Expiratory Aerosol pH: The Overlooked Driver of Airborne Virus Inactivation.

Respiratory viruses, including influenza virus and SARS-CoV-2, are transmitted by the airborne route. Air filtration and ventilation mechanically reduce the concentration of airborne viruses and are necessary tools for disease mitigation. However, they ignore the potential impact of the chemical environment surrounding aerosolized viruses, which determines the aerosol pH. Atmospheric aerosol gravitates toward acidic pH, and enveloped viruses are prone to inactivation at strong acidity levels. Yet, the acidity of expiratory aerosol particles and its effect on airborne virus persistence have not been examined. Here, we combine pH-dependent inactivation rates of influenza A virus (IAV) and SARS-CoV-2 with microphysical properties of respiratory fluids using a biophysical aerosol model. We find that particles exhaled into indoor air (with relative humidity ≥ 50%) become mildly acidic (pH ∼ 4), rapidly inactivating IAV within minutes, whereas SARS-CoV-2 requires days. If indoor air is enriched with nonhazardous levels of nitric acid, aerosol pH drops by up to 2 units, decreasing 99%-inactivation times for both viruses in small aerosol particles to below 30 s. Conversely, unintentional removal of volatile acids from indoor air may elevate pH and prolong airborne virus persistence. The overlooked role of aerosol acidity has profound implications for virus transmission and mitigation strategies.

Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity.

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.

Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense.

Host interferons (IFNs) powerfully restrict viruses through the action of several hundred IFN-stimulated gene (ISG) products, many of which remain uncharacterized. Here, using RNAi screening, we identify several ISG restriction factors with previously undescribed contributions to IFN-mediated defense. Notably, RABGAP1L, a Tre2/Bub2/Cdc16 (TBC)-domain-containing protein involved in regulation of small membrane-bound GTPases, robustly potentiates IFN action against influenza A viruses (IAVs). Functional studies reveal that the catalytically active TBC domain of RABGAP1L promotes antiviral activity, and the RABGAP1L proximal interactome uncovered its association with proteins involved in endosomal sorting, maturation, and trafficking. In this regard, RABGAP1L overexpression is sufficient to disrupt endosomal function during IAV infection and restricts an early post-attachment, but pre-fusion, stage of IAV cell entry. Other RNA viruses that enter cells primarily via endocytosis are also impaired by RABGAP1L, while entry promiscuous SARS-CoV-2 is resistant. Our data highlight virus endocytosis as a key target for host defenses.

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir.

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC50 value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

An antiviral trap made of protein nanofibrils and iron oxyhydroxide nanoparticles.

Minimizing the spread of viruses in the environment is the first defence line when fighting outbreaks and pandemics, but the current COVID-19 pandemic demonstrates how difficult this is on a global scale, particularly in a sustainable and environmentally friendly way. Here we introduce and develop a sustainable and biodegradable antiviral filtration membrane composed of amyloid nanofibrils made from food-grade milk proteins and iron oxyhydroxide nanoparticles synthesized in situ from iron salts by simple pH tuning. Thus, all the membrane components are made of environmentally friendly, non-toxic and widely available materials. The membrane has outstanding efficacy against a broad range of viruses, which include enveloped, non-enveloped, airborne and waterborne viruses, such as SARS-CoV-2, H1N1 (the influenza A virus strain responsible for the swine flu pandemic in 2009) and enterovirus 71 (a non-enveloped virus resistant to harsh conditions, such as highly acidic pH), which highlights a possible role in fighting the current and future viral outbreaks and pandemics.

SARS-CoV-2 variants reveal features critical for replication in primary human cells.

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.

Interferon system deficiencies exacerbating severe pandemic virus infections.

Pandemics are caused by novel pathogens to which pre-existing antibody immunity is lacking. Under these circumstances, the body must rely on innate interferon-mediated defenses to limit pathogen replication and allow development of critical humoral protection. Here, we highlight studies on disease susceptibility during H1N1 influenza and COVID-19 (SARS-CoV-2) pandemics. An emerging concept is that genetic and non-genetic deficiencies in interferon system components lead to uncontrolled virus replication and severe illness in a subset of people. Intriguingly, new findings suggest that individuals with autoantibodies neutralizing the antiviral function of interferon are at increased risk of severe COVID-19. We discuss key questions surrounding how such autoantibodies develop and function, as well as the general implications of diagnosing interferon deficiencies for personalized therapies.

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir (preprint)

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we screened by high-throughput docking 6,000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3CL main protease inhibitor in vitro (IC50 value of 29 M) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication in Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our study shows that combining computational and cellular screening is an efficient means to identify existing drugs with repurposing potential as antiviral compounds. Future studies should aim at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

Antiviral Activity of Type I, II, and III Interferons Counterbalances ACE2 Inducibility and Restricts SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is a recently emerged respiratory coronavirus that has infected >23 million people worldwide with >800,000 deaths. Few COVID-19 therapeutics are available, and the basis for severe infections is poorly understood. Here, we investigated properties of type I (ß), II (γ), and III (λ1) interferons (IFNs), potent immune cytokines that are normally produced during infection and that upregulate IFN-stimulated gene (ISG) effectors to limit virus replication. IFNs are already in clinical trials to treat COVID-19. However, recent studies highlight the potential for IFNs to enhance expression of host angiotensin-converting enzyme 2 (ACE2), suggesting that IFN therapy or natural coinfections could exacerbate COVID-19 by upregulating this critical virus entry receptor. Using a cell line model, we found that beta interferon (IFN-ß) strongly upregulated expression of canonical antiviral ISGs, as well as ACE2 at the mRNA and cell surface protein levels. Strikingly, IFN-λ1 upregulated antiviral ISGs, but ACE2 mRNA was only marginally elevated and did not lead to detectably increased ACE2 protein at the cell surface. IFN-γ induced the weakest ISG response but clearly enhanced surface expression of ACE2. Importantly, all IFN types inhibited SARS-CoV-2 replication in a dose-dependent manner, and IFN-ß and IFN-λ1 exhibited potent antiviral activity in primary human bronchial epithelial cells. Our data imply that type-specific mechanisms or kinetics shape IFN-enhanced ACE2 transcript and cell surface levels but that the antiviral action of IFNs against SARS-CoV-2 counterbalances any proviral effects of ACE2 induction. These insights should aid in evaluating the benefits of specific IFNs, particularly IFN-λ, as repurposed therapeutics.IMPORTANCE Repurposing existing, clinically approved, antiviral drugs as COVID-19 therapeutics is a rapid way to help combat the SARS-CoV-2 pandemic. Interferons (IFNs) usually form part of the body's natural innate immune defenses against viruses, and they have been used with partial success to treat previous new viral threats, such as HIV, hepatitis C virus, and Ebola virus. Nevertheless, IFNs can have undesirable side effects, and recent reports indicate that IFNs upregulate the expression of host ACE2 (a critical entry receptor for SARS-CoV-2), raising the possibility that IFN treatments could exacerbate COVID-19. Here, we studied the antiviral- and ACE2-inducing properties of different IFN types in both a human lung cell line model and primary human bronchial epithelial cells. We observed differences between IFNs with respect to their induction of antiviral genes and abilities to enhance the cell surface expression of ACE2. Nevertheless, all the IFNs limited SARS-CoV-2 replication, suggesting that their antiviral actions can counterbalance increased ACE2.