Screening and Identification of Host Proteins Interacting with PDCoV S1-CTD

The C-terminal domain (CTD) of porcine deltacoronavirus S1 subunit is the main region which induces the neutralizing antibody. S1-CTD was expressed by HEK-293T eukaryotic expression system and purified, and porcine ileal epithelium cells membrane proteins were extracted to investigate porcine host proteins that interact with it. Thirty-two suspected interacting host proteins were obtained by co-inmunprecipitation (Co-IP) and mass spectrometry. Eukaryotic expression plasmid of KIF1 binding protein (KIFBP) was constructed, and the interaction between KIFBP and S1-CTD was identified by Co-IP and laser confocal microscopy. All results proved that KIFBP interacted with S1-CTD and co-located in cytoplasm. Further research indicated that overexpression of KIFBP could effectively reduce the viral mRNA level and the viral titer in which the mRNA level decreased by about 70%, and the viral titer decreased by 101.6TCID50. In conclusion, a host protein KIFBP interacting with PDCoV S1-CTD was screened and identified in this study which provides a theoretical basis for understanding the pathogenesis of PDCoV.

Preclinical studies of antiviral activity of the RPH-137 fusion protein and molnupiravir against COVID-19

Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment. The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection. Materials and methods: the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung-body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann–Whitney test. Results: RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells;the EC50 values of RPH-137 amounted to 4.69 μg/mL (21.3 nM) and 16.24 μg/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 μg/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection. Conclusions: both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research.

In vitro evaluation of the activity of teriflunomide against SARS-CoV-2 and the human coronaviruses 229E and OC43

Previous data have suggested an antiviral effect of teriflunomide, including against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the agent underlying the ongoing COVID-19 pandemic. We undertook an in vitro investigation to evaluate the inhibitory activity of teriflunomide against SARS-CoV-2 in a cell-based assay. Teriflunomide was added to Vero (kidney epithelial) cells that had been infected with SARS-CoV-2. A nucleocapsid immunofluorescence assay was performed to examine viral inhibition with teriflunomide and any potential cytotoxic effect. The 50% effective concentration (EC(50)) for teriflunomide against SARS-CoV-2 was 15.22 μM. No cytotoxicity was evident for teriflunomide in the Vero cells (i.e., the 50% cytotoxic concentration [CC(50)] was greater than the highest test concentration of 100 μM). The data were supported by additional experiments using other coronaviruses and human cell lines. In the SARS-CoV-2-infected Vero cells, the prodrug leflunomide had an EC(50) of 16.49 μM and a CC(50) of 54.80 μM. Our finding of teriflunomide-mediated inhibition of SARS-CoV-2 infection at double-digit micromolar potency adds to a growing body of evidence for a broad-ranging antiviral effect of teriflunomide.

Nitric-oxide enriched plasma-activated water inactivates 229E coronavirus and alters antiviral response genes in human lung host cells

The ongoing pandemic caused by the novel coronavirus, SARS-CoV-2, is influencing global health. Moreover, there is a major threat of future coronaviruses affecting the entire world in a similar, or even more dreadful, manner. Therefore, effective and biocompatible therapeutic options against coronaviruses are urgently needed. To address this challenge, medical specialists require a well-informed and safe approach to treating human coronaviruses (HCoVs). Herein, an environmental friendly approach for viral inactivation, based on plasma technology, was considered. A microwave plasma system was employed for the generation of the high amount of gaseous nitric oxide to prepare nitric oxide enriched plasma-activated water (NO-PAW), the effects of which on coronaviruses, have not been reported to date. To determine these effects, alpha-HCoV-229E was used in an experimental model. We found that NO-PAW treatment effectively inhibited coronavirus infection in host lung cells, visualized by evaluating the cytopathic effect and expression level of spike proteins. Interestingly, NO-PAW showed minimal toxicity towards lung host cells, suggesting its potential for therapeutic application. Moreover, this new approach resulted in viral inactivation and greatly improved the gene levels involved in host antiviral responses. Together, our findings provide evidence of an initiation point for further progress toward the clinical development of antiviral treatments, including such coronaviruses. © 2022 The Authors

Evaluation of the use of VitaPCRTM SARS-CoV-2 Assay for the confirmation of samples tested positive with LIAISON SARS-CoV-2 Ag assay

Background At the Urbino hospital, all patients tested with antigen tests for SARS-CoV-2, if positive, are confirmed with a molecular test. Among the molecular tests in use in our laboratory, VitaPCRTM SARS-CoV-2 Assay is the fastest and easiest to use, as it takes about half an hour from preparation to the result and does not require a biosecurity laboratory, because performed from inactivated samples. Aim of the study Given the rapidity of VitaPCRTM SARS-CoV-2 Assay, it was decided to use it for the confirmation of samples found positive to LIAISON SARS-CoV-2 Ag test. This last test is performed by dedicated tubes with inactivating buffer based on detergent, therefore it does not interfere in the processes of extraction and amplification of nucleic acids. Materials and methods For evaluation, 48 samples tested positive with LIAISON SARS-CoV-2 Ag (Diasorin) test were analyzed, coming from various departments of the Urbino hospital, and confirmed by SimplexaTM COVID-19 Direct (Diasorin) molecular test. From the tubes of all the samples, 200 uL were taken and transferred to dedicated tubes for analysis with VitaPCRTM SARS-CoV-2 Assay (Menarini) to be analyzed, after eliminating 2.2 mL of buffer. Results VitaPCRTM SARS-CoV-2 Assay was able to detect SARSCoV- 2 RNA in all 48 tested antigenically positive samples. In particular, even in all positive samples with low values of TCID50/mL (<1000), 11 out of 48, results with Ct<28.8 were found, although not maintaining a direct correspondence between quantity of antigen and quantity of detected RNA. Conclusions The results obtained, despite requiring an increase in the number of cases, demonstrate the potential use of VitaPCRTM SARS-CoV-2 Assay for the confirmation of positive samples directly from the inactivated buffer used for LIAISON SARS-CoV-2 Ag assay. VitaPCRTM instrument, given the speed, simplicity of use and the possibility of use outside biosafety laboratories, it is an excellent diagnostic tool to support the confirmation procedures of samples tested positive for the SARSCoV- 2 N antigen.

Analytical sensitivity of COVID-19 rapid antigen tests: A case for a robust reference standard.

Aggressive diagnostic testing remains an indispensable strategy for health and aged care facilities to prevent the transmission of SARS-CoV-2 in vulnerable populations. The preferred diagnostic platform has shifted towards COVID-19 rapid antigen tests (RATs) to identify the most infectious individuals. As such, RATs are being manufactured faster than at any other time in our history yet lack the relevant quantitative analytics required to inform on absolute analytical sensitivity enabling manufacturers to maintain high batch-to-batch reproducibility, and end-users to accurately compare brands for decision making. Here, we describe a novel reference standard to measure and compare the analytical sensitivity of RATs using a recombinant GFP-tagged nucleocapsid protein (NP-GFP). Importantly, we show that the GFP tag does not interfere with NP detection and provides several advantages affording streamlined protein expression and purification in high yields as well as faster, cheaper and more sensitive quality control measures for post-production assessment of protein solubility and stability. Ten commercial COVID-19 RATs were evaluated and ranked using NP-GFP as a reference standard. Analytical sensitivity data of the selected devices as determined with NP-GFP did not correlate with those reported by the manufacturers using the median tissue culture infectious dose (TCID50) assay. Of note, TCID50 discordance has been previously reported. Taken together, our results highlight an urgent need for a reliable reference standard for evaluation and benchmarking of the analytical sensitivity of RAT devices. NP-GFP is a promising candidate as a reference standard that will ensure that RAT performance is accurately communicated to healthcare providers and the public.

Evaluation of a simple ultrafiltration method for concentration of infective canine parvovirus and feline coronavirus from cell culture supernatants.

Enrichment of viral infectious titers following its propagation by cell culture is desirable for various experimental studies. The performance of an ultrafiltration (UF) process to concentrate infectious titers of non-enveloped Canine parvovirus 2 (CPV-2) and enveloped Feline coronavirus (FCoV) obtained from cell culture supernatants was evaluated in this study, and compared with ultracentrifugation (UC) process. A mean gain of > 1.0 log10 TCID50/mL was obtained for CPV-2 with UF, which was comparable with the gain obtained by UC. On the other hand, the gain was lower (0.7-1.0 log10 TCID50/mL) for FCoV with UF in contrast to UC (> 2.0 log10 TCID50/mL). However, the lower retentate volume following UC (∼120 fold) compared to that following UF (∼10 fold) for either of the viruses suggests a trend of increased infectious titer retention in UF concentrates relative to UC concentrates. The simplistic UF process evaluated here thus has the potential for use in applications requiring increased infectious titers of CPV-2 and FCoV.

ASSURED-compliant point-of-care diagnostics for the detection of human viral infections

Viral disease outbreaks have been always a threat to global public health making affordable, rapid and accurate diagnostics highly important tools to slow down the spread of viruses and decrease the mortality rate. Point-of-care (POC) diagnostics have emerged as a strong tool for the diagnosis of viral infections, especially in countries where health-care systems are inadequate to provide proper services to all citizens. According to the World Health Organization, an ideal POC diagnostic must be Affordable, Sensitive, Specific, User-friendly, Rapid/Robust, Equipment-free and Deliverable (ASSURED). This review surveys carefully each ASSURED criterion and identifies where existing viral POC diagnostics fail to meet these criteria. Given the widespread concern with plastic pollution, we also propose the addition of 'disposability' to the existing ASSURED criteria and consider the letter “D” as the representative of both Deliverable and Disposable. Next, the POC tests used for the diagnosis of several common human viral infections which met all the ASSURED criteria (ASSURED-compliant) are described in detail. Finally, the future of ASSURED-compliant POC diagnostics, capable of generating comparable results to the viral diagnostic gold standards, is discussed.

Differential Infectivity of Original and Delta Variants of SARS-CoV-2 in Children Compared to Adults.

Although children of all ages are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, they have not been implicated as major drivers of transmission thus far. However, it is still unknown if this finding holds true with new variants of concern (VOC), such as Delta (B.1.617.2). This study aimed to examine differences in both viral RNA (as measured by cycle threshold [CT]) and viable-virus levels from children infected with Delta and those infected with original variants (OV). Furthermore, we aimed to compare the pediatric population infection trends to those in adults. We obtained 690 SARS-CoV-2 RT-PCR positive nasopharyngeal swabs from across Manitoba, Canada, which were further screened for mutations characteristic of VOC. Aliquots of sample were then provided for TCID50 (50% tissue culture infective dose) assays to determine infectious titers. Using a variety of statistical analyses we compared CT and infectivity of VOC in different age demographics. Comparing 122 Delta- to 175 OV-positive nasopharyngeal swab samples from children, we found that those infected with Delta are 2.7 times more likely to produce viable SARS-CoV-2 with higher titers (in TCID50 per milliliter), regardless of viral RNA levels. Moreover, comparing the pediatric samples to 130 OV- and 263 Delta-positive samples from adults, we found only that the Delta pediatric culture-positive samples had titers (TCID50 per milliliter) similar to those of culture-positive adult samples. IMPORTANCE These important findings show that children may play a larger role in viral transmission of Delta than for previously circulating SARS-CoV-2 variants. Additionally, they may suggest a mechanism for why Delta has evolved to be the predominant circulating variant.

Comparison among plaque assay, tissue culture infectious dose (TCID) 50 and real-time RT-PCR for SARS-CoV-2 variants quantification

Background and Objectives: SARS-CoV-2 variants of concern (VOC) and interest (VOI) pose a significant threat to public health because the rapid change in the SARS-CoV-2 genome can alter viral phenotypes such as virulence, transmissi-bility and the ability to evade the host response. Hence, SARS-CoV-2 quantification techniques are essential for timely diagnosis and follow-up. Besides, they are vital to understanding viral pathogenesis, antiviral evaluation, and vaccine de-velopment. Materials and Methods: Five isolates of SARS-CoV-2: D614G strain (B.1), three VOC (Alpha, Gamma and Delta), and one VOI (Mu) were used to compare three techniques for viral quantification, plaque assay, median tissue culture infectious dose (TCID) and real-time RT-PCR. 50 Results: Plaque assay showed viral titers between 0.15 ± 0.01×107 and 1.95 ± 0.09×107 PFU/mL while viral titer by TCID 50 assay was between 0.71 ± 0.01×106 to 4.94 ± 0.80×106 TCID /mL for the five SARS-CoV-2 isolates. The PFU/mL titer 50 obtained by plaque and the calculated from TCID assays differed by 0.61 log10, 0.59 log10, 0.59 log10 and 0.96 log10 50 for Alfa, Gamma, Delta, and Mu variants (p≤0.0007), respectively. No differences were observed for the D614G strain. Real-time PCR assay exhibited titers ranging from 0.39 ± 0.001×108 to 3.38 ± 0.04×108 RNA copies/µL for all variants. The relation between PFU/mL and RNA copies/mL was 1:29800 for D614G strain, 1:11700 for Alpha, 1:8930 for Gamma, 1:12500 for Delta, and 1:2950 for Mu. Conclusion: TCID assay was comparable to plaque assay for D614G but not for others SARS-CoV-2 variants. Our data 50 demonstrated a correlation among PFU/mL and E gene RNA copies/µL, units of measure commonly used to quantify the viral load in diagnostic and research fields. The results suggest that the proportion of infectious virions in vitro changes de-pending on the SARS-CoV-2 variant, being Mu, the variant reaching a higher viral titer with fewer viral copies.

TREK-1 Potassium Channel Activation Protects Against Influenza-A Induced Lung Injury

Introduction and Rationale: No targeted therapies exist that improve the outcomes of patients with Acute Respiratory Distress Syndrome (ARDS), in part to the multifactorial etiology of this devastating disease. Infectious agents remain the most common initiating insults, and besides SARS-CoV-2, Influenza-A virus (IAV) is responsible for more ARDS cases and deaths than any other agent. In fact, IAV increases the risk of mortality in ARDS patients three-fold, and accounts for almost half of all ARDS deaths. We recently identified TREK-1 potassium channels on epithelial cells as important regulators of alveolar inflammation and barrier function, two hallmarks of ARDS, and found that pharmacological activation of TREK-1 protects against hyperoxia-induced lung injury. However, whether TREK-1 channels convey similar protection in a clinically more relevant IAVinduced lung injury model, remains unknown. Methods: We infected adult C57BL/6 wildtype mice intra-tracheally (i.t.) with IAV (PR8 strain;TCID50 400), followed by once-daily i.t. injections (days 5, 6 and 7 post-IAV) with the novel TREK-1 activating compounds ML335 (60mcg/kg), BL1249 (100mcg/kg), or a vehicle control, to create a clinically-relevant treatment model. To evaluate the role of epithelial cells in this model, we infected primary human alveolar epithelial cells (HAEC) with IAV (0.01 pfu) for 24 hours. Endpoint analysis consistent in quantification of quasi-static lung compliance;BAL fluid total protein, cell counts, and ROS concentrations;cytokine levels in BAL fluid and cell supernatants;and HAEC viability (XTT assay). In addition, we measured alterations in epithelial potassium currents (fluorometric FLIPR assays) and in IAV-induced signaling cascades (real-time PCR) following IAV infection and treatment with our TREK-1 activators. Results: Oncedaily treatment of mice with the TREK-1 activating compounds ML335 or BL1249 following IAV infection improved lung compliance, and BAL fluid total protein levels, cell counts, IL-6, CXCL-10, MIP-1alpha, and TNF-alpha concentrations, but not ROS, CCL-2 or IL-10 levels. In HAEC, TREK-1 activation improved IAV-induced IL-6, CXCL-10, and CCL-2 levels, while MIP-1alpha, TNF-alpha and IL-10 levels remained unchanged. XTT assays confirmed that in our model IAV infection did not cause significant cell death. Interestingly, IAV infection decreased HAEC potassium currents, which could be counteracted by TREK-1 activation and cell hyperpolarization. Finally, TREK-1 activationmediated cell hyperpolarization inhibited TLR4- and TNFSF13-mediated downstream signaling in IAV-infected HAEC, whereas NFkB, RIG1, TLR3, and TLR7 signaling was not affected. Conclusions: TREK-1 potassium channel activation may represent a novel approach to protect against IAV-induced acute lung injury.

Zebrafish embryos to study BSL-3 viruses and antiviral molecules

Emerging viruses are currently a burden with the recent SARS-CoV-2 pandemic resulting in more than 6 million death worldwide. Other viruses such as Arboviruses, transmitted by mosquitoes, can also emerge easily and represent a threat for humans and animals. These viruses are often RNA enveloped viruses and require to be studied in a BSL-3 (Bio-Safety- Laboratory of level 3 security), underlying the need to develop simple and rapid antiviral screenings. In recent years, the zebrafish has become a powerful tool in the biomedical sector to study viral infection and immunity. The optical transparency of the zebrafish embryo offers a major advantage for real time imaging of the infection process, and study hostpathogen interactions at subcellular levels in living systems using fluorescently labelled pathogens. While zebrafish embryos have been shown to be a successful vertebrate model to study a large panel of human disease, this model has been very little explored to study BSL-3 pathogen infection and propagation. This project was created to develop the zebrafish infection model for emerging BSL-3 viruses, and evolve towards high-content screening methods for antiviral molecules. Thanks to the setup of a microinjection system under a laminar flow hood in the BSL-3, we were able to inject in zebrafish embryos several emerging viruses, including the Chikungunya virus (CHIKV), Dengue virus (DENV) and SARS-CoV-2. Using fluorescently tagged viruses, infection was monitored in real time in vivo and confirmed with classical virology methods (RT-qPCR, plaque assays, TCID50). We then tested several nucleoside analogues described for their antiviral activity in vitro for these different viruses and we were able to validate the antiviral effect of some of these molecules in infected embryos. Altogether, our zebrafish infection model will provide us a better understanding of in vivo infection and propagation of these emergent BSL-3 viruses, and will be used as an intermediate model between in vitro antiviral screens and in vivo screens in mammals.

Rational development of tools to appropriately respond to SARSCoV- 2 needs

For many years, our laboratory has been developing cellular models for the study of human pathogenic viruses with RNA genomes, in order to study the replication of these pathogens, to propose new therapeutic pathways, to screen and test inhibitors. In response to the COVID-19 outbreak, we have set up the tools for the study of SARS-CoV-2 replication. First, clinical and reference SARS-CoV-2 strains have been successfully isolated and amplified using Vero E6 cells in the BSL3 facility of Bordeaux University (UB'L3, www.mfp.cnrs.fr/wp/larecherche/ andevir/ubl3/). We set up the monitoring of SARS-CoV-2 replication using conventional RT-qPCR quantification as well as evaluation of the cytopathic effect by microscopic observation or content analysis. Using VERO cells, we are now able to precisely titer viral supernatant (determination of the TCID50) and screen for potential antiviral molecule (determination of EC50 and CC50). We have developed a full-length Spike sequencing based on a Sanger approach1 as well as whole genome sequencing by nanopore technology, allowing the tracking of emerging variants. In parallel, we developed various other models to study SARS-CoV-2 replication including Calu-3 cells, modified human cells expressing Ace2 (e.g. 293T, U2OS) or even more complex cellular models (reconstituted human airway epithelium, vessels) according to the biological question to resolve. As an example, bronchial epithelia reconstituted from biopsies of adult or child donors were used to evaluate the inflammatory response upon SARS-CoV-2 infection in an age-dependent manner [2] (see poster G. Beucher). Similarly reconstituted blood vessels were used to study the impact of SARS-CoV- 2 infection on the vascular system and determine whether clinical observations (blood brain barrier damages, myocarditis) are due to direct infection of cells or indirect effects. Finally, we evaluate the efficacy of different chemical or physical processes for viral inactivation in air or on surfaces.

PROTECTIVE EFFICACY of A GASTROINTESTINAL SARS-CoV-2 VACCINE

Background: SARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations;however no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Thus far, we assessed whether oral administration of live SARS-CoV-2 in non-human primates might offer prophylactic benefits. Methods: In this study, we assessed the immunogenicity of gastrointestinal (GI) delivery of SARS-CoV-2 and the protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge in rhesus macaques. Esophagogastroduodenoscopy (EGD) administration of 106 50% Tissue Culture Infectious Dose (TCID50) of SARS-CoV-2 elicited low levels of serum neutralizing antibodies (NAb), which correlated with modestly diminished viral loads in nasal swabs (NS) and Bronchoalveolar Lavage (BAL) post-challenge. In addition, mucosal NAb titers from the rectal swabs (RS), NS, and BAL and Spike-specific T-cell responses appear to be below the limit of detection post-vaccination. Replicating virus was only observed in 44% of macaques and on limited number of dates post vaccination, suggesting limited, if any, productive infection in the GI tract. Results: We demonstrate that GI delivery of live 1x106 TCID50 SARS-CoV-2 elicited modest immune responses and provided partial protection against intranasal and intratracheal challenge with SARS-CoV-2. Moreover, serum neutralizing antibody titers correlated with protective efficacy. Conclusion: These data provide proof-of-concept that an orally administered vaccine can protect against respiratory SARS-CoV-2 challenge, but the limited immunogenicity and protective efficacy observed here suggests that the oral vaccine approach will require optimization.

RESTRICTED INFECTION of MACROPHAGES by SARS-CoV-2 INDUCES PROINFLAMMATORY RESPONSES

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with immune hyperactivation and high levels of proinflammatory cytokines. Extensive lung infiltration by CD169+ inflammatory monocytes and presence of activated CD169+ alveolar macrophages suggest monocyte/macrophages are key drivers of severe morbidity and mortality. In this study, we determined whether CD169 mediated ACE2-independent SARS-CoV-2 entry and restricted viral genome replication in macrophages triggers pro-inflammatory cytokine expression. Methods: Monocyte-derived macrophages (MDMs) and PMA-differentiated THP-1 macrophages engineered to constitutively express CD169, ACE2, or CD169 and ACE2 were infected with USA-WA1/2020/SARS-CoV-2 isolate with or without Remdesivir pre-treatment. To identify mechanism of innate immune activation, nucleic acid sensing pathways were selectively depleted in CD169+ macrophages. Extent of viral genomic (gRNA) and sub-genomic (sgRNA) expression and induction of pro-inflammatory cytokines was determined by qRT-PCR and single molecule RNA FISH analysis. Viral protein expression and infectious virus particle production was determined by immunofluorescence analysis and TCID50. Results: While productive virus infection (viral protein expression and infectious virus particle release) was only observed in ACE2+ macrophages, SARS-CoV-2 N or S expression and infectious virus production was not observed in CD169+ macrophages. Co-expression of ACE2 and CD169 significantly enhanced infectious virus production and spread. Interestingly, smFISH and RT-qPCR analysis revealed CD169+ cells express cytosolic negative-strand gRNA and positive strand sgRNA. Importantly, CD169-mediated SARS-CoV-2 infection of macrophages and expression of viral mRNAs led to induction of pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, despite lack of viral protein expression in CD169+ macrophages. Pre-treatment with Remdesivir blocked de novo expression of viral mRNAs and induction of inflammatory cytokines in CD169-dependent infection of macrophages. Furthermore, knockdown of cytosolic RLRs (RIG-I and MDA-5) or MAVS significantly attenuated inflammatory cytokine expression in CD169+ macrophages, confirming that nucleic acid sensing of restricted cytosolic viral mRNA expression in macrophages triggers innate immune activation. Conclusion: These results suggest that restricted SARS-CoV-2 infection of CD169+ macrophages contributes to COVID-19-associated hyperinflammatory cytokine response.

GALECTIN-9 INDUCES SARS-CoV-2 REPLICATION and INFLAMMATION in AIRWAY EPITHELIAL CELLS

Background: Galectin-9 (Gal-9) is a β-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis. Multiple recent reports demonstrate that plasma levels of Gal-9 are elevated in the setting of severe COVID-19 disease. However, a causal role of Gal-9 in SARS-CoV-2 pathology remains to be elucidated. Here, we determined the impact of Gal-9 on SARS-CoV-2 replication and pro-inflammatory signaling in immortalized and primary human airway epithelial cells (AECs). Methods: Dose-dependent cytotoxicity of recombinant human Gal-9 in the Calu-3 AEC line was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 lineage P.1 (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the nucleocapsid (N) gene, immunofluorescence assay (IFA) of N protein, and titration of supernatant (TCID50). Viral entry was measured using luciferase activity of VSV-SARS-CoV-2 S-ΔG-Luciferase reporter pseudovirus. ACE2 and TMPRSS2 cell-surface expression were measured by flow cytometry. Pro-inflammatory factors (IL-6, IL-8, and TNFα) were detected by RT-PCR. Total RNA-seq was used to evaluate Gal-9 effects on the host transcriptome. Groups were compared by Student's t-test, and differential expression analyses were performed using DESeq2. Results: Gal-9 reached 50% cytotoxicity in Calu-3 cells at 597 nM. Gal-9 significantly increased SARS-CoV-2 expression (8.1 to 25.5 fold;p<0.0001) and infectious virus release (1.9 to 17.8 fold;p<0.038) in a dose-dependent manner in Calu-3 cells. Pseudovirus entry into Calu-3 cells was enhanced by Gal-9 (2.4 to 5.6 fold;p<0.0016), and the enhanced entry was inhibited by anti-ACE2 antibody (p<0.0027). Cell surface ACE2 and TMPRSS2 expression were unaffected by Gal-9. Gal-9 treatment accelerated virus-induced expression of IL-6, IL-8, and TNFα (p<0.018) in Calu-3 cells. Gal-9 increased SARS-CoV-2 production (p=0.03) and pro-inflammatory factor expression (p<0.05) in primary AECs (N=5 donors). RNA-seq data revealed that Gal-9 significantly induced IL-17, EIF2, IL-8 and IL-6 signaling pathways in the setting of SARS-CoV-2 infection. Conclusion: Gal-9 facilitates SARS-CoV-2 entry, replication, and virus-induced pro-inflammatory signaling in AECs ex vivo. Our data suggest that pharmacologic manipulation of Gal-9 should be explored as a SARS-CoV-2 therapeutic strategy.

IFITM DEPENDENCY of SARS-CoV-2 VARIANTS of CONCERN

Background: We recently showed that genuine SARS-CoV-2 hijacks endogenously expressed interferon-induced transmembrane proteins, especially IFITM2, as entry cofactors for efficient infection (Prelli Bozzo, Nchioua et al., Nat. Com., 2021). This came as a surprise, since IFITMs have been reported to inhibit entry of numerous enveloped viruses, including SARS-CoV-2. However, most data were obtained using IFITM overexpression and pseudoparticle infection assays. In our initial study, we used a SARS-CoV-2 strain isolated in the Netherlands in February 2020 (NL-02-2020). Since then several "variants of concern" (VOCs) have emerged that show increased transmission fitness and evasion of vaccine-induced immunity. These VOCs contain various alterations in their Spike (S) proteins that may alter their dependency on entry cofactors. Here, we examined whether SARS-CoV-2 VOCs, including the currently dominating Delta variant, still depend on IFITMs for efficient infection and replication. Methods: To determine the role of IFITMs in infection of SARS-CoV-2 VOCs, we silenced IFITM1, 2, or 3 expression in Calu-3 cells using siRNAs and infected them with NL-02-2020 as well VOCs B.1.1.7, B.1.351, P.1 and B.1.617.2, also referred to as Alpha, Beta, Gamma and Delta variants, respectively. Viral entry and replication were quantified by qRT-PCR as well as TCID50 analysis. In addition, we determined the inhibitory effect of an α-IFITM2 antibody on VOC infection in iPSC-derived human alveolar epithelial type 2 (iAT2) cells. Results: Depletion of IFITM2 reduced viral RNA production from 31-(B.1.1.7) to 754-fold (P.1). In comparison, KD of IFITM1 generally had little effect, while silencing of IFITM3 resulted in 2-to 20-fold reduction of viral RNA yields by the four VOCs. An antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iAT2 cells. Conclusion: Endogenously expressed IFITM proteins (especially IFITM2) are important cofactors for entry and replication of SARS-CoV-2 VOCs, including the Delta variant that currently dominates the COVID-19 pandemic.

CETYLPYRIDINIUM CHLORIDE MOUTHWASHES to REDUCE the SHEDDING of VIABLE SARS-CoV-2

Background: SARS-CoV-2 is spread via airborne transmission. Mouthwashes containing virucidal compounds can help reduce viral spread. Here we show that cetylpyridinium chloride (CPC), a quaternary ammonium present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by disrupting viral membranes both in vitro and in vivo. Methods: We tested the capacity of CPC-containing mouthwashes to inhibit SARS-CoV-2 entry into target cells by using a luciferase-based assay with a reporter lentivirus pseudotyped with the SARS-CoV-2 spike protein. The replication-competent SARS-CoV-2 B.1.1.7 and D614G variants were also assayed. Viral envelope disruption by CPC's virucidal effect was measured by dynamic light-scattering analyses (DSL). We confirmed these results by modifying an ELISA that detects the SARS-CoV-2 nucleocapsid (NC), which was used in the absence of its own lysis buffer. The effect of CPC in the saliva of individuals with CoVID-19 was assessed in a double-blind, placebo-controlled, randomized clinical trial. SARS-CoV-2 positive patients were randomized to gargle either water or 0.07% CPC mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load by RT-PCR and the NC protein levels by ELISA, both in saliva at 1h and 3h post-intervention. Results: CPC-containing mouthwashes inhibited SARS-CoV-2 viral fusion in vitro in a dose-dependent manner and decreased more than a 1000 times the viral TCID50 in target cells, regardless of the variant tested. The ELISA and the DSL analyses pointed to the effective disruption of the integrity of viral membranes after treatment with CPC. The clinical study performed with 105 patients showed no significant differences in viral RNA load at 1h and 3h post-treatment in saliva between placebo and CPC-treated groups. However, the levels of SARS-CoV-2 NC protein of lysed viruses were significantly higher in the CPC group at 1h and 3h post-intervention. Conclusion: CPC decreased more than a 1000 times the infectivity of SARS-CoV-2 in vitro and was effective against different SARS-CoV-2 variants. In CoVID-19 patients, the use of a 0.07% CPC mouthwash correlated with a statistically significant increase of NC protein levels in saliva, indicating enhanced disruption of viral particles. CPC-containing mouth rinses can represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.

Study of Riamilovir Activity Against SARS-CoV-2 Infection In Syrian Hamsters

The activity of the antimetabolic drug Riamilovir (TriazavirinR) was studied on a model of SARS-CoV-2 infection on Syrian hamsters. Infectious process was caused by the intranasal administration of the virus accumulated in the Vero-B culture with a concentration of 4.25×104 TCID50, in a volume of 26 μl/hamster. The effects of the drug at a dose of 20 mg/kg intraperitoneally daily in the midst of the infectious process were traced to accelerate the clearance of the virus in the lungs, prevent body weight loss and the severity of pulmonary edema, as well as preserve the mass of the spleen. The protective effects of Riamilovir on the structure of the lungs and brain are shown, it is suggested that the drug has the ability to penetrate the blood-brain barrier. It was concluded that Riamilovir has antiviral activity against SARS-CoV-2.

Quantification of Infectious SARS-CoV-2 by the 50% Tissue Culture Infectious Dose Endpoint Dilution Assay.

A number of viral quantification methods are used to measure the concentration of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the traditional plaque-based assay allows for direct enumeration of replication competent lytic virions and remains the gold standard for the quantification of infectious virus, the 50% tissue culture infectious dose (TCID50) endpoint dilution assay allows for a more rapid, large-scale analysis of experimental samples. In this chapter, we describe a well-established TCID50 assay protocol to measure the SARS-CoV-2 infectious titer in viral stocks, in vitro cell or organoid models, and animal tissue. We also present alternative assays for scoring the cytopathic effect of SARS-CoV-2 in cell culture and comparable methods to calculate the 50% endpoint by serial dilution.