Lessons Learned from In-Person Conferences in the Times of COVID-19.

Scientific societies and conference secretariats have recently resumed in-person meetings after a long pause owing to the COVID-19 pandemic. Some safety measures continue to be implemented at these in-person events to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With increased numbers of waves of infection, caused by the emergence of SARS-CoV-2 variants, additional information is needed to ensure maximal safety at in-person events. The MEX-DART case study was conducted at the in-person Hep-DART 2021 conference, which was held in Los Cabos, Mexico, in December 2021. Many COVID-19 safety measures were implemented, and incidence of SARS-CoV-2 infection during the conference was tested onsite. In this study, we highlight the specific conditions and safety measures set in place at the conference. In addition to vaccination requirements, social distancing, and mask wearing, daily rapid testing was implemented for the duration of the conference. At the end of the 4-day meeting, none of the 166 delegates (and family members attending the conference) had tested antigen positive for SARS-CoV-2. Two delegates tested positive in the week after the conference; the timing of their positive test result suggests that they contracted the virus during their travels home or during postconference vacationing. We believe that this model can serve as a helpful template for organizing future in-person meetings in the era of COVID-19 and any other respiratory virus pandemics of the future. While the outcomes of this case study are encouraging, seasonal surges in respiratory virus infections such as SARS-CoV-2, RSV, and influenza virus incidence suggest that continued caution is warranted.

TREM2+ and interstitial-like macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques.

The immunopathological mechanisms driving the development of severe COVID-19 remain poorly defined. Here, we utilize a rhesus macaque model of acute SARS-CoV-2 infection to delineate perturbations in the innate immune system. SARS-CoV-2 initiates a rapid infiltration of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generate a longitudinal scRNA-Seq dataset of airway cells, and map these subsets to corresponding populations in the human lung. SARS-CoV-2 infection elicits a rapid recruitment of two macrophage subsets: CD163+MRC1-, and TREM2+ populations that are the predominant source of inflammatory cytokines. Treatment with baricitinib (Olumiant®), a JAK1/2 inhibitor is effective in eliminating the influx of non-alveolar macrophages, with a reduction of inflammatory cytokines. This study delineates the major lung macrophage subsets driving airway inflammation during SARS-CoV-2 infection.

Synthesis of 4'-Substituted Carbocyclic Uracil Derivatives and Their Monophosphate Prodrugs as Potential Antiviral Agents.

Over the past decades, both 4'-modified nucleoside and carbocyclic nucleoside analogs have been under the spotlight as several compounds from either family showed anti-HIV, HCV, RSV or SARS-CoV-2 activity. Herein, we designed compounds combining these two features and report the synthesis of a series of novel 4'-substituted carbocyclic uracil derivatives along with their corresponding monophosphate prodrugs. These compounds were successfully prepared in 19 to 22 steps from the commercially available (-)-Vince lactam and were evaluated against a panel of RNA viruses including SARS-CoV-2, influenza A/B viruses and norovirus.

Botanical inhibitors of SARS-CoV-2 viral entry: a phylogenetic perspective.

Throughout the SARS-CoV-2 pandemic, the use of botanical dietary supplements in the United States has increased, yet their safety and efficacy against COVID-19 remains underexplored. The Quave Natural Product Library is a phylogenetically diverse collection of botanical and fungal natural product extracts including popular supplement ingredients. Evaluation of 1867 extracts and 18 compounds for virus spike protein binding to host cell ACE2 receptors in a SARS-CoV-2 pseudotyped virus system identified 310 extracts derived from 188 species across 76 families (3 fungi, 73 plants) that exhibited ≥ 50% viral entry inhibition activity at 20 µg/mL. Extracts exhibiting mammalian cytotoxicity > 15% and those containing cardiotoxic cardiac glycosides were eliminated. Three extracts were selected for further testing against four pseudotyped variants and infectious SARS-CoV-2 and were then further chemically characterized, revealing the potent (EC50 < 5 µg/mL) antiviral activity of Solidago altissima L. (Asteraceae) flowers and Pteridium aquilinum (L.) Kuhn (Dennstaedtiaceae) rhizomes.

Synthesis of Novel N4-Hydrocytidine Analogs as Potential Anti-SARS-CoV-2 Agents.

Coronavirus disease 2019 (COVID-19) is an emerging global pandemic with severe morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molnupiravir, an ester prodrug form of N4-hydroxycytidine (NHC), was recently emergency-use approved for the treatment of early SARS-CoV-2 infections. Herein, we report the synthesis and evaluation of a series of novel NHC analogs.

An ACE2-IgG4 Fc Fusion Protein Demonstrates Strong Binding to All Tested SARS-CoV-2 Variants and Reduced Lung Inflammation in Animal Models of SARS-CoV-2 and Influenza.

Background: The continued emergence of SARS-CoV-2 variants has caused concern that a constantly evolving virus will escape vaccines and antibody therapies. New approaches are needed. Methods: We created and manufactured an ACE2 extracellular domain (ECD) fragment Fc fusion drug candidate, G921, and engineered the compound for enhanced delivery of drug to peripheral tissues by minimizing the size of the ACE2 ECD and by incorporating an Fc domain to enhance transcytosis. G921 was assessed for binding, neutralization, in vivo anti-inflammatory effect, and pharmacokinetic profile. Results: G921 was expressed as an IgG4 Fc fusion protein presenting two ACE2 domains to ACE2 ligands while avoiding risk of infection via antibody-dependent enhancement. G921 strongly binds to the SARS-CoV-2 Wuhan-Hu-1 spike protein and demonstrates further diminished off rate to the spike protein from each of the currently identified variants of concern. G921 demonstrates ACE2 enzymatic activity comparable to positive control and binding to the neonatal Fc receptor (FcRn) without binding to low affinity Fc-gamma receptors (FcγRs). G921 is effective in a concentration-dependent manner in a focus reduction neutralization assay with EC50=16.3±4.2 µg/mL without cytotoxicity in Vero E6 cells when tested at 200 µg/mL in an MTS cell proliferation assay. G921 demonstrates statistically significant reduction of lung inflammation in relevant models of both SARS-CoV-2 and influenza. The pharmacokinetic profile demonstrated dose-dependent exposure with a multi-day half-life in monkeys and rats. Conclusion: G921 data are consistent with both antiviral and anti-inflammatory modes of action. G921 is a novel approach for the prevention and treatment of COVID-19 and possible other diseases characterized by deficiency of ACE2.

Assessment of a Computational Approach to Predict Drug Resistance Mutations for HIV, HBV and SARS-CoV-2.

Viral resistance is a worldwide problem mitigating the effectiveness of antiviral drugs. Mutations in the drug-targeting proteins are the primary mechanism for the emergence of drug resistance. It is essential to identify the drug resistance mutations to elucidate the mechanism of resistance and to suggest promising treatment strategies to counter the drug resistance. However, experimental identification of drug resistance mutations is challenging, laborious and time-consuming. Hence, effective and time-saving computational structure-based approaches for predicting drug resistance mutations are essential and are of high interest in drug discovery research. However, these approaches are dependent on accurate estimation of binding free energies which indirectly correlate to the computational cost. Towards this goal, we developed a computational workflow to predict drug resistance mutations for any viral proteins where the structure is known. This approach can qualitatively predict the change in binding free energies due to mutations through residue scanning and Prime MM-GBSA calculations. To test the approach, we predicted resistance mutations in HIV-RT selected by (-)-FTC and demonstrated accurate identification of the clinical mutations. Furthermore, we predicted resistance mutations in HBV core protein for GLP-26 and in SARS-CoV-2 3CLpro for nirmatrelvir. Mutagenesis experiments were performed on two predicted resistance and three predicted sensitivity mutations in HBV core protein for GLP-26, corroborating the accuracy of the predictions.

Identification of Botanical Viral Entry Inhibitors for SARS‐CoV‐2

The examination of biodiversity across the world has historically been a critical part of drug development and led to the discovery of common medications for many medical issues including pain management, cancer, heart disease, and infections. During the SARS‐CoV‐2 pandemic, the use of natural supplements in the United States has increased. The efficacy of these natural products to prevent SARS‐CoV‐2 infection and the safety of their use remains unexplored;therefore, more research must be done to determine which supplements have antiviral properties. The Quave Natural Product Library (QNPL) is a collection of over 2,000 botanical and fungal extracts and includes the 40 most used natural supplements in the United States. Collection of the biological samples for the library requires field expeditions to areas throughout the world with high levels of biodiversity. Each of these extracts were tested in a SARS‐CoV‐2 pseudotyped virus system to determine which extracts inhibit viral entry, specifically the virus spike protein binding to host cells ACE2 receptors. Mammalian cell cytotoxicity assays were run in parallel. Evaluation of 1,887 extracts and 18 single compounds from the QNPL against SARS‐CoV‐2 identified. 317 extracts derived from 134 species across 76 families (1 lichen, 2 fungi, 73 plant families) exhibited ≥50% inhibition activity in the wild type spike pseudotyped model at 20 µg/mL. Of these bioactive extracts, 129 extracts derived from 95 plant species exhibited ≥85% inhibition activity and ≤15% cytotoxicity in the wild‐type model. Once these 129 extracts were identified, an interesting pattern emerged indicating many hits were from species that are known to be cardiotoxic due to rich composition of cardiac glycosides. For further selection and testing, we reviewed each extract and consulted the literature to eliminate extracts with those properties or similar compounds, which narrowed down our interest to 8 extracts. These extracts were further validated in a concentration‐response assay in a pseudotyped virus model. The EC50 values of the top 3 extracts were all under 10 µg/mL. These 3 extracts all exhibited activity (≥85% inhibition activity) in the wildtype and variant pseudotyped models. Testing in live SARS‐CoV‐2 confirmed antiviral activity from 2 of 3 extracts, Plant Aflowers and Plant B roots. Further chemical characterization of the major metabolites of these two hits was performed using MS/MS fragmentation data compared with the literature, in silico prediction, and web‐based databases. The results revealed phenylpropanoids, flavonoids, triterpenes, glycosidic terpenes, and fatty acids as the major chemical classes. The next steps of this study seek to identify and isolate purified bioactive compounds to further understand their role in SARS‐CoV‐2 inhibition.

Baricitinib attenuates the proinflammatory phase of COVID-19 driven by lung-infiltrating monocytes.

SARS-CoV-2-infected subjects are generally asymptomatic during initial viral replication but may suffer severe immunopathology after the virus has receded and monocytes have infiltrated the airways. In bronchoalveolar lavage fluid from severe COVID-19 patients, monocytes express mRNA encoding inflammatory mediators and contain SARS-CoV-2 transcripts. We leverage a human small airway model of infection and inflammation, whereby primary blood monocytes transmigrate across SARS-CoV-2-infected lung epithelium to characterize viral burden, gene expression, and inflammatory mediator secretion by epithelial cells and monocytes. In this model, lung-infiltrating monocytes acquire SARS-CoV-2 from the epithelium and upregulate expression and secretion of inflammatory mediators, mirroring in vivo data. Combined use of baricitinib (Janus kinase inhibitor) and remdesivir (nucleoside analog) enhances antiviral signaling and viral clearance by SARS-CoV-2-positive monocytes while decreasing secretion of proneutrophilic mediators associated with acute respiratory distress syndrome. These findings highlight the role of lung-infiltrating monocytes in COVID-19 pathogenesis and their importance as a therapeutic target.

Elimination of Aicardi-Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication.

The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi-Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR-Cas9 gene KO or lentiviral viral protein X-mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2-infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.

Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies.

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Lethal mutagenesis as an antiviral strategy.

[Figure: see text].

ß-d-N4-hydroxycytidine Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells.

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in genomes of RNA viruses during viral replication. ß-d-N4-hydroxycytidine (NHC, initial metabolite of molnupiravir) is >100-fold more active than ribavirin or favipiravir against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate highly active mutagenic ribonucleosides may hold risk for the host.

Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern.

To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 CT values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.

Contemporary Approaches to the Discovery and Development of Broad-Spectrum Natural Product Prototypes for the Control of Coronaviruses.

The pressing need for SARS-CoV-2 controls has led to a reassessment of strategies to identify and develop natural product inhibitors of zoonotic, highly virulent, and rapidly emerging viruses. This review article addresses how contemporary approaches involving computational chemistry, natural product (NP) and protein databases, and mass spectrometry (MS) derived target-ligand interaction analysis can be utilized to expedite the interrogation of NP structures while minimizing the time and expense of extraction, purification, and screening in BioSafety Laboratories (BSL)3 laboratories. The unparalleled structural diversity and complexity of NPs is an extraordinary resource for the discovery and development of broad-spectrum inhibitors of viral genera, including Betacoronavirus, which contains MERS, SARS, SARS-CoV-2, and the common cold. There are two key technological advances that have created unique opportunities for the identification of NP prototypes with greater efficiency: (1) the application of structural databases for NPs and target proteins and (2) the application of modern MS techniques to assess protein-ligand interactions directly from NP extracts. These approaches, developed over years, now allow for the identification and isolation of unique antiviral ligands without the immediate need for BSL3 facilities. Overall, the goal is to improve the success rate of NP-based screening by focusing resources on source materials with a higher likelihood of success, while simultaneously providing opportunities for the discovery of novel ligands to selectively target proteins involved in viral infection.

Correlation of SARS-CoV-2 Subgenomic RNA with Antigen Detection in Nasal Midturbinate Swab Specimens.

Among symptomatic outpatients, subgenomic RNA of severe acute respiratory syndrome coronavirus 2 in nasal midturbinate swab specimens was concordant with antigen detection but remained detectable in 13 (82.1%) of 16 nasopharyngeal swab specimens from antigen-negative persons. Subgenomic RNA in midturbinate swab specimens might be useful for routine diagnostics to identify active virus replication.

Comparison of anti-SARS-CoV-2 activity and intracellular metabolism of remdesivir and its parent nucleoside.

Remdesivir, a monophosphate prodrug of nucleoside analog GS-441524, is widely used for the treatment of moderate to severe COVID-19. It has been suggested to use GS-441524 instead of remdesivir in the clinic and in new inhalation formulations. Thus, we compared the anti-SARS-CoV-2 activity of remdesivir and GS-441524 in Vero E6, Vero CCL-81, Calu-3, Caco-2 â€‹cells, and anti-HCoV-OC43 activity in Huh-7 â€‹cells. We also compared the cellular pharmacology of these two compounds in Vero E6, Vero CCL-81, Calu-3, Caco-2, Huh-7, 293T, BHK-21, 3T3 and human airway epithelial (HAE) cells. Overall, remdesivir exhibited greater potency and superior intracellular metabolism than GS-441524 except in Vero E6 and Vero CCL-81 â€‹cells.

Multidisciplinary assessment of the Abbott BinaxNOW SARS-CoV-2 point-of-care antigen test in the context of emerging viral variants and self-administration.

While there has been significant progress in the development of rapid COVID-19 diagnostics, as the pandemic unfolds, new challenges have emerged, including whether these technologies can reliably detect the more infectious variants of concern and be viably deployed in non-clinical settings as "self-tests". Multidisciplinary evaluation of the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW, a widely used rapid antigen test, included limit of detection, variant detection, test performance across different age-groups, and usability with self/caregiver-administration. While BinaxNOW detected the highly infectious variants, B.1.1.7 (Alpha) first identified in the UK, B.1.351 (Beta) first identified in South Africa, P.1 (Gamma) first identified in Brazil, B.1.617.2 (Delta) first identified in India and B.1.2, a non-VOC, test sensitivity decreased with decreasing viral loads. Moreover, BinaxNOW sensitivity trended lower when devices were performed by patients/caregivers themselves compared to trained clinical staff, despite universally high usability assessments following self/caregiver-administration among different age groups. Overall, these data indicate that while BinaxNOW accurately detects the new viral variants, as rapid COVID-19 tests enter the home, their already lower sensitivities compared to RT-PCR may decrease even more due to user error.