Replicating-Competent VSV-Vectored Pseudotyped Viruses.

Vesicular stomatitis virus (VSV) is prototype virus in the family of Rhabdoviridae. Reverse genetic platform has enabled the genetic manipulation of VSV as a powerful live viral vector. Replicating-competent VSV is constructed by replacing the original VSV glycoprotein gene with heterologous envelope genes. The resulting recombinant viruses are able to replicate in permissive cells and incorporate the foreign envelope proteins on the surface of the viral particle without changing the bullet-shape morphology. Correspondingly, the cell tropism of replicating-competent VSV is determined by the foreign envelope proteins. Replicating-competent VSVs have been successfully used for selecting critical viral receptors or host factors, screening mutants that escape therapeutic antibodies, and developing VSV-based live viral vaccines.

Pseudotyped Viruses for Coronaviruses.

Seven coronaviruses have been identified that can infect humans, four of which usually cause mild symptoms, including HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1, three of which are lethal coronaviruses, named severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus 2. Pseudotyped virus is an important tool in the field of human coronavirus research because it is safe, easy to prepare, easy to detect, and highly modifiable. In addition to the application of pseudotyped viruses in the study of virus infection mechanism, vaccine, and candidate antiviral drug or antibody evaluation and screening, pseudotyped viruses can also be used as an important platform for further application in the prediction of immunogenicity and antigenicity after virus mutation, cross-species transmission prediction, screening, and preparation of vaccine strains with better broad spectrum and antigenicity. Meanwhile, as clinical trials of various types of vaccines and post-clinical studies are also being carried out one after another, the establishment of a high-throughput and fully automated detection platform based on SARS-CoV-2 pseudotyped virus to further reduce the cost of detection and manual intervention and improve the efficiency of large-scale detection is also a demand for the development of SARS-CoV-2 pseudotyped virus.

A novel hamster model of SARS-CoV-2 respiratory infection using a pseudotyped virus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a biosafety level (BSL)-3 pathogen; therefore, its research environment is limited. Pseudotyped viruses that mimic the infection of SARS-CoV-2 have been widely used for in vitro evaluation because they are available in BSL-2 containment laboratories. However, in vivo application is inadequate. Therefore, animal models instigated with animal BSL-2 will provide opportunities for in vivo evaluation. Hamsters (6-10-week-old males) were intratracheally inoculated with luciferase-expressing vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotyped virus. The lungs were harvested 24-72 h after inoculation and luminescence was measured using an in vivo imaging system. Lung luminescence after inoculation with the SARS-CoV-2 pseudotyped virus increased in a dose-dependent manner and peaked at 48 h. The VSV-G (envelope G) pseudotyped virus also induced luminescence; however, a 100-fold concentration was required to reach a level similar to that of the SARS-CoV-2 pseudotyped virus. The SARS-CoV-2 pseudotyped virus is applicable to SARS-CoV-2 respiratory infections in a hamster model. Because of the single-round infectious virus, the model can be used to study the steps from viral binding to entry, which will be useful for future research on SARS-CoV-2 entry without using live SARS-CoV-2 or transgenic animals.

The Extracts of Polygonum cuspidatum Root and Rhizome Block the Entry of SARS-CoV-2 Wild-Type and Omicron Pseudotyped Viruses via Inhibition of the S-Protein and 3CL Protease.

COVID-19, resulting from infection by the SARS-CoV-2 virus, caused a contagious pandemic. Even with the current vaccines, there is still an urgent need to develop effective pharmacological treatments against this deadly disease. Here, we show that the water and ethanol extracts of the root and rhizome of Polygonum cuspidatum (Polygoni Cuspidati Rhizoma et Radix), a common Chinese herbal medicine, blocked the entry of wild-type and the omicron variant of the SARS-CoV-2 pseudotyped virus into fibroblasts or zebrafish larvae, with IC50 values ranging from 0.015 to 0.04 mg/mL. The extracts were shown to inhibit various aspects of the pseudovirus entry, including the interaction between the spike protein (S-protein) and the angiotensin-converting enzyme II (ACE2) receptor, and the 3CL protease activity. Out of the chemical compounds tested in this report, gallic acid, a phytochemical in P. cuspidatum, was shown to have a significant anti-viral effect. Therefore, this might be responsible, at least in part, for the anti-viral efficacy of the herbal extract. Together, our data suggest that the extracts of P. cuspidatum inhibit the entry of wild-type and the omicron variant of SARS-CoV-2, and so they could be considered as potent treatments against COVID-19.

GMI, a protein from Ganoderma microsporum, induces ACE2 degradation to alleviate infection of SARS-CoV-2 Spike-pseudotyped virus.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) induces a global serious pandemic and is responsible for over 4 million human deaths. Currently, although various vaccines have been developed, humans can still get SARS-CoV-2 infection after being vaccinated. Therefore, the blocking of SARS-CoV-2 infection may be potential therapeutic strategies. Ganoderma microsporum immunomodulatory protein (GMI), a small fungal protein, is cloned from Ganoderma microsporum. It exhibits anti-cancer and immunomodulatory functions. Currently, it is still unclear whether GMI involves in interfering with viral infection. PURPOSE: This study aimed to examine the potential functions and mechanisms of GMI on inhibiting SARS-CoV-2 pseudovirus infection. METHODS: The effects of GMI were examined in vitro on ACE2 overexpressing HEK293T (HEK293T/ACE2) cells exposed to SARS-CoV-2 Spike lentiviral pseudovirus encoding a green fluorescent protein (GFP) gene. The infection efficacy was determined using fluorescence microscopy and flow cytometry. The protein level of ACE2 was verified by Western blot. The effects of GMI on cell viability of HEK293T/ACE2 and lung epithelial WI38-2RA cells were determined by MTT assay. Mice received GMI via nebulizer. RESULTS: GMI did not affect the cell viability of HEK293T/ACE2, WI38-2RA and macrophages. Functional studies showed that GMI inhibited GFP expressing SARS-CoV-2 pseudovirus from infecting HEK293T/ACE2 cells. GMI slightly interfered the interaction between ACE2 and Spike protein. GMI interacted with S2 domain of Spike protein. Specifically, GMI dramatically reduced ACE2 expression in HEK293T/ACE2 and WI38-2RA cells. Mechanistically, GMI induced ACE2 degradation via activating protein degradation system, including proteasome and lysosome. Abolishing proteasome and lysosome by MG132 and bafilomycin A1, respectively, rescued GMI-reduced ACE2 levels. In addition, GMI triggered dynamin and lipid raft-mediated ACE2 endocytosis. ACE2 levels were downregulated in the lung tissue after the mice inhaling GMI. CONCLUSIONS: GMI prevents SARS-CoV-2 pseudovirus infection via induction of ACE2 degradation in host cells. Our findings suggest that GMI will be a potential prevention agent to alleviate SARS-CoV-2 infection.

Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain.

Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.

The antigenicity of SARS-CoV-2 Delta variants aggregated 10 high-frequency mutations in RBD has not changed sufficiently to replace the current vaccine strain.

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants' spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011-2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10-100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

The significant immune escape of pseudotyped SARS-CoV-2 variant Omicron.

The emergence of Omicron/BA.1 has brought new challenges to fight against SARS-CoV-2. A large number of mutations in the Spike protein suggest that its susceptibility to immune protection elicited by the existing COVID-19 infection and vaccines may be altered. In this study, we constructed the pseudotyped SARS-CoV-2 variant Omicron. The sensitivity of 28 serum samples from COVID-19 convalescent patients infected with SARS-CoV-2 original strain was tested against pseudotyped Omicron as well as the other variants of concern (VOCs, Alpha, Beta, Gamma, Delta) and variants of interest (VOIs, Lambda, Mu). Our results indicated that the mean neutralization ED50 of these sera against Omicron decreased to 66, which is about 8.4-folds compared to the D614G reference strain (ED50 = 556), whereas the neutralization activity of other VOC and VOI pseudotyped viruses decreased only about 1.2-4.5-folds. The finding from our in vitro assay suggest that Omicron variant may lead to more significant escape from immune protection elicited by previous SARS-CoV-2 infection and perhaps even by existing COVID-19 vaccines.

Cross-Reactive Antibodies to SARS-CoV-2 and MERS-CoV in Pre-COVID-19 Blood Samples from Sierra Leoneans.

Many countries in sub-Saharan Africa have experienced lower COVID-19 caseloads and fewer deaths than countries in other regions worldwide. Under-reporting of cases and a younger population could partly account for these differences, but pre-existing immunity to coronaviruses is another potential factor. Blood samples from Sierra Leonean Lassa fever and Ebola survivors and their contacts collected before the first reported COVID-19 cases were assessed using enzyme-linked immunosorbent assays for the presence of antibodies binding to proteins of coronaviruses that infect humans. Results were compared to COVID-19 subjects and healthy blood donors from the United States. Prior to the pandemic, Sierra Leoneans had more frequent exposures than Americans to coronaviruses with epitopes that cross-react with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). The percentage of Sierra Leoneans with antibodies reacting to seasonal coronaviruses was also higher than for American blood donors. Serological responses to coronaviruses by Sierra Leoneans did not differ by age or sex. Approximately a quarter of Sierra Leonian pre-pandemic blood samples had neutralizing antibodies against SARS-CoV-2 pseudovirus, while about a third neutralized MERS-CoV pseudovirus. Prior exposures to coronaviruses that induce cross-protective immunity may contribute to reduced COVID-19 cases and deaths in Sierra Leone.

Age-Dependent Reduction in Neutralization against Alpha and Beta Variants of BNT162b2 SARS-CoV-2 Vaccine-Induced Immunity.

Vaccines against severe acute respiratory syndrome coronavirus-2 have been introduced. To investigate the relationship between vaccine-induced humoral immunity and patient age, we measured antibody levels and neutralization in vaccinated sera. Sera from 13 to 17 days after the second dose of the BNT162b2 vaccine were collected from health care workers at the University of Toyama (n = 740). Antibody levels were measured by the anti-receptor binding domain antibody test (anti-RBD test), and neutralization against wild-type (WT), α- and ß-variant pseudotyped viruses were assayed using a high-throughput chemiluminescent reduction neutralizing test (htCRNT; positivity cutoff, 50% neutralization at serum dilution 1:100). Basic clinical characteristics were obtained from questionnaires. Antibodies were confirmed in all participants in both the anti-RBD test (median, 2,112 U/ml; interquartile range [IQR], 1,275 to 3,390 U/ml) and the htCRNT against WT (median % inhibition, >99.9; IQR, >99.9 to >99.9). For randomly selected sera (n = 61), 100.0% had positive htCRNT values against the α- and ß-derived variants. Among those who answered the questionnaire (n = 237), the values of the anti-RBD test were negatively correlated with age in females (P < 0.01). An age-dependent decline in neutralization was observed against the variants but not against the wild-type virus (wild type, P = 0.09; α, P < 0.01; ß, P < 0.01). The neutralizing activity induced by BNT162b2 was obtained not only against the wild-type virus, but also against the variants; however, there was an age-dependent decrease in the latter. Age-related heterogeneity of vaccine-acquired immunity is a concern in preventive strategies in the era dominated by variants. IMPORTANCE Since mRNA vaccines utilize wild-type SARS-CoV-2 spike protein as an antigen, there are potential concerns about acquiring immunity to variants of this virus. The neutralizing activity in BNT162b2-vaccinated individuals was higher against the wild-type virus than against its variants; this effect was more apparent in older age groups. This finding suggests that one of the weaknesses of the mRNA vaccine is the high risk of variant infection in the elderly population. Because the elderly are at a higher risk of SARS-CoV-2 infection, the age-dependent decline of neutralization against viral variants should be considered while planning vaccination programs that include boosters.

Correlation of the Commercial Anti-SARS-CoV-2 Receptor Binding Domain Antibody Test with the Chemiluminescent Reduction Neutralizing Test and Possible Detection of Antibodies to Emerging Variants.

Serological tests are beneficial for recognizing the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). To identify protective immunity, optimization of the chemiluminescent reduction neutralizing test (CRNT) is critical. Whether commercial antibody tests have comparable accuracy is unknown. Serum samples were obtained from COVID-19 patients (n = 74), SARS-CoV-2 PCR-negative (n = 179), and suspected healthy individuals (n = 229) before SARS-CoV-2 variants had been detected locally. The convalescent phase was defined as the period after day 10 from disease onset or the episode of close contact. The CRNT using pseudotyped viruses displaying the wild-type (WT) spike protein and a commercial anti-receptor-binding domain (RBD) antibody test were assayed. Serology for the B.1.1.7 and B.1.351 variants was also assayed. Both tests concurred for symptomatic COVID-19 patients in the convalescent phase. They clearly differentiated between patients and suspected healthy individuals (sensitivity: 95.8% and 100%, respectively; specificity: 99.1% and 100%, respectively). Anti-RBD antibody test results correlated with neutralizing titers (r = 0.31, 95% confidence interval [CI] 0.22-0.38). Compared with the WT, lower CRNT values were observed for the variants. Of the samples with ≥100 U/mL by the anti-RBD antibody test, 77.8% and 88.9% showed ≥50% neutralization against the B.1.1.7 and the B.1.351 variants, respectively. Exceeding 100 U/mL in the anti-RBD antibody test was associated with neutralization of variants (P < 0.01). The CRNT and commercial anti-RBD antibody test effectively classified convalescent COVID-19 patients. Strong positive results with the anti-RBD antibody test can reflect neutralizing activity against emerging variants. IMPORTANCE This study provides a diagnostic evidence of test validity, which can lead to vaccine efficacy and proof of recovery after COVID-19. It is not easy to know neutralization against SARS-CoV-2 in the clinical laboratory because of technical and biohazard issues. The correlation of the quantitative anti-receptor-binding domain antibody test, which is widely available, with neutralizing test indicates that we can know indirectly the state of acquisition of functional immunity against wild and variant-type viruses in the clinical laboratory.

Reduced sensitivity of the SARS-CoV-2 Lambda variant to monoclonal antibodies and neutralizing antibodies induced by infection and vaccination.

Severe acute respiratory syndrome coronavirus 2 variants have continued to emerge in diverse geographic locations with a temporal distribution. The Lambda variant containing multiple mutations in the spike protein, has thus far appeared mainly in South America. The variant harbours two mutations in the receptor binding domain, L452Q and F490S, which may change its infectivity and antigenicity to neutralizing antibodies. In this study, we constructed 10 pseudoviruses to study the Lambda variant and each individual amino acid mutation's effect on viral function, and used eight cell lines to study variant infectivity. In total, 12 monoclonal antibodies, 14 convalescent sera, and 23 immunized sera induced by mRNA vaccines, inactivated vaccine, and adenovirus type 5 vector vaccine were used to study the antigenicity of the Lambda variant. We found that compared with the D614G reference strain, Lambda demonstrated enhanced infectivity of Calu-3 and LLC-MK2 cells by 3.3-fold and 1.6-fold, respectively. Notably, the sensitivity of the Lambda variant to 5 of 12 neutralizing monoclonal antibodies, 9G11, AM180, R126, X593, and AbG3, was substantially diminished. Furthermore, convalescent- and vaccine-immunized sera showed on average 1.3-2.5-fold lower neutralizing titres against the Lambda variant. Single mutation analysis revealed that this reduction in neutralization was caused by L452Q and F490S mutations. Collectively, the reduced neutralization ability of the Lambda variant suggests that the efficacy of monoclonal antibodies and vaccines may be compromised during the current pandemic.

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction.

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

Antiviral drugs suppress infection of 2019-nCoV spike pseudotyped virus by interacting with ACE2 protein.

The outbreak of coronavirus disease 2019 (COVID-19) has induced a large number of deaths worldwide. Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for the 2019 novel coronavirus (2019-nCoV) to infect the host cells. Therefore, ACE2 may be an important target for the prevention and treatment of COVID-19. The aim of this study was to investigate the inhibition effect of valaciclovir hydrochloride (VACV), zidovudine (ZDV), saquinavir (SQV), and efavirenz (EFV) on 2019-nCoV infection. The results of molecule docking and surface plasmon resonance showed that VACV, ZDV, SQV, and EFV could bind to ACE2 protein, with the KD value of (4.33 ± 0.09) e-8 , (6.29 ± 1.12) e-6 , (2.37 ± 0.59) e-5 , and (4.85 ± 1.57) e-5 M, respectively. But only ZDV and EFV prevent the 2019-nCoV spike pseudotyped virus to enter ACE2-HEK293T cells with an EC50 value of 4.30 ± 1.46 and 3.92 ± 1.36 µM, respectively. ZDV and EFV also have a synergistic effect on preventing entry of virus into cells. In conclusion, ZDV and EFV suppress 2019-nCoV infection of ACE2-HEK293T cells by interacting with ACE2.

Investigating interactions between chloroquine/hydroxychloroquine and their single enantiomers and angiotensin-converting enzyme 2 by a cell membrane chromatography method.

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS-CoV-2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high-expression angiotensin-converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin-converting enzyme 2 receptor. Molecular docking assay and detection of SARS-CoV-2 spike pseudotyped virus entry into angiotensin-converting enzyme 2-HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin-converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R-Chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S-chloroquine or the racemate, R-chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R-hydroxychloroquine showed better inhibitory effects than S-hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R-chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability and inhibitory effects compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS-CoV-2 pseudovirus from entering cells was weaker than R-hydroxychloroquine/hydroxychloroquine (racemate).

Replication and single-cycle delivery of SARS-CoV-2 replicons.

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.

Application of pseudotyped virus particles to monitor Ebola virus and SARS-CoV-2 viral entry in human cell lines.

Experimental work on highly pathogenic viruses such as Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus-2 requires high-level biosafety facilities. Here, we provide a detailed step-by-step protocol which details the production and application of replication-incompetent murine leukemia virus-based pseudotyped particles to monitor and quantify the viral entry efficiency in human cell lines under biosafety level-2 conditions. We describe the use of viral particles encoding luciferase gene and the quantification of transduction efficiency by measuring luciferase activity. For complete details on the use and execution of this protocol, please refer to Imre et al. (2021).

Potential antiviral activity of isorhamnetin against SARS-CoV-2 spike pseudotyped virus in vitro.

Coronavirus Disease 2019 (COVID-19) cases and deaths are still rising worldwide, there is currently no effective treatment for severe inflammation and acute lung injury caused by new coronavirus (SARS-COV-2) infection. Therapies to prevent or treat COVID-19, including antiviral drug and several vaccines, are still being development. Human angiotensin-converting enzyme 2 (ACE2), expressing in lung, has been confirmed to be a receptor for SARS-COV-2 infection, interventions for attachment of spike protein of SARS-CoV-2 to ACE2 may be a potential approach to prevent viral infections and it is considered as a potential target for drug development. In this study, we observed that seabuckthorn and its flavonoid compounds quercetin and isorhamnetin were shown strong retention to ACE2 overexpression HEK293 (ACE2h ) cells by CMC analysis. Based on drug receptor interaction analysis and viral entry studies in vitro, we evaluated the interaction of two flavonoid compounds and ACE2 as well as the inhibitory effect of the two compounds on viral entry. Surface plasmon resonance assay proved the effect that isorhamnetin bound to the ACE2, and its affinity (KD value) was at the micromolar level, that was, 2.51 ± 0.68 µM. Viral entry studies in vitro indicated that isorhamnetin inhibited SARS-CoV-2 spike pseudotyped virus entering ACE2h cells. Based on promising in vitro results, we proposed isorhamnetin to be a potential therapeutic candidate compound against COVID-19.