Rapid Pseudovirus for SARS-CoV-2: Ha-CoV-2

A newly developed hybrid alphavirus-SARS-CoV-2 pseudovirion

Ha-CoV-2 is a newly developed hybrid 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. Ha-CoV-2 represents a major technology advancement in the development of SARS-CoV-2 pseudoviruses, and serves as platforms for rapid and robust quantification of neutralizing antibodies, viral mutants, and antiviral drugs (Dabbagh et al., 2021; He et al., 2021).

Virongy offers pre-assembled Ha-CoV-2 reporter pseudovirions and derived S protein variants. 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.

Applications

  • Screening and quantification of anti-SARS-CoV-2 neutralizing antibodies
  • Anti-SARS-CoV-2 drug screening
  • Quantification of SARS-CoV-2 viral mutants
  • Identification of host co-factors and restriction factors of SARS-CoV-2

 

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 4x105 HEK293T(ACE2/TMPRSS2) target cells in a 96-well plate. Luciferase assay was performed at 12 hours post-infection.

 

Pre-assembled Ha-CoV-2 reporter pseudovirions

Catalog No. Products Price1
HaCoV2Luc-01 5 x 200 µL of HA-CoV-2(Luc) particles, Firefly Luciferase Reporter $400.00
HaCoV2Luc-02 10 x 200 µL of HA-CoV-2(Luc) particles, Firefly Luciferase Reporter $760.00
HaCoV2Luc-03 50 x 200 µL of HA-CoV-2(Luc) particles, Firefly Luciferase Reporter $3600.00
HaCoV2Luc-04 100 x 200 µL of HA-CoV-2(Luc) particles, Firefly Luciferase Reporter $6800.00
HaCoV2Luc-10C 5 x 100 µL of HA-CoV-2(Luc) particles 10X concentrated, Firefly Luciferase Reporter $750.00
HaCoV2Luc-60C 5 x 100 µL of HA-CoV-2(Luc) particles 60X concentrated, Firefly Luciferase Reporter $990.00
HaCoV2GFP-10C 5 x 100 µL of HA-CoV-2(GFP) particles 10X concentrated, Green Fluorescent Protein Reporter $750.00
HaCoV2GFP-60C 5 x 100 µL of HA-CoV-2(GFP) particles 60X concentrated, Green Fluorescent Protein Reporter $990.00
HaCoV2RFP-10C 5 x 100 µL of HA-CoV-2(RFP) particles 10X concentrated , DsRed-Express2 Fluorescent Protein Reporter $750.00
HaCoV2RFP-60C 5 x 100 µL of HA-CoV-2(RFP) particles 60X concentrated , DsRed-Express2 Fluorescent Protein Reporter $990.00
1Academic and government price. Others inquire.
Technical Support Email: info@virongy.com

Example of product Certificate of Analysis

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

SARS-CoV-2 Spikes Available Spike Mutations GISAID Accession number
Alpha (B.1.1.7)

Spike A570D, Spike D614G, Spike D1118H, Spike H69del, Spike N501Y, Spike P681H, Spike S982A, Spike T716I, Spike V70del, Spike Y145del EPI_ISL_581117
(B.1.1.207)

Spike D614G, Spike E484K, Spike P681H EPI_ISL_778908
(B.1.1.298)

Spike D614G, Spike H69del, Spike I692V, Spike M1229I, Spike V70del, Spike Y453F EPI_ISL_616802
(B.1.2)

Spike D614G, Spike E484K, Spike G446V, Spike Y453F EPI_ISL_833413
(B.1.258)
Spike D614G, Spike H69del, Spike N439K, Spike V70del EPI_ISL_755592
Beta (B.1.351) Spike A243del, Spike A701V, Spike D80A, Spike D215G, Spike D614G, Spike E484K, Spike K417N, Spike L242del, Spike L244del, Spike N501Y EPI_ISL_678597
Epsilon (B.1.429)
Spike A222V, Spike D614G, Spike L452R, Spike S13I, Spike W152C EPI_ISL_847764
(B.1.494)
Spike A262S, Spike D614G, Spike D796Y, Spike H49Y, Spike L452R, Spike N501Y, Spike P681R, Spike Q613H EPI_ISL_826591
Gamma (P.1)Spike D138Y, Spike D614G, Spike E484K, Spike H655Y, Spike K417T, Spike L18F, Spike N501Y, Spike P26S, Spike R190S, Spike T20N, Spike T1027I, Spike V1176F EPI_ISL_833136
Kappa (B.1.617.1) Spike E154K, Spike L452R, Spike E484Q, Spike D614G, Spike P681R, Spike Q1071H EPI_ISL_1663368

Eta (B.1.525)
Spike A67V, Spike D614G, Spike E484K, Spike F888L, Spike H69del, Spike Q52R, Spike Q677H, Spike V70del, Spike Y144del EPI_ISL_1198845
Epsilon (B.1.427/429)
Spike D614G, Spike E484K, Spike L452R, Spike S13I, Spike W152C EPI_ISL_1444002
Delta (B.1.617.2)
Spike T19R, Spike del157/158, Spike L452R, Spike T478K, Spike D614G, Spike P681R, Spike D950N Derived from EPI_ISL_1841283
Delta Plus (AY.2)
Spike T19R, Spike V70F, Spiked del157/158, Spike A222V, Spike K417N, Spike L452R, Spike T478K, Spike D614G, Spike P681R, Spike D950N Derived from
EPI_ISL_2929979
Lambda (C.37)Spike G75V, Spike T76I, Spike R246N, Spike del247/253, Spike L452Q, Spike F490S, Spike D614G, Spike T859N Derived from:
EPI_ISL_2921276
Mu (B.1.621)Spike T95I, Spike Y144S, Spike Y145N, Spike R346K, Spike E484K, Spike N501Y, Spike D614G, Spike P681H, Spike D950NDerived from:
EPI_ISL_2178402
Delta Plus (AY4.2)Spike T19R, Spike T95I, Spike G142D, Spike Y145H, Spike E156G, Spike del157/158, Spike A222V, Spike L452R, Spike T478K, Spike D614G, Spike P681R, Spike D950NDerived from
EPI_ISL_6777023
Omicron (B.1.1.529)Spike A67V, Spike H69del,Spike V70del, Spike T95I, Spike G142D, Spike V143del, Spike Y144del, Spike Y145del, Spike N211del, Spike L212I, Spike ins214EPE, Spike G339D, Spike S371L, Spike S373P, Spike S375F, Spike K417N, Spike N440K, Spike G446S, Spike S477N, Spike T478K, Spike E484A, Spike Q493R, Spike G496S, Spike Q498R, Spike N501Y, Spike Y505H, Spike T547K, Spike D614G, Spike H655Y, Spike N679K, Spike P681H, Spike N764K, Spike D796Y, Spike N856K, Spike Q954H, Spike N969K, Spike L981F

Derived from:
EPI_ISL_6640919
BA.2 Spike T19I, Spike L24del, Spike P25del, Spike P26del, Spike A27S, Spike G142D, Spike V213G, Spike G339D, Spike S371F, Spike S373P, Spike S375F, Spike T376A, Spike D405N, Spike R408S, Spike K417N, Spike N440K, Spike S477N, Spike T478K, Spike E484A, Spike Q493R, Spike Q498R, Spike N501Y, Spike Y505H, Spike D614G, Spike H655Y, Spike N679K, Spike P681H, Spike N764K, Spike D796Y, Spike Q954H, Spike N969K Derived from:
EPI_ISL_9670541
AY.4/BA.1Spike T19R, Spike A27S, Spike T95I, Spike G142D, Spike E156G, Spike F157del, Spike R158del, Spike ins214EPE, Spike N211del, Spike L212I, Spike G339D, Spike S371L, Spike S373P, Spike S375F Spike K417N, Spike N440K, Spike G446S, Spike S477N, Spike T478K, Spike E484A, Spike Q493R, Spike G496S, Spike Q498R, Spike N501Y, Spike Y505H, Spike T547K, Spike D614G, Spike H655Y, Spike N679K, Spike P681H, Spike N764K, Spike D796Y, Spike N856K, Spike Q954H, Spike L981F, Spike N969K EPI_ISL_10819657
BA.4 and BA.5 (Share the Same Spike Mutations) Spike T19I, Spike L24S, Spike del25/27, Spike del69/70, Spike G142D, Spike V213G, Spike G339D,Spike S371F, Spike S373P, Spike S375F, Spike T376A, Spike D405N, Spike R408S, Spike K417N, Spike N440K, Spike L452R, Spike S477N, Spike T478K, Spike E484A, Spike F486V, Spike Q498R, Spike N501Y, Spike Y505H, Spike D614G, Spike H655Y, Spike N679K, Spike P681H, Spike N764K, Spike D796Y, Spike Q954H, Spike N969KDerived from: EPI_ISL_12464782

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.