The high mutation rate at the D614G hotspot-furin cleavage site region increases the priming efficiency of the Spike protein by furin protease: analysis of Indonesian SARS-CoV-2 G614 variants obtained during the early COVID-19 pandemic.

D614G mutation plays a significant role in the transmissibility of SARS-CoV-2. Identification of other mutations related to D614G mutation within the Spike protein is pivotal as they might contribute to the pathogenicity of SARS-CoV-2. This study aims to analyze the mutation rate of furin cleavage site (FCS) region of Indonesian origin SARS-CoV-2 and to predict the effect of mutation against Spike priming efficiency by furin. A total of 375 sequences of Indonesian isolates obtained during the early pandemic were used for mutation analysis. Mutation analysis includes mutation pattern, variability, frequency of mutation, amino acid conservation, and mutation rate. The effect of mutation against Spike priming efficiency by furin protease from eight sequences with mutation in the FCS region was analyzed by protein-protein docking. We showed that mutations related to the G614 variant were increasing through time, in contrast to the D614 variant. The FCS region at the position 675-692 contained the most variable (66.67%) as well as the highest mutation frequency (85.92%) and has been observed to be the hotspot mutations linked to the D614G mutation. The D614G hotspot-FCS region (residue 600-700) had the highest amino acid change per site (20.8%) as well as the highest mutation rate as 1.34 × 10-2 substitution per site per year (95% CI 1.79 × 10-3-2.74 × 10-2), compared with other Spike protein regions. Mutations in the FCS region were the most common mutation found after the D614G mutation. These mutations were predicted to increase the Spike priming efficiency by furin. Thus, this study elucidates the importance of D614G mutation to other mutations located in the FCS region and their significance to Spike priming efficiency by furin. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00827-w.

Clinical problems associated with the spike protein D614G mutation in SARS-CoV-2

Covid-19 is the most famous disease around the world due to its perniciousness. At present, more and more countries and institutions try to develop a new vaccine and find an antidote to Covid-19. However, SARS-CoV-2 possesses a characteristic of rapid variation. This paper mainly focuses on the spike protein D614G mutation in SARS-CoV-2, and concludes some clinical problems caused by D614G mutation. To conclude, the D614G mutation could enhance the dissemination of Covid-19 but does not enhance its severity. Vaccine development has not been affected. More and more detect methods are surging. Mutation has caused some influence in spike protein. And mutation will lead to Chemosensory Dysfunction. © 2023 SPIE.

A polyethylene glycol enhanced ligation-triggered self-priming isothermal amplification for the detection of SARS-CoV-2 D614G mutation.

We presented a polyethylene glycol (PEG) enhanced ligation-triggered self-priming isothermal amplification (PEG-LSPA) for the detection D614G mutation in S-glycoprotein of SARS-CoV-2. PEG was employed to improve the ligation efficiency of this assay by constructing a molecular crowding environment. Two hairpin probes (H1 and H2) were designed to contain 18 nt and 20 nt target binding site at their 3' end and 5' end, respectively. In presence of target sequence, it complemented with H1 and H2 to trigger ligation by ligase under molecular crowding condition to form ligated H1-H2 duplex. Then 3' terminus of the H2 would be extended by DNA polymerase under isothermal conditions to form a longer extended hairpin (EHP1). 5' terminus of EHP1 with phosphorothioate (PS) modification could form hairpin structure due to the lower Tm value. The resulting 3' end overhang would also fold back as a new primer to initiate the next round of polymerization, resulting in the formation of a longer extended hairpin (EHP2) containing two target sequence domains. In the circle of LSPA, long extended hairpin (EHPx) containing numerous target sequence domains was produced. The resulting DNA products can be monitored in real-time fluorescence signaling. Our proposed assay owns an excellent linear range from 10 fM to 10 nM with a detection limit down to 4 fM. Thus, this work provides a potential isothermal amplification method for monitoring mutations in SARS-CoV-2 variants.

Do SARS-CoV-2 Variants Differ in Their Neuropathogenicity?

Neurological complications associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are a huge societal problem. Although the neuropathogenicity of SARS-CoV-2 is not yet fully understood, there is evidence that SARS-CoV-2 can invade and infect cells of the central nervous system. Kong et al. (https://doi.org/10.1128/mbio.02308-22) shows that the mechanism of virus entry into astrocytes in brain organoids and primary astrocytes differs from entry into respiratory epithelial cells. However, how SARS-CoV-2 enters susceptible CNS cells and whether there are differences among SARS-CoV-2 variants is still unclear. In vivo and in vitro models are useful to study these important questions and may reveal important differences among SARS-CoV-2 variants in their neuroinvasive, neurotropic, and neurovirulent potential. In this commentary we address how this study contributes to the understanding of the neuropathology of SARS-CoV-2 and its variants.

Construction of Multi-Epitopes Vaccine Candidate against SARS-CoV-2 D614G Variant

COVID-19 caused by the SARS-CoV-2 virus has become a real threat due to the emergence of new variants which are more deadly with higher infectivity. Vaccine constructs that target specific SARS-CoV-2 variants are needed for stemming COVID-19 fatality. The spike (S) glycoprotein is the major antigenic component that triggers the host immune response. Reverse vaccinology strategy was applied to the S protein of COVID-19 variant D614G to identify highly ranked antigenic proteins. In this study, a multi-epitope synthetic gene was designed using computational strategies for the COVID-19 D614G variant. The SARS-CoV-2 D614G variant protein sequence was retrieved from the NCBI database. The prediction of linear B-cell epitopes was carried out using Artificial Neural Network (ANN)-based ABCpred and BepiPred 2.0 software. The top 15 highly antigenic epitopes sequences were then selected. Propred 1 and Propred servers were used to identify major histocompatibility complex (MHC) class I and class II binding epitopes within pre-determined B-cell epitopes to predict T-cell epitopes. The top 5 MHC class I and class II were selected. Further in-silico testing for its solubility, allergenicity, antigenicity, and other physiochemical properties was analyzed using Bpred. The constructed gene was subjected to assembly PCR and the gene product was confirmed by Sanger sequencing. The findings from this study suggested that a highly antigenic specific region of the SARS-CoV-2 D614G variant can be predicted in-silico and amplified using the assembly PCR method. The designed synthetic gene was shown to elicit specific humoral and cell-mediated immune responses towards the SARS-CoV-2 variants. © 2022 Penerbit Universiti Kebangsaan Malaysia. All rights reserved.

Comparison of Clinical Characteristics and Outcomes of Hospitalized Patients Infected with the D614G Strain or Alpha Variant of COVID-19 in Taiwan: A Multi-Center Cohort Study.

Objective: Direct comparison of the clinical traits of coronavirus disease 2019 (COVID-19) in strain D614G, which originated from Wuhan, China, and the Alpha variant, which contains 17 mutations, infected patients could help physicians distinguish between strains and make clinical decisions accordingly. This study sought to compare the clinical characteristics and outcomes of the D614G strain and Alpha variant of SARS-COV-2 and identify the predictors for viral RNA clearance and in-hospital mortality in patients with COVID-19. Methods: This study recruited consecutive patients from four hospitals between March 1, 2020, and July 31, 2021. Demographic characteristics, laboratory results, and clinical outcomes were determined. Results: Among the 239 enrolled patients, 11.2% (27/239) were infected with strain D614G and 88.7% (212/239) were infected with the Alpha variant. There were no significant differences in disease progression, rate of respiratory failure, subsequent development of acute respiratory distress syndrome (ARDS), acute kidney injury, cardiac injury, duration of stay in the intensive care unit or hospital, discharge rate, mortality rate, or viral RNA clearance time between the two groups. Multivariate Cox regression revealed that antibiotic therapy reduced the risk of delayed viral RNA clearance (hazard ratio [HR], 0.26; 95% confidence interval [CI], 0.13-0.55), while autoimmune disease increased the risk of delayed viral RNA clearance (HR, 3.98; 95% CI, 1.21-13.04). Elderly patients (age > 65 years) and patients with a history of cerebrovascular accident (CVA) were at increased risk of in-hospital mortality (HR, 5.14; 95% CI, 1.06-24.72 and HR, 3.62; 95% CI, 1.25-10.42, respectively). Conclusions: There were no significant differences between the D614G strain and Alpha variant of COVID-19 in terms of clinical characteristics and outcomes. However, factors affecting viral RNA clearance and the risk of in-hospital mortality were identified. These results could help to inform the future prioritization of resource allocation and identify patients in need of intense monitoring.

Candidate Multi-Epitope Vaccine against Corona B.1.617 Lineage: In Silico Approach.

Various mutations have accumulated since the first genome sequence of SARS-CoV2 in 2020. Mutants of the virus carrying the D614G and P681R mutations in the spike protein are increasingly becoming dominant all over the world. The two mutations increase the viral infectivity and severity of the disease. This report describes an in silico design of SARS-CoV-2 multi-epitope carrying the spike D614G and P681R mutations. The designed vaccine harbors the D614G mutation that increases viral infectivity, fitness, and the P681R mutation that enhances the cleavage of S to S1 and S2 subunits. The designed multi-epitope vaccine showed an antigenic property with a value of 0.67 and the immunogenicity of the predicted vaccine was calculated and yielded 3.4. The vaccine construct is predicted to be non-allergenic, thermostable and has hydrophilic nature. The combination of the selected CTL and HTL epitopes in the vaccine resulted in 96.85% population coverage globally. Stable interactions of the vaccine with Toll-Like Receptor 4 were tested by docking studies. The multi-epitope vaccine can be a good candidate against highly infecting SARS-CoV-2 variants.

Association of Clinical Features with Spike Glycoprotein Mutations in Iranian COVID-19 Patients.

BACKGROUND: Mutations in spike glycoprotein, a critical protein of SARS-CoV-2, could directly impact pathogenicity and virulence. The D614G mutation, a non-synonymous mutation at position 614 of the spike glycoprotein, is a predominant variant circulating worldwide. This study investigated the occurrence of mutations in the crucial zone of the spike gene and the association of clinical symptoms with spike mutations in isolated viruses from Iranian patients infected with SARS-CoV-2 during the second and third waves of the COVID-19 epidemic in Isfahan, the third-largest city in Iran. METHODS: The extracted RNA from 60 nasopharyngeal samples of COVID-19 patients were subjected to cDNA synthesis and RT-PCR (in three overlapping fragments). Each patient's reverse transcriptase polymerase chain reaction (RT-PCR) products were assembled and sequenced. Information and clinical features of all sixty patients were collected, summarized, and analyzed using the GENMOD procedure of SAS 9.4. RESULTS: Analysis of 60 assembled sequences identified nine nonsynonymous mutations. The D614G mutation has the highest frequency among the amino acid changes. In our study, in 31 patients (51.66%), D614G mutation was determined. For all the studied symptoms, no significant relationship was observed with the incidence of D614G mutation. CONCLUSIONS: D614G, a common mutation among several of the variants of SARS-CoV-2, had the highest frequency among the studied sequences and its frequency increased significantly in the samples of the third wave compared to the samples of the second wave of the disease.

Why Does the Omicron Variant Largely Spare Olfactory Function? Implications for the Pathogenesis of Anosmia in Coronavirus Disease 2019.

The omicron variant of severe acute respiratory syndrome coronavirus 2 causes much less olfactory dysfunction than the previous variants. There are several potential mechanisms for how omicron may change tissue tropism and spare olfactory function. The new mutations make omicron more hydrophobic and alkaline than previous variants, which may reduce penetration of the mucus layer. Overall, the new mutations minimally change receptor binding affinity, but entry efficiency into host cells is reduced in cells expressing transmembrane serine protease 2 (TMPRSS2). Because the support cells in the olfactory epithelium abundantly express TMPRSS2, these main target cells in the olfactory epithelium may become infected less by the new omicron variant.

The First Three Months of COVID-19: Epidemiological Evidence for Two SARS-CoV-2 Strains Spreading and Implications for Prevention Strategies.

About a month after the COVID-19 epidemic peaked in Mainland China and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) migrated to Europe and then the USA, the epidemiological data began to provide important insights into the risks associated with the disease and the effectiveness of intervention strategies such as travel restrictions and lockdowns ("social distancing"). Respiratory diseases, including the 2003 severe acute respiratory syndrome (SARS) epidemic, remain only about two months in any given population, although peak incidence and lethality can vary. The epidemiological data suggested that at least two strains of SARS-CoV-2 had evolved during the first months of the epidemic while the virus migrated from Mainland China to Europe. South Korea (SK), Iran, Italy (IT), and Italy's neighbors were then hit by the more dangerous "SKII" variant. While the first epidemic in continental Asia was about to end and in Europe about to level off, the more recent epidemic in the younger US population was still increasing, albeit not exponentially anymore. The same models that help us to understand the epidemic also help us to choose prevention strategies. The containment of high-risk people, such as the elderly with comorbidities, and reducing disease severity, by either vaccination, reduction of comorbidities (seen as risk factors already in Italy), or early treatment of complications, are the best strategies against a respiratory virus disease (RVD). Lockdowns can be effective during the month following the peak incidence of infections when the exponential increase of cases ends (the window of opportunity). From the standard susceptible-infectious-resistant (SIR) model used, containing low-risk people too early, instead, merely prolongs the time the virus needs to circulate until the incidence is high enough to reach "herd immunity." Containing low-risk people too late is also not helpful, unless to prevent a rebound if containment started too early.

A Deletion Encompassing the Furin Cleavage Site in the Spike Encoding Gene Does Not Alter SARS-CoV-2 Replication in Lung Tissues of Mink and Neutralization by Convalescent Human Serum Samples.

SARS-CoV-2 has been shown to lose the furin polybasic cleavage site (FCS) following adaptation on cell culture. Deletion occurring in this region, which may include also the FCS flanking regions, seem not to affect virus replication in vitro; however, a chimeric SARS-CoV-2 virus without the sole FCS motif has been associated with lower virulence in mice and lower neutralization values. Moreover, SARS-CoV-2 virus lacking the FCS was shed to lower titers from experimentally infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. In this study, we investigated the replication kinetics and cellular tropism of a SARS-CoV-2 isolate carrying a 10-amino acid deletion in the spike protein spanning the FCS in lung ex vivo organ cultures of mink. Furthermore, we tested the neutralization capabilities of human convalescent SARS-CoV-2 positive serum samples against this virus. We showed that this deletion did not significantly hamper neither ex vivo replication nor neutralization activity by convalescent serum samples. This study highlights the importance of the preliminary phenotypic characterization of emerging viruses in ex vivo models and demonstrates that mink lung tissues are permissive to the replication of a mutant form of SARS-CoV-2 showing a deletion spanning the FCS. Notably, we also highlight the need for sequencing viral stocks before any infection study as large deletions may occur leading to the misinterpretation of results.

Analysis of Mutational Profiles of SARS-CoV-2 Structural and Non-Structural Proteins with Emphasis on Spike Protein Variants

SARS-CoV-2 has very recently posed a potential threat to humanity due to its very rapid rate of mutations and repairing mechanism. The spread of this virus is considered to have occurred in Wuhan, China in December 2019. Characterized by high rates of transmission, the virus is constantly evolving towards attaining higher rates of stability and transmissibility through acquiring mutations in its genome. Therefore, this study aims to analyse the mutational profiles of SARS-CoV-2 isolates. Analysis of the mutational profiles in individual SARS-CoV-2 proteins will allow us to look into the rates of mutations associated with each protein. Frequently mutated residues have been identified in this research by aligning 688 SARS-CoV-2 nucleotide sequences, which were downloaded from NCBI (National Center For Biotechnology Information) repository. Further, mutational frequencies of these mutated residues have been studied, which is instrumental in identifying the proteins that are resistant to changes, as well as the ones that have a greater proclivity towards incorporating mutations. © 2022, Walailak University. All rights reserved.

Enhanced antibody responses in fully vaccinated individuals against pan-SARS-CoV-2 variants following Omicron breakthrough infection.

Omicron has become the globally dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, creating additional challenges due to its ability to evade neutralization. Here, we report that neutralizing antibodies against Omicron variants are undetected following COVID-19 infection with ancestral or past SARS-CoV-2 variant viruses or after two-dose mRNA vaccination. Compared with two-dose vaccination, a three-dose vaccination course induces broad neutralizing antibody responses with improved durability against different SARS-CoV-2 variants, although neutralizing antibody titers against Omicron remain low. Intriguingly, among individuals with three-dose vaccination, Omicron breakthrough infection substantially augments serum neutralizing activity against a broad spectrum of SARS-CoV-2 variants, including Omicron variants BA.1, BA.2, and BA.5. Additionally, after Omicron breakthrough infection, memory T cells respond to the spike proteins of both ancestral and Omicron SARS-CoV-2 by producing cytokines with polyfunctionality. These results suggest that Omicron breakthrough infection following three-dose mRNA vaccination induces pan-SARS-CoV-2 immunity that may protect against emerging SARS-CoV-2 variants of concern.

Transmission Dynamics and Genomic Epidemiology of Emerging Variants of SARS-CoV-2 in Bangladesh.

With the progression of the global SARS-CoV-2 pandemic, the new variants have become more infectious and continue spreading at a higher rate than pre-existing ones. Thus, we conducted a study to explore the epidemiology of emerging variants of SARS-CoV-2 that circulated in Bangladesh from December 2020 to September 2021, representing the 2nd and 3rd waves. We collected new cases and deaths per million daily data with the reproduction rate. We retrieved 928 SARS-CoV-2 sequences from GISAID and performed phylogenetic tree construction and mutation analysis. Case counts were lower initially at the end of 2020, during January-February and April-May 2021, whereas the death toll reached the highest value of 1.587 per million on the first week of August and then started to decline. All the variants (α, ß, δ, η) were prevalent in the capital city, Dhaka, with dispersion to large cities, such as Sylhet and Chattogram. The B.1.1.25 lineage was prevalent during December 2020, but the B.1.617.2/δ variant was later followed by the B.1.351/ß variant. The phylogeny revealed that the various strains found in Bangladesh could be from numerous countries. The intra-cluster and inter-cluster communication began in Bangladesh soon after the virus arrived. The prominent amino acid substitution was D614G from December 2020 to July 2021 (93.5 to 100%). From February-April, one of the VOC's important mutations, N501Y substitution, was also estimated at 51.8%, 76.1%, and 65.1% for the α, ß and γ variants, respectively. The γ variant's unique mutation K417T was detected only at 1.8% in February. Another frequent mutation was P681R, a salient feature of the δ variant, detected in June (88.2%) and July (100%). Furthermore, only one γ variant was detected during the entire second and third wave, whereas no η variant was observed in this period. This rapid growth in the number of variants identified across Bangladesh shows virus adaptation and a lack of strict quarantine, prompting periodic genomic surveillance to foresee the spread of new variants, if any, and to take preventive measures as soon as possible.

Full-genome sequencing and mutation analysis of SARS-CoV-2 isolated from Makassar, South Sulawesi, Indonesia.

Introduction: A global surge in SARS-CoV-2 cases is occurring due to the emergence of new disease variants, and requires continuous adjustment of public health measures. This study aims to continuously monitor and mitigate the impact of SARS-CoV-2 through genomic surveillance, to determine the emergence of variants and their impact on public health. Methods: Data were collected from 50 full-genome sequences of SARS-CoV-2 isolates from Makassar, South Sulawesi, Indonesia. Mutation and phylogenetic analysis was performed of SARS-CoV-2 from Makassar, South Sulawesi, Indonesia. Results: Phylogenetic analysis showed that two samples (4%) were of the B.1.319 lineage, while the others (96%) were of the B.1.466.2 lineage. Mutation analysis of the spike (S) protein region showed that the most common mutation was D614G (found in 100% of the sequenced isolates), followed by N439K (98%) and P681R (76%). Several mutations were also identified in other genomes with a high frequency, including P323L (nsp12), Q57H (ns3-orf3a), and T205I (nucleoprotein). Conclusion: Our findings highlight the importance of continuous genomic surveillance to identify new viral mutations and variants with possible impacts on public health.

SARS-CoV-2 phase I transmission and mutability linked to the interplay of climatic variables: a global observation on the pandemic spread.

The study aims to determine the impact of global meteorological parameters on SARS-COV-2, including population density and initiation of lockdown in twelve different countries. The daily trend of these parameters and COVID-19 variables from February 15th to April 25th, 2020, were considered. Asian countries show an increasing trend between infection rate and population density. A direct relationship between the time-lapse of the first infected case and the period of suspension of movement controls the transmissivity of COVID-19 in Asian countries. The increase in temperature has led to an increase in COVID-19 spread, while the decrease in humidity is consistent with the trend in daily deaths during the peak of the pandemic in European countries. Countries with 65°F temperature and 5 mm rainfall have a negative impact on COVID-19 spread. Lower oxygen availability in the atmosphere, fine droplets of submicron size together with infectious aerosols, and low wind speed have contributed to the increase in total cases and mortality in Germany and France. The onset of the D614G mutation and subsequent changes to D614 before March, later G614 in mid-March, and S943P, A831V, D839/Y/N/E in April were observed in Asian and European countries. The results of the correlation and factor analysis show that the COVID-19 cases and the climatic factors are significantly correlated with each other. The optimum meteorological conditions for the prevalence of G614 were identified. It was observed that the complex interaction of global meteorological factors and changes in the mutational form of CoV-2 phase I influenced the daily mortality rate along with other comorbid factors. The results of this study could help the public and policymakers to create awareness of the COVID-19 pandemic.

Clinical, Virological, Immunological, and Genomic Characterization of Asymptomatic and Symptomatic Cases With SARS-CoV-2 Infection in India.

Purpose: The current global pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to the investigation with clinical, biochemical, immunological, and genomic characterization from patients to understand the pathophysiology of viral infection. Methods: Samples were collected from six asymptomatic and six symptomatic SARS-CoV-2-confirmed hospitalized patients in Bhubaneswar, Odisha, India. Clinical details, biochemical parameters, and treatment regimen were collected from a hospital; viral load was determined by RT-PCR; and the levels of cytokines and circulating antibodies in plasma were assessed by Bio-Plex and isotyping, respectively. In addition, whole-genome sequencing of viral strains and mutational analysis were carried out. Results: Analysis of the biochemical parameters highlighted the increased levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), serum SGPT, serum SGOT, and ferritin in symptomatic patients. Symptomatic patients were mostly with one or more comorbidities, especially type 2 diabetes (66.6%). The virological estimation revealed that there was no significant difference in viral load of oropharyngeal (OP) samples between the two groups. On the other hand, viral load was higher in plasma and serum samples of symptomatic patients, and they develop sufficient amounts of antibodies (IgG, IgM, and IgA). The levels of seven cytokines (IL-6, IL-1α, IP-10, IL-8, IL-10, IFN-α2, IL-15) were found to be highly elevated in symptomatic patients, while three cytokines (soluble CD40L, GRO, and MDC) were remarkably higher in asymptomatic patients. The whole-genome sequence analysis revealed that the current isolates were clustered with 19B, 20A, and 20B clades; however, 11 additional changes in Orf1ab, spike, Orf3a, Orf8, and nucleocapsid proteins were acquired. The D614G mutation in spike protein is linked with higher virus replication efficiency and severe SARS-CoV-2 infection as three patients had higher viral load, and among them, two patients with this mutation passed away. Conclusions: This is the first comprehensive study of SARS-CoV-2 patients from India. This will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection and thereby advance the implementation of effective disease control strategies.

High-throughput drug screening allowed identification of entry inhibitors specifically targeting different routes of SARS-CoV-2 Delta and Omicron/BA.1.

The Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has continuously evolved, resulting in the emergence of several variants of concern (VOCs). To study mechanisms of viral entry and potentially identify specific inhibitors, we pseudotyped lentiviral vectors with different SARS-CoV-2 VOC spike variants (D614G, Alpha, Beta, Delta, Omicron/BA.1), responsible for receptor binding and membrane fusion. These SARS-CoV-2 lentiviral pseudoviruses were applied to screen 774 FDA-approved drugs. For the assay we decided to use CaCo2 cells, since they equally allow cell entry through both the direct membrane fusion pathway mediated by TMPRSS2 and the endocytosis pathway mediated by cathepsin-L. The active molecules which showed stronger differences in their potency to inhibit certain SARS-CoV-2 VOCs included antagonists of G-protein coupled receptors, like phenothiazine-derived antipsychotic compounds such as Chlorpromazine, with highest activity against the Omicron pseudovirus. In general, our data showed that the various VOCs differ in their preferences for cell entry, and we were able to identify synergistic combinations of inhibitors. Notably, Omicron singled out by relying primarily on the endocytosis pathway while Delta preferred cell entry via membrane fusion. In conclusion, our data provide new insights into different entry preferences of SARS-CoV-2 VOCs, which might help to identify new drug targets.