CoV-SARS 229E, using a standard ASTM suspension test, with dilution and contact times applicable to recommended mouthwash use. Results: the mouthwash formulated with 0.07% Cetylpyridinium Chloride exhibited virucidal effects providing a [≥]3.0 log reduction HCoV-229E viral count. Mouthwashes containing 15.7% ethanol, 0.2% zinc sulphate heptahydrate and a mix of enzymes and proteins did not demonstrate substantive virucidal activity in this test. Conclusion: mouthwash containing 0.07% Cetylpyridinium Chloride warrants further laboratory and clinical assessment to determine their potential benefit in reducing the risk of SARS-CoV-2.

Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir (preprint)

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3'-exonuclease that recognizes mismatches. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models (preprint)

More than a million people have now died from COVID-19, because of infection with the SARS-CoV-2 coronavirus. Currently, the FDA has approved remdesivir, an inhibitor of SARS-CoV-2 replication, to treat COVID-19, though very recent data from WHO showed little if any COVID19 protective effect. Here we report that ethacridine, a safe and potent antiseptic use in humans, effectively inhibits SARS-CoV-2, at very low concentrations (EC50 ~ 0.08 M). Ethacridine was identified through a high-throughput screening of an FDA-approved drug library in living cells using a fluorescent assay. Interestingly, the main mode of action of ethacridine is to inactivate virus particles, preventing binding to the host cells. Thus, our work has identified a potent drug with a distinct mode of action against SARS-CoV-2.

Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice (preprint)

The SARS-CoV-2 virus caused one of the severest pandemic around the world. The vaccine development for urgent use became more of an issue during the pandemic. An inactivated virus formulated vaccines such as Hepatitis A, inactivated polio, and influenza has been proven to be a reliable approach for immunization for long years. In this pandemic, we produced an inactivated SARS-CoV-2 vaccine candidate by modification of the oldest but the most experienced method that can be produced quickly and tested easily rather than the recombinant vaccines. Here, we optimized an inactivated virus vaccine which includes the gamma irradiation process for the inactivation as an alternative to classical chemical inactivation methods so that there is no extra purification required. Also, we applied the vaccine candidate (OZG-38.61.3) using the intradermal route in mice which decreased the requirement of a higher concentration of inactivated virus for proper immunization unlike most of the classical inactivated vaccine treatments. Thus, the novelty of our vaccine candidate (OZG-38.61.3) is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. We first determined the efficiency and safety dose (either 1013 or 1014 viral copy per dose) of the OZG-38.61.3 in Balb/c mice. Next, to test the immunogenicity and protective efficacy of the OZG-38.61.3, we immunized human ACE2-encoding transgenic mice and infected them with a dose of infective SARS-CoV-2 virus for the challenge test. We showed that the vaccinated mice showed lowered SARS-CoV-2 viral copy number in oropharyngeal specimens along with humoral and cellular immune responses against the SARS-CoV-2, including the neutralizing antibodies similar to those shown in Balb/c mice without substantial toxicity. This study encouraged us towards a new promising strategy for inactivated vaccine development (OZG-38.61.3) and the Phase 1 clinical trial for the COVID-19 pandemic.

SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic coronavirus to spill over to humans in less than 20 years, after SARS-CoV-1 in 2002-2003 and Middle East respiratory syndrome (MERS)-CoV in 2012. SARS-CoV-2 is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to severe lung injury and death in the most severe cases. The COVID-19 pandemic is currently a major health issue worldwide. Immune dysregulation characterized by altered innate cytokine responses is thought to contribute to the pathology of COVID-19 patients, which is a testimony of the fundamental role of the innate immune response against SARS-CoV-2. Here, we further characterized the host cell antiviral response against SARS-CoV-2 by using primary human airway epithelia and immortalized model cell lines. We mainly focused on the type I and III interferon (IFN) responses, which lead to the establishment of an antiviral state through the expression of IFN-stimulated genes (ISGs). Our results demonstrate that both primary airway epithelial cells and model cell lines elicit a robust immune response characterized by a strong induction of type I and III IFN through the detection of viral pathogen molecular patterns (PAMPs) by melanoma differentiation associated gene (MDA)-5. However, despite the high levels of type I and III IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication, whereas IFN pre-treatment strongly inhibited viral replication and de novo production of infectious virions. Taken together, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.

The IMEx Coronavirus interactome: an evolving map of Coronaviridae-Host molecular interactions (preprint)

The current Coronavirus Disease 2019 (COVID-19) pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has spurred a wave of research of nearly unprecedented scale. Among the different strategies that are being used to understand the disease and develop effective treatments, the study of physical molecular interactions enables studying fine-grained resolution of the mechanisms behind the virus biology and the human organism response. Here we present a curated dataset of physical molecular interactions, manually extracted by IMEx Consortium curators focused on proteins from SARS-CoV-2, SARS-CoV-1 and other members of the Coronaviridae family. Currently, the dataset comprises over 2,200 binarized interactions extracted from 86 publications. The dataset can be accessed in the standard formats recommended by the Proteomics Standards Initiative (HUPO-PSI) at the IntAct database website (www.ebi.ac.uk/intact), and will be continuously updated as research on COVID-19 progresses.