Pseudotyped VSV-SARS-CoV-2 S-ΔG-mCherry is a replication-restricted, recombinant pseudotyped VSV particles containing SARS-CoV-2 spike protein. Because the infectivity of Pseudotyped VSV-SARS-CoV-2 S-ΔG-mCherry is restricted to a single round of replication, the pseudotypes can be handled using BSL-2 containment practices. The pseudotype VSV particles encode mCherry in their pVSV-ΔG vector. When the VSV pseudotypes infect the target cells, mCherry expression is proportional to the number of cells that were infected.
Nature
Virus
Application Notes
We recommended to use 10-20 uL pseudotyped virus per 1E+04 293T cells for in vitro assay.
Due to differences in cell status,the best infection conditions and MOI should be determined by the end user.The virus can be diluted with cell culture medium if needed.
Storage
Store at -80°C. Multiple freeze/thaw cycles not recommended. When using the virus, transfer the virus from the -80 ° C refrigerator and melt it in an ice bath.
Ship
Frozen on dry ice
Citations
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Vasoactive Intestinal Peptide (VIP) in COVID-19 Therapy—Shedding of ACE2 and TMPRSS2 via ADAM10
International Journal of Molecular Sciences2025 MarPubMed ID: 40141308Read Article
Applications: NeutralizationReactive species: Unspecified reactive species
"Abstract: Patients infected with SARS-CoV-2 may develop mild respiratory symptoms but also Acute Respiratory Distress Syndrome (ARDS). Additionally, severe systemic inflammation contributes to morbidity and mortality. The SARS-CoV-2 virus enters the cell by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, followed by cleavage by transmembrane serine protease 2 (TMPRSS2). Vasoactive intestinal peptide (VIP) is known for its immune-modulating effects by suppressing the release of pro-inflammatory cytokines and enhancing regulatory T-cells. Furthermore, it has been tested in SARS-CoV-2-related clinical trials. We set out to investigate its role in the setting of SARS-CoV-2 infection in vitro. Epithelial cells (CaCo-2) were stimulated with SARS-CoV-2 spike protein, treated with native VIP and analyzed to investigate the mRNA and surface expression of ACE2 and TMPRSS2, the enzyme activity of TMPRSS2 and the infection rate by a SARS-CoV-2 pseudovirus. VIP downregulated ACE2 and TMPRSS2 mRNA and surface expression. Beyond these direct effects, VIP mediates the shedding of surface-expressed ACE2 and TMPRSS2 via upregulation of a sheddase protease (ADAM10). Functionally, these dual mechanisms of VIP-mediated downregulation of proteins involved in SARS-CoV-2 cell entry resulted in a reduced infection rate by the SARS-CoV-2 pseudovirus. These data imply that VIP hampers viral entry mechanisms based on SARS-CoV-2 and the linkage to ADAM10 may stimulate research in other indications beyond SARS-CoV-2."Article snippet: Adherent cells were infected with SARS-CoV-2 pseudovirus (Pseudotyped VSV-SARS-CoV-2 SΔG-mCherry, Creative Diagnostics, Shirley, NY, USA).
Figure 1. Pretreatment with VIP reduces the infection rate of epithelial cells with a non-pathogenic SARS-CoV-2 pseudovirus.
Background
High-risk pathogens (risk group 3 and 4) are required to be handled and cultured in Biosafety Level (BSL) -3 and BSL-4 laboratories, many countries lack the infrastructure and resources. In order to be able to study high-risk pathogens under BSL-2 laboratory conditions, researchers have developed pseudotyped viruses (PVs) for exploring the characterization or mechanism of high-risk viruses, which are amplification-defective viruses that can infect host cells in the same way as wild-type viruses but have only one replication cycle and do not have the same impact on the human body as high-risk viruses. PVs mainly comes from retroviruses and rhabdoviruses, and it is used to study the functions of viral fusion proteins, including life cycle initiation, host and cell tendency, viral pathogenesis and pathways into cells. Vesicular stomatitis virus (VSV), which is often used as an enveloped virus to create PVs carrying surface proteins of foreign viruses, is a negatively polarized enveloped RNA virus with low pathogenicity. Its genome size is 11 kb and contains a nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase protein (L).
The SARS-CoV-2 virus consists of a coronavirus backbone and a viral membrane containing the spike S glycoprotein. During the development of SARS-CoV-2 PV, the S protein was incorporated into other processed viral backbones, and the resulting recombinant pseudotyped viruses express the S protein and bind to the SARS-CoV-2 receptor, hACE2, rather than interacting with the host virus receptor. VSV has been widely used as the core of SARS-CoV-2 pseudotyped viruses and can integrate with other reporter proteins. This VSV-SARS-CoV-2 has no replication ability compared to wild-type SARS-CoV-2 and can only undergo one infection cycle in infected host cells. In addition it is essentially free of toxic components and can be treated by BSL-2 laboratories. Different yields of pseudotyped viruses can be obtained by selecting different packaging cell lines, including Vero, BHK-21, and 293T cells, with Vero cells producing the highest titers of pseudotyped viruses. Overall, VSV-SARS-CoV-2 is relatively safe and reliable, with detection results similar to those of live viruses, and can be used for high-throughput assays, but it should also be noted that when expressing S protein pseudotyped viruses have different kinetic characteristics compared to live viruses, which may lead to detection bias.
Figure 1. General neutralization assay procedures by using live virus, pseudovirus as epitope sources (Source: Septisetyani EP, et al. 2021)
References
1. Septisetyani EP, et al. SARS-CoV-2 Antibody Neutralization Assay Platforms Based on Epitopes Sources: Live Virus, Pseudovirus, and Recombinant S Glycoprotein RBD. Immune Netw. 2021 Nov 23;21(6):e39.
2. Dong T, et al. Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives. Chem Sci. 2023 May 3;14(23):6149-6206.
3. Salazar-García M, et al. Pseudotyped Vesicular Stomatitis Virus-Severe Acute Respiratory Syndrome-Coronavirus-2 Spike for the Study of Variants, Vaccines, and Therapeutics Against Coronavirus Disease 2019. Front Microbiol. 2022 Jan 14;12:817200.
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References
High-titer manufacturing of SARS-CoV-2 Spike-pseudotyped VSV in stirred-tank bioreactors
Mol Ther Methods Clin Dev
Authors: Todesco HM, Gafuik C, John CM, Roberts EL, Borys BS, Pawluk A, Kallos MS, Potts KG, Mahoney DJ.
The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×106 cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×108 plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 106 PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.
Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry
Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole-genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using live viruses and cellular lethality for selection, these screens can identify an overwhelming number of genes without specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole-genome screen and counter-screen strategy based on a pseudoviral platform that allowed identification of genes specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and vesicular stomatitis virus glycoprotein (VSV-G) mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed the identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein packaged into the pseudovirus was used to bypass retro-transcription and integration by directly activating a floxed fluorescent protein reporter upon entry reduced the number of gene hits and increase specificity for viral entry. Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and low-density lipoprotein receptors, respectively, and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry-associated genes. Validation of a few hits demonstrates that this approach distinguishes envelope-specific host factors from genes affecting reporter expression. Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication.
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Figure 1. Pretreatment with VIP reduces the infection rate of epithelial cells with a non-pathogenic SARS-CoV-2 pseudovirus. 
Figure 1. General neutralization assay procedures by using live virus, pseudovirus as epitope sources
