In Vitro Evaluation and Mitigation of Niclosamide Liabilities as a COVID-19 Treatment.

Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including B.1.1.7. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future. ImportanceThere is still an urgent need for effective anti-SARS-CoV-2 therapeutics due to waning vaccine efficacy, the emergence of variants of concern, and limited efficacy of existing antivirals. One potential therapeutic option is niclosamide, an FDA approved anthelmintic compound that has shown promising anti-SARS-CoV-2 activity in cell-based assays. Unfortunately, there are significant barriers for the clinical utility of niclosamide as a COVID-19 therapeutic. Our work emphasizes these limitations by showing that niclosamide has high cytotoxicity at antiviral concentrations, variable potency against variants of concern, and significant polypharmacology as a result of its activity as a nonspecific protonophore. Some of these clinical limitations can be mitigated, however, through structural modifications to the niclosamide scaffold, which we demonstrate through a preliminary structure activity relationship analysis. Overall, we show that niclosamide is not a suitable candidate for the treatment of COVID-19, but that structural analogs with improved drug properties may have higher clinical-translational potential.

ARF6 is an important host factor for SARS-CoV-2 infection in vitro

SARS-CoV-2 is a newly emerged beta-coronavirus that enter cells via two routes, direct fusion at the plasma membrane or endocytosis followed by fusion with the late endosome/lysosome. While the viral receptor, ACE2, multiple entry factors, and the mechanism of fusion of the virus at the plasma membrane have been extensively investigated, viral entry via the endocytic pathway is less understood. By using a human hepatocarcinoma cell line, Huh-7, which is resistant to the antiviral action of the TMPRSS2 inhibitor camostat, we discovered that SARS-CoV-2 entry is not dependent on dynamin but dependent on cholesterol. ADP-ribosylation factor 6 (ARF6) has been described as a host factor for SARS-CoV-2 replication and it is involved in the entry and infection of several pathogenic viruses. Using CRISPR-Cas9 genetic deletion, we observed that ARF6 is important for SARS-CoV-2 uptake and infection in Huh-7. This finding was corroborated using a pharmacologic inhibitor, whereby the ARF6 inhibitor NAV-2729 showed a dose-dependent inhibition of viral infection. Importantly, NAV-2729 reduced SARS-CoV-2 viral loads also in more physiologic models of infection: Calu-3 and kidney organoids. This highlighted the importance of ARF6 in multiple cell contexts. Together, these experiments points to ARF6 as a putative target to develop antiviral strategies against SARS-CoV-2.

Identification of ACE2 modifiers by CRISPR screening

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. We identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2 in HuH7 cells. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry and suggest potential targets for therapeutic development.

SARS-CoV-2 Total and Subgenomic RNA Viral Load in Hospitalized Patients

Understanding viral load in patients infected with SARS-CoV-2 is critical to epidemiology and infection control. Previous studies have demonstrated that SARS-CoV-2 RNA can be detected for many weeks after symptom onset. The clinical significance of this finding is unclear and, in most patients, likely does not represent active infection. There are, however, patients who shed infectious virus for weeks. Detection of subgenomic RNA transcripts expressed by SARS-CoV-2 has been proposed to represent productive infection and may be a tractable marker for monitoring infectivity. Here, we use RT-PCR to quantify total and subgenomic nucleocapsid (N) and envelope (E) transcripts in 190 SARS-CoV-2 positive samples collected on hospital admission. We relate these findings to duration of symptoms. We find that all transcripts decline at the same rate; however, subgenomic E becomes undetectable before other transcripts. In Kaplan-Meier analysis the median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic RNA compared to total RNA suggesting subgenomic transcript copy number is highly dependent on copy number of total transcripts. The mean difference between total N and subgenomic N is 16-fold (4.0 cycles) and the mean difference between total E and sub-genomic E is 137-fold (7.1 cycles). This relationship is constant over duration of symptoms allowing prediction of subgenomic copy number from total copy number. Although Subgenomic E is undetectable at a time that may more closely reflect the duration of infectivity, its utility in determining active infection may be no more useful than a copy number threshold determined for total transcripts.

Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background "noise". Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.

Prolonged SARS-CoV-2 replication in an immunocompromised patient

We describe a case of chronic COVID-19 in a patient with lymphoma and associated B-cell immunodeficiency. Viral cultures and sequence analysis demonstrate ongoing replication of infectious SARS-CoV-2 virus for at least 119 days. The patient had three admissions related to COVID-19 over a four-month period and was treated twice with remdesivir and convalescent plasma with resolution of symptoms. The lack of seroconversion and prolonged course illustrate the importance of humoral immunity in resolving SARS-CoV-2 infection. This case highlights challenges in managing immunocompromised hosts, who may act as persistent shedders and sources of transmission.

The Effect of Ultraviolet C Radiation Against SARS-CoV-2 Inoculated N95 Respirators

Since March 31st, 2020, during the height of the pandemic, we have decontaminated thousands of 3M 1860 respirators with Ultraviolet C (UVC) for our frontline workers. There is no published peer-reviewed data regarding the dose required to effectively disinfect SARS-CoV-2 on N95 filtering facepiece respirators (FFRs). Four different locations (facepiece and strap) on 5 different N95 FFR models (3M 1860, 8210, 8511, 9211; Moldex 1511) were inoculated with a 10 L drop of SARS-CoV-2 viral stock (8 x 107 TCID50/mL). The outside-facing and wearer-facing surfaces of the respirators were each irradiated with a dose of 1.5 J/cm2 UVC (254 nm). Viable SARS-CoV-2 was quantified by a median tissue culture infectious dose assay (TCID50). UVC delivered using a dose of 1.5 J/cm2, to each side, was an effective method of decontamination for the facepieces of 3M 1860 and Moldex 1511, and for the straps of 3M 8210 and the Moldex 1511. This dose is an appropriate decontamination method to facilitate reuse of respirators for healthcare personnel when applied to certain models/materials. Increasing the dose may improve decontamination for the other models and straps; however, UVC radiation can degrade certain polymers in a dose dependent manner, and the effects may vary greatly between different models. Therefore, fit-testing of UVC decontaminated respirators must be performed each time a new model and/or dose is introduced into the healthcare system.

Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10-15 years from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 FDA-approved compounds and clinical candidates, we identified 17 dose-responsive compounds with in vitro antiviral efficacy in human liver Huh7 cells and confirmed antiviral efficacy in human colon carcinoma Caco-2, human prostate adenocarcinoma LNCaP, and in a physiologic relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein classically found in secretory fluids, including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19. IMPORTANCESince its emergence in China in December 2019, SARS-CoV-2 has caused a global pandemic. Repurposing of FDA-approved drugs is a promising strategy for identifying rapidly deployable treatments for COVID-19. Herein, we developed a pipeline for quantitative high-throughput image-based screening of SARS-CoV-2 infection in human cells that led to the identification of several FDA-approved drugs and clinical candidates with in vitro antiviral activity.

Recommendations for enterovirus diagnostics and characterisation within and beyond Europe.

Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5'noncoding regions (5'NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5'NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden.