Rapid Alpha-Pseudoviruses for Coronavirus Research

Product Description

A rapid alpha-pseudovirus for SARS-CoV-2 and other coronavirus species

The hybrid alpha-pseudovirus for SARS-CoV-2 (Ha-CoV-2) is a newly developed SARS-CoV-2 virus-like particle (VLP) that encapsulates an alphavirus-derived RNA genome for rapid report expression (luciferase or GFP) in target cells (Hetrick et al., 2022). Different from commonly used S protein pseudotyped lenti- or vesicular stomatitis virus (VSV)-psedoviruses, Ha-CoV-2 is assembled with all 4 structural proteins (S, M, N, and E) of SARS-CoV-2, and contains a reporter genome derived from an alphavirus-based vector for rapid (6 hours) and robust expression of reporter genes. The alphavirus vector does not contain any of the structural proteins from the authentic virus and the alpha-pseudovirus particles are single-cycle.  Ha-CoV-2 represents a major technological advancement in the development of SARS-CoV-2 pseudoviruses and serves as a platform for rapid and robust quantification of neutralizing antibodies, viral mutants, and antiviral drugs (Dabbagh et al., 2021; He et al., 2021). Virongy has now expanded its library of rapid alpha-pseudoviruses to other viruses within the coronavirus family.

Applications

• Screening and quantification of anti-coronavirus-neutralizing antibodies
• Anti-coronavirus drug screening
• Quantification of viral mutants’ infectivity
• Identification of host co-factors and restriction factors of coronaviruses

Virongy offers pre-assembled Ha-CoV-2 and other coronaviral alpha-pseudovirus particles.  We also help customers to perform antiviral drug screening and quantification of neutralizing antibodies. For more information, please contact us by email: info@virongy.com

Fig. 1. Schematic of the assembly of Ha-CoV-2 particles.

Major advances

• Faster speed – As fast as 6 hours for the detection of reporter expression for Ha-CoV-2 versus 2-3 days for S protein pseudotyped lentivirus.
• More robust reporter signal – Ha-CoV-2 takes advantage of the rapid and robust gene expression capacity of alphavirus vector for reporter expression.
• High fidelity in particle structure – Ha-CoV-2 contains all 4 SARS-CoV-2 structural proteins (S, M, E, and N), but no structural proteins from other viruses. In contrast, lenti- and VSV- pseudoviruses contain only the S protein from SARAS-CoV-2, and multiple structural proteins from other viruses, such as HIV-1 Gag and Pol.
• Strong correlation (coefficient value r² = 0.87) with wild-type SARS-CoV-2 in neutralizing antibody assays (Hetrick et al., 2020).

Fig. 2. Comparison of Ha-CoV-2 with S protein pseudotyped lentivirus in a time course of infection and reporter expression.

HEK293T(ACE2/TMPRESS2) cells were used as the target cell.

Fig. 3. Neutralizing antibody activity measured with Ha-CoV-2(Luc).

50 μl of Ha-CoV-2(Luc) (Cat# HaCoV2Luc-01) was incubated with serially diluted anti-serum for 1 hour at 37°C. The virus-antibody complex was used to infect 4×10⁵ HEK293T(ACE2/TMPRSS2) target cells in a 96-well plate. Luciferase assay was performed at 12 hours post-infection.

Example of product Certificate of Analysis

We also offer pre-assembled Ha-CoV-2 particles with the following S protein variants:

Suggested protocol

(For the infection of target cells in 12-well plate. For more protocol details in other formats, such as in 96-well plate, please contact info@virongy.com)

• Count target cells, g. HEK293T(ACE2/TMPRSS2), to be infected, and seed 1 x 10⁵ cells per well in 0.5 ml culture medium in each well of 12-well plate. Culture cells overnight. Note: Cell viability should be ≥ 80%.
• Before infection, remove medium, add 300 μl fresh medium.
• Add 100 μl Ha-CoV-2 virus, and infect for 2-6 hours. *
• Wash cells by adding 1 ml fresh medium.
• After washing, add 1 ml fresh complete medium (DMEM + 10% FBS).
• Culture cells for 12-18 hours to read signals (e.g. luciferase assay).

* if performing neutralizing antibody assays, incubate virus with antibodies at 37°C for 1 hour, then add the virus-antibody complex to cells for infection.

Ha-CoV-2  are intended for Research Use Only and are not for diagnostic or therapeutic purposes or uses in humans or animals.

 References

Dabbagh, D., He, S., Hetrick, B., Chilin, L., Andalibi, A., and Wu, Y. (2021). Identification of the SHREK family of proteins as broad-spectrum host antiviral factors. bioRxiv, doi: https://doi.org/10.1101/2021.02.02.429469

He, S., Waheed, A.A., Hetrick, B., Dabbagh, D., Akhrymuk, I.V., Kehn-Hall, K., Freed, E.O., and Wu, Y. (2021). PSGL-1 Inhibits the Incorporation of SARS-CoV and SARS-CoV-2 Spike Glycoproteins into Pseudovirions and Impairs Pseudovirus Attachment and Infectivity. Viruses 13, 46.

Hetrick, B., Chilin, L. D., He, S., Dabbagh, D., Alem, F., Narayanan, A., … & Wu, Y. (2022). Development of a hybrid alphavirus-SARS-CoV-2 pseudovirion for rapid quantification of neutralization antibodies and antiviral drugs. Cell Reports Methods, 100181.