Monoclonal Antibodies Specific for SARS-CoV-2 Spike Protein Suitable for Multiple Applications for Current Variants of Concern.

The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spawned an ongoing demand for new research reagents and interventions. Herein we describe a panel of monoclonal antibodies raised against SARS-CoV-2. One antibody showed excellent utility for immunohistochemistry, clearly staining infected cells in formalin-fixed and paraffin embedded lungs and brains of mice infected with the original and the omicron variants of SARS-CoV-2. We demonstrate the reactivity to multiple variants of concern using ELISAs and describe the use of the antibodies in indirect immunofluorescence assays, Western blots, and rapid antigen tests. Finally, we illustrate the ability of two antibodies to reduce significantly viral tissue titers in K18-hACE2 transgenic mice infected with the original and an omicron isolate of SARS-CoV-2.

Warmer ambient air temperatures reduce nasal turbinate and brain infection, but increase lung inflammation in the K18-hACE2 mouse model of COVID-19.

Warmer climatic conditions have been associated with fewer COVID-19 cases. Herein we infected K18-hACE2 mice housed at the standard animal house temperature of ∼22 °C, or at ∼31 °C, which is considered to be thermoneutral for mice. On day 2 post infection, RNA-Seq analyses showed no significant differential gene expression lung in lungs of mice housed at the two temperatures, with almost identical viral loads and type I interferon responses. There was also no significant difference in viral loads in lungs on day 5, but RNA-Seq and histology analyses showed clearly elevated inflammatory signatures and infiltrates. Thermoneutrality thus promoted lung inflammation. On day 2 post infection mice housed at 31 °C showed reduced viral loads in nasal turbinates, consistent with increased mucociliary clearance at the warmer ambient temperature. These mice also had reduced virus levels in the brain, and an ensuing amelioration of weight loss and a delay in mortality. Warmer air temperatures may thus reduce infection of the upper respiratory track and the olfactory epithelium, resulting in reduced brain infection. Potential relevance for anosmia and neurological sequelae in COVID-19 patients is discussed.

IFITM3 is a host restriction factor that inhibits porcine transmissible gastroenteritis virus infection.

Interferon-induced transmembrane proteins (IFITMs) play an important role in the innate immune response triggered by viral infection. Transmissible gastroenteritis virus (TGEV) causes severe diarrhea, vomiting and dehydration in piglets, resulting in huge economic losses to the swine industry. In this study, we showed that IFITM3 inhibits the replication of TGEV and interferes with the binding of TGEV to PK15 cells. Moreover, the inhibitory effect of IFITM3 on TGEV circumvents the upregulation of inflammatory cytokines. Subsequently, we found that the M22A mutant loses part of the antiviral effect of IFITM3 on TGEV; in contrast, the K24A mutant enhances the antiviral effect of IFITM3. Notably, our data shows a synergistic effect between IFITM3 and CQ, which further amplifies the antiviral effect against TGEV.

Mouse models of COVID-19 recapitulate inflammatory pathways rather than gene expression.

How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. Herein we use RNA-Seq data and bioinformatics approaches to analyze the transcriptional responses in SARS-CoV-2 infected lungs, comparing 4 human studies with the widely used K18-hACE2 mouse model, a model where hACE2 is expressed from the mouse ACE2 promoter, and a model that uses a mouse adapted virus and wild-type mice. Overlap of single copy orthologue differentially expressed genes (scoDEGs) between human and mouse studies was generally poor (≈15-35%). Rather than being associated with batch, sample treatment, viral load, lung damage or mouse model, the poor overlaps were primarily due to scoDEG expression differences between species. Importantly, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between species. As immunity and immunopathology are the focus of most studies, these mouse models can thus be viewed as representative and relevant models of COVID-19.

Establishment of interferon membrane protein IFITML knockdown cells and investigation of its effect on the replication of PRV, PCV2 and TGEV

To establish a J2-KD (knockdown) cell line stably expressing interfered IFITM1 and study the effect of interference with IFITMI gene on the infection of PCV2, PRV and TGEV. Gene cloning tech- niques were used to constructed pLKO. l-EGFP-Puro-IFITMI recombinant vector, which was co-transfected into 293 FT cells with lentiviral packaging plasmids psPAXZ and pMDZ. G to produce green fluorescent protein labeled lentiviruses expression IFITMlshRNA, the viral supernatant was collected at 48 hours after post transfection. J2 cells were infected with the harvested lentiviruses, screened by puromycin and cloned via cell limited dilution. Real-time PCR identify that the cell lines with stable interference with IFITMl gene were obtained, and via MTT method verify that interference with IFITMI expression had no effect on the growth of J2 cells, the successfully constructed J2 stable cell line interfere with IFITMl expression was named as JZ-KD. PRV, PCV2 and TGEV infected J2-KD cells, respectively. Using real-time fluorescence quantitative PCR detect virus replication. The results showed that J2-KD cell line was successfully generated with interfered IFITMl expression;the copy number of PCV2 and TGEV were in- creased, while PRV was decreased in J'Z-KD cell. Indicating that the interference of IFITMI gene expression markedly inhibited the replication of PRV while promoted that. of TGEV and PCV2, providing a basis for further study on the function of porcine IFITMI protein and elucidates its antiviral mechanism.

Evolution of ACE2-independent SARS-CoV-2 infection and mouse adaption after passage in cells expressing human and mouse ACE2.

Human ACE2 Human angiotensin converting enzyme 2 (hACE2) is the key cell attachment and entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe the emergence of a SARS-CoV-2 strain capable of ACE2-independent infection and the evolution of mouse-adapted (MA) SARS-CoV-2 by in vitro serial passaging of virus in co-cultures of cell lines expressing hACE2 and mACE2. MA viruses evolved with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. MA viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates and caused characteristic lung histopathology. One MA virus also evolved to replicate efficiently in several ACE2-negative cell lines across several species, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ACE2 knockout cells. An E484D substitution is likely involved in ACE2-independent entry and has appeared in only ≈0.003 per cent of human isolates globally, suggesting that it provided no significant selection advantage in humans. ACE2-independent entry reveals a SARS-CoV-2 infection mechanism that has potential implications for disease pathogenesis, evolution, tropism, and perhaps also intervention development.

Cold atmospheric plasma for preventing infection of viruses that use ACE2 for entry.

Rational: The mutating SARS-CoV-2 potentially impairs the efficacy of current vaccines or antibody-based treatments. Broad-spectrum and rapid anti-virus methods feasible for regular epidemic prevention against COVID-19 or alike are urgently called for. Methods: Using SARS-CoV-2 virus and bioengineered pseudoviruses carrying ACE2-binding spike protein domains, we examined the efficacy of cold atmospheric plasma (CAP) on virus entry prevention. Results: We found that CAP could effectively inhibit the entry of virus into cells. Direct CAP or CAP-activated medium (PAM) triggered rapid internalization and nuclear translocation of the virus receptor, ACE2, which began to return after 5 hours and was fully recovered by 12 hours. This was seen in vitro with both VERO-E6 cells and human mammary epithelial MCF10A cells, and in vivo. Hydroxyl radical (·OH) and species derived from its interactions with other species were found to be the most effective CAP components for triggering ACE2 nucleus translocation. The ERα/STAT3(Tyr705) and EGFR(Tyr1068/1086)/STAT3(Tyr705) axes were found to interact and collectively mediate the effects on ACE2 localization and expression. Conclusions: Our data support the use of PAM in helping control SARS-CoV-2 if developed into products for nose/mouth spray; an approach extendable to other viruses utilizing ACE2 for host entry.

Pathophysiological Response to SARS-CoV-2 Infection Detected by Infrared Spectroscopy Enables Rapid and Robust Saliva Screening for COVID-19.

Fourier transform infrared (FTIR) spectroscopy provides a (bio)chemical snapshot of the sample, and was recently used in proof-of-concept cohort studies for COVID-19 saliva screening. However, the biological basis of the proposed technology has not been established. To investigate underlying pathophysiology, we conducted controlled infection experiments on Vero E6 cells in vitro and K18-hACE2 mice in vivo. Potentially infectious culture supernatant or mouse oral lavage samples were treated with ethanol or 75% (v/v) Trizol for attenuated total reflectance (ATR)-FTIR spectroscopy and proteomics, or RT-PCR, respectively. Controlled infection with UV-inactivated SARS-CoV-2 elicited strong biochemical changes in culture supernatant/oral lavage despite a lack of viral replication, determined by RT-PCR or a cell culture infectious dose 50% assay. Nevertheless, SARS-CoV-2 infection induced additional FTIR signals over UV-inactivated SARS-CoV-2 infection in both cell and mouse models, which correspond to aggregated proteins and RNA. Proteomics of mouse oral lavage revealed increased secretion of kallikreins and immune modulatory proteins. Next, we collected saliva from a cohort of human participants (n = 104) and developed a predictive model for COVID-19 using partial least squares discriminant analysis. While high sensitivity of 93.48% was achieved through leave-one-out cross-validation, COVID-19 patients testing negative on follow-up on the day of saliva sampling using RT-PCR was poorly predicted in this model. Importantly, COVID-19 vaccination did not lead to the misclassification of COVID-19 negatives. Finally, meta-analysis revealed that SARS-CoV-2 induced increases in the amide II band in all arms of this study and in recently published cohort studies, indicative of altered ß-sheet structures in secreted proteins. In conclusion, this study reveals a consistent secretory pathophysiological response to SARS-CoV-2, as well as a simple, robust method for COVID-19 saliva screening using ATR-FTIR.

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions.

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives.

BACKGROUND: The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19. The initial step to identifying potential candidates usually involves in vitro screening that includes standard cytotoxicity controls. Under-appreciated is that viable, but stressed or otherwise compromised cells, can also have a reduced capacity to replicate virus. A refinement proposed herein for in vitro drug screening thus includes a simple growth assay to identify drug concentrations that cause cellular stress or "cytomorbidity", as distinct from cytotoxicity or loss of viability. METHODS: A simple rapid bioassay is presented for antiviral drug screening using Vero E6 cells and inhibition of SARS-CoV-2 induced cytopathic effects (CPE) measured using crystal violet staining. We use high cell density for cytotoxicity assays, and low cell density for cytomorbidity assays. RESULTS: The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. In contrast, nitazoxanide, oleuropein, cyclosporine A and ribavirin all showed no ability to inhibit SARS-CoV-2 CPE. Hydroxychloroquine, cyclohexamide, didemnin B, γ-mangostin and linoleic acid were all able to inhibit viral CPE at concentrations that did not induce cytotoxicity. However, these drugs inhibited CPE at concentrations that induced cytomorbidity, indicating non-specific anti-viral activity. CONCLUSIONS: We describe the methodology for a simple in vitro drug screening assay that identifies potential anti-viral drugs via their ability to inhibit SARS-CoV-2-induced CPE. The additional growth assay illustrated how several drugs display anti-viral activity at concentrations that induce cytomorbidity. For instance, hydroxychloroquine showed anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities. The cytomorbidity assay can therefore rapidly exclude potential false positives.

Targeting novel LSD1-dependent ACE2 demethylation domains inhibits SARS-CoV-2 replication.

Treatment options for COVID-19 remain limited, especially during the early or asymptomatic phase. Here, we report a novel SARS-CoV-2 viral replication mechanism mediated by interactions between ACE2 and the epigenetic eraser enzyme LSD1, and its interplay with the nuclear shuttling importin pathway. Recent studies have shown a critical role for the importin pathway in SARS-CoV-2 infection, and many RNA viruses hijack this axis to re-direct host cell transcription. LSD1 colocalized with ACE2 at the cell surface to maintain demethylated SARS-CoV-2 spike receptor-binding domain lysine 31 to promote virus-ACE2 interactions. Two newly developed peptide inhibitors competitively inhibited virus-ACE2 interactions, and demethylase access to significantly inhibit viral replication. Similar to some other predominantly plasma membrane proteins, ACE2 had a novel nuclear function: its cytoplasmic domain harbors a nuclear shuttling domain, which when demethylated by LSD1 promoted importin-α-dependent nuclear ACE2 entry following infection to regulate active transcription. A novel, cell permeable ACE2 peptide inhibitor prevented ACE2 nuclear entry, significantly inhibiting viral replication in SARS-CoV-2-infected cell lines, outperforming other LSD1 inhibitors. These data raise the prospect of post-exposure prophylaxis for SARS-CoV-2, either through repurposed LSD1 inhibitors or new, nuclear-specific ACE2 inhibitors.