We've updated our Privacy Policy to make it clearer how we use your personal data.

We use cookies to provide you with a better experience. You can read our Cookie Policy here.

Leveraging Pseudoviruses in the Face of the COVID-19 Pandemic

Leveraging Pseudoviruses in the Face of the COVID-19 Pandemic

Leveraging Pseudoviruses in the Face of the COVID-19 Pandemic

Leveraging Pseudoviruses in the Face of the COVID-19 Pandemic

Credit: Pixabay.
Read time:

Want a FREE PDF version of This Article?

Complete the form below and we will email you a PDF version of "Leveraging Pseudoviruses in the Face of the COVID-19 Pandemic"

First Name*
Last Name*
Email Address*
Company Type*
Job Function*
Would you like to receive further email communication from Technology Networks?

Technology Networks Ltd. needs the contact information you provide to us to contact you about our products and services. You may unsubscribe from these communications at any time. For information on how to unsubscribe, as well as our privacy practices and commitment to protecting your privacy, check out our Privacy Policy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic and had a devastating impact on the lives and livelihood of humans since December 2019. However, the incredible efforts of scientists and public health agencies worldwide has resulted in the emergency use authorization and rapid deployment of antibody-based countermeasures, including therapeutic monoclonal antibodies (mAbs), convalescent plasma therapy and messenger RNA, inactivated and viral-vectored vaccines. Success of antibody-based counter measures is dependent upon the ability of an antibody to neutralize SARS-CoV-2 infection by binding to the SARS-CoV-2 spike (S) glycoprotein, with which it normally binds angiotensin-converting enzyme 2 (ACE2) on host cells to achieve cell entry. The effectiveness of antibodies is typically measured in a neutralization assay. This assay, however, requires SARS-CoV-2 isolation and handling in a Biosafety Level 3 (BSL-3) laboratory following BSL-3 practices as per CDC biosafety guidelines. BSL-3 laboratories are limited to a few institutions, restricting the scope for evaluating the success of antibody-based counter measures. One viable alternative would be to use a pseudovirus instead of SARS-CoV-2 in these neutralization assays. Pseudoviruses can be handled safely in routine BSL-2 laboratories that are widely available and used at a massive scale, opening opportunities for therapeutic advancement.

Definition of a pseudovirus

Pseudoviruses are synthetic chimeras that consist of a surrogate viral core, derived from a parent virus, and an envelope glycoprotein on its surface derived from a heterologous virus.1 The parent virus used to generate a pseudovirus is typically a rhabdovirus (e.g., vesicular stomatitis virus (VSV)) or a retrovirus (e.g., murine leukemia virus (MLV) or human immunodeficiency virus 1 (HIV-1))1. The parent viral genome is modified to delete essential genes required for replication, including the native envelope glycoprotein gene, and instead, a reporter gene coding for luciferase or fluorescent protein is inserted.1 Pseudoviruses are therefore defective for undergoing a complete replication cycle and can only undergo a single infection cycle.1

Figure 1: 
Pseudoviruses expressing SARS-CoV-2 S glycoprotein bind ACE2 receptor on the surface of host cells where TMPRSS2 protease cleaves S resulting in conformational change and fusion of viral and host cell membranes. Upon fusion, the genome is released into the cell cytoplasm and the reporter encoded by the genome is expressed, the activity of which is quantified by means of an instrument.

Upon successful pseudovirus infection of host cells, the reporter luciferase or fluorescent protein is expressed, the activity of which can be quantified using a luminometer or a fluorimeter respectively (Figure 1). Since the entry step for infection of a host cell is governed by the envelope glycoprotein on its surface, it is imperative that the host cells used for pseudovirus infection express appropriate receptors that bind the envelope glycoprotein of interest. As such, pseudoviruses are excellent surrogates to study functions pertaining to envelope glycoproteins and viral entry processes. Viral envelope glycoproteins that mediate entry into host cells are one of the main targets of the immune system in generating antibodies that neutralize viral entry (Figure 2).1,2 Therefore, viral envelope glycoproteins are of interest in vaccine and mAb development for many viruses of pandemic and epidemic potential (e.g., influenza, Ebola, and COVID-19).

Figure 2: 
Neutralization assay employing pseudoviruses expressing SARS-CoV-2  S glycoprotein. Neutralizing antibodies targeting the S glycoprotein prevent ACE2 receptor binding and therefore, subsequent steps of fusion, genome release and reporter gene expression.

Pseudovirus platforms

Pseudoviruses are of great advantage in evaluating the efficacy of vaccines and mAbs being used in animal models and clinical trials. Several SARS-CoV-2 S pseudoviruses based on VSV, MLV and HIV platforms have been developed and are currently being used worldwide to evaluate the success of antibody-based countermeasures.

VSV platform

For making pseudoviruses using a VSV platform, initially a VSV
ΔG-G* pseudovirus must be produced. This is achieved by transfecting two plasmids, one coding for the VSV core genome lacking the native glycoprotein and containing the reporter gene (VSVΔG), and the other coding for VSV envelope glycoprotein (G*), into HEK293T cells amenable for transfection.3 This results in the production of VSVΔG-G* pseudoviruses that can then be used to infect HEK293T cells previously transfected with a plasmid expressing SARS-CoV-2 S glycoprotein. The VSVΔG-G* pseudovirus genome contains all the core components necessary for replication and a reporter gene, but lacks the envelope glycoprotein required for assembly and budding of pseudoviruses.3 The spike glycoprotein expressed separately on the plasma membrane thus facilitates assembly and budding of VSVΔG-S pseudoviruses. Any carryover of G* from the VSVΔG-G* pseudovirus used for infection is prevented by the addition of a neutralizing antibody directed against G*. The VSVΔG-S pseudoviruses can then be directly used for the infection of HEK293T cells expressing the ACE2 receptor (HEK293T-ACE2) or Vero cells that permit S binding and entry. The general timeline to produce VSVΔG-S pseudovirus is five to six days.3

MLV and HIV platform

The MLV and HIV platforms employ three plasmids for transfection into HEK293T cells:

-          A plasmid encoding MLV/HIV core genes, gag and pol, but lacking the MLV/HIV envelope glycoprotein env gene

-          A transfer vector plasmid encoding firefly luciferase or green fluorescent protein (GFP) reporter gene, Ψ-RNA packaging signal, along with 5'- and 3'-flanking long terminal repeat (LTR) regions

-           A plasmid encoding the envelope glycoprotein of interest, in this case the SARS-CoV-2 S.

Two days after transfection, pseudovirus can be collected and purified for infection of host cells. The general timeline to produce MLV-S/HIV-S pseudovirus is two days.

Advantages and limitations of pseudoviruses

As described below, pseudoviruses offer several advantages and a few limitations compared to native viruses classified as BSL-3 agents.


Pseudoviruses are easily scalable, genetically stable and inherently safer than native viruses.1, 4

Pseudoviruses permit the study of cell entry mechanisms of novel emerging viruses and viruses that cannot be cultivated in cell culture.4

Host cell entry process and target cell tropism can be quantified by means of reporter genes (Figure 1).1

The study of cell entry by highly pathogenic viruses for which BSL -3 or -4 facilities (of which there is limited accessibility) would be required is made accessible. As pseudoviruses are defective for replication, they can be safely handled in a routine BSL-2 facility.4

Stable cell lines expressing or lacking a gene of interest can be generated.1

Viral vectors and vaccines for gene delivery can be produced.4

The efficacy of vaccines and therapeutic monoclonal antibodies can be evaluated by quantifying neutralizing antibodies in a neutralization assay (Figure 2).1

The therapeutic potential of virus entry inhibitors can be studied.1


The envelope surface glycoprotein density on a pseudovirus might not necessarily reflect the original density on the native virus.

Although several studies have observed a significant correlation between neutralization of pseudovirus and native virus, some studies nevertheless reported a lack of correlation between them.5

Pseudoviruses generated using the HIV platform cannot be used to evaluate the success of antibody-based counter measures in HIV-infected individuals on anti-retroviral therapy (ART). ART involves nucleoside analogs that might interfere with reverse transcription and integration steps of reporter genes in the neutralization assay thus leading to false positives.1 Thus, an alternative platform based on VSV or MLV must be used.

Using pseudoviruses in the fight against SARS-CoV-2

Neerukonda et al. have developed a facile and highly efficient SARS-CoV-2 pseudovirus system that is based on an HIV platform.1 Although several SARS-CoV-2 pseudovirus platforms exist, the system developed by Neerukonda et al. comparatively achieves greater levels of pseudovirus infection in a relatively short period of time, which allows screening of antibody-based counter measures in vaccinated or infected individuals at a massive scale.1 For the purpose of pseudovirus infection, they engineered a HEK293T cell line that stably expresses an ACE2 receptor and TMPRSS2 protease (HEK293T-ACE2/TMPRSS2) which supports superior levels of pseudovirus infection compared to any other cell types currently available.1 This assay,
published in PLOSONE, is a major advancement and is currently being employed by scientists and public health agencies worldwide for rapid screening and testing of antibody-based counter measures against existing and emerging SARS-CoV-2 variants. Furthermore, this system is also amenable for studying other viruses of human health importance including influenza, Ebola and Hepatitis B and C.


1.         Neerukonda, S.N.; Vassell, R.; Herrup, R.; Liu, S.; Wang, T.; Takeda, K.; Yang, Y.; Lin, T.-L.; Wang, W.; Weiss, C.D. Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2. PLOS ONE. 2021, 16, e0248348. doi:10.1371/journal.pone.0248348

2.         Nath Neerukonda, S.; Vassell, R.; Weiss, C.D. Neutralizing Antibodies Targeting the Conserved Stem Region of Influenza Hemagglutinin. Vaccines. 2020, 8, 382. doi:10.3390/vaccines8030382

3.         Zettl, F.; Meister, T.L.; Vollmer, T.; Fischer, B.; Steinmann, J.; Krawczyk, A.; V’kovski, P.; Todt, D.; Steinmann, E.; Pfaender, S., et al. Rapid Quantification of SARS-CoV-2-Neutralizing Antibodies Using Propagation-Defective Vesicular Stomatitis Virus Pseudotypes. Vaccines. 2020, 8, 386. doi:10.3390/vaccines8030386

4.         Millet, J.K.; Whittaker, G.R. Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection. Bio Protoc. 2016, 6, e2035, doi:10.21769/BioProtoc.2035

5.         Wang, P.; Nair, M.S.; Liu, L.; Iketani, S.; Luo, Y.; Guo, Y.; Wang, M.; Yu, J.; Zhang, B.; Kwong, P.D., et al. Antibody Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7. Nature. 20

Meet The Author
Karen Steward PhD
Karen Steward PhD
Senior Science Writer