Over the past two decades, the world has faced several infectious disease outbreaks. Ebola, Influenza A (H1N1), SARS, MERS and Zika virus have had a massive global impact in terms of economic disruption, strain on local and global public health resources and, above all, human health (By The Elsevier Community - March 25, 2020).
Figure 1: WHO Coronavirus (COVID-19) Dashboard
At the end of 2019, COVID-19, caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), posed a serious threat to global public health and social stability1. According to the World Health Organization (WHO), as of April 20, 2021, more than 504 million cases and 6.2 million deaths have been confirmed globally, and the numbers continue to increase every day (https://covid19.who.int/). For this situation, there is an urgent need to understand the nature and infection mechanism of the virus. However, consistent with other studies on emerging and severe infectious disease pathogens, due to the high pathogenicity and infectivity of SARS-CoV-2, all studies using live viruses to evaluate the effect of related products must be at Biosafety Level 3 (BSL-3 ) laboratory2. This has prevented most research laboratories around the world from conducting SARS-CoV-2 research, severely hindering the development of vaccines and related drugs. Therefore, there is an urgent need for a pathogen model that replaces live viruses and reduces the level of biosafety, resulting in a new immunological technology-pseudovirus system.
Figure 2: 3D structure of SARS-CoV-2
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What is a SARS-CoV-2 pseudovirus?
Pseudovirus refers to a retrovirus that integrates the envelope glycoprotein of another virus to form an exogenous viral envelope, while the genome still retains the genome characteristics of the retrovirus itself3. The generation process of the pseudovirus is shown in Figure 3. The plasmid with one viral outer membrane protein gene and the plasmid with another viral backbone gene are co-transfected into packaging cells (such as 293T cells). The outer membrane protein is expressed by the outer membrane protein gene on the surface of the packaging cell. Meanwhile, the gene of another virus is transcribed and translated in the packaging cell to assemble into virus particles. When the virus particle buds, it will be packaged by the outer membrane protein expressed on the cell surface, that is, a pseudovirus is formed.
Figure 3: The production process of the pseudovirus3
Create SARS-CoV-2 pseudoviruses
Firstly, the pseudovirus internal nucleic acid is a defective genome and cannot express pseudovirus particle surface proteins, so the virus surface proteins need to be achieved by additional plasmid transfection or stable expression in cell lines. Studies have shown that the SARS-CoV-2 enters cells through the binding of the Spike protein to the human angiotensin-converting enzyme 2 (hACE2) receptor4 (Figure 4). Therefore, the membrane protein selected for the construction of the new coronavirus pseudovirus is the S protein.
Figure 4: The binding of the S protein to the hACE2 receptor5
Secondly, the backbone vector of the pseudovirus contains gene sequences such as viral transcription, packaging, and integration, and provides all the proteins except the membrane protein for the pseudovirus. The most commonly used enveloped virus pseudovirus packaging systems include (Figure 5): lentivirus (such as HIV) vector packaging system, vesicular stomatitis virus packaging system (VSV) and murine leukemia virus packaging system (MLV system)6.
Finally, since the pseudovirus usually only performs one round of infection and does not have the ability to replicate and proliferate, the pseudovirus system can be made to carry a reporter gene, thereby facilitating subsequent qualitative and quantitative research. Commonly used reporter genes are firefly luciferase (Fluc), Renilla luciferase (Gluc), green/red fluorescent protein (GFP/RFP), β-galactosidase (Lac Z) and secreted alkaline phosphate enzyme (SEAP), etc.7
Figure 5: The schematic of acquiring different pseudotyped viruses(PVs) based on different packaging systems8
Application of SARS-CoV-2 pseudoviruses
At present, SARS-CoV-2 pseudoviruses play a key role in the study of virus invasion mechanism, screening of antiviral drugs, evaluation of vaccine titer, determination of neutralizing antibody ability and so on8.
1. Vaccine potency evaluation
In terms of vaccine potency evaluation, in both preclinical and clinical stages, the determination of neutralizing antibodies in serum after vaccination by pseudoviruses neutralization test is an essential experiment. A number of vaccines, including S RBD subunit protein vaccine developed by Sichuan University, full-length S glycoprotein vaccine developed by Novavax, mRNA vaccine of Pfizer and Moderna , adenovirus vector vaccine developed by Johnson and Oxford University and inactivated vaccine developed by Sinovac Biotech and Beijing Institute of biological products, relied on pseudoviruses system to evaluate the neutralization ability of antibody-induced after vaccination.
2. Neutralizing antibodies quantification
Currently, the Spike-containing SARS-CoV-2 pseudovirus is the most developed pseudovirus system. As various studies have confirmed, SARS-CoV-2 uses ACE2 as a host cell surface receptor, and the interaction between S protein and ACE2 mainly mediates its entry into target cells9. Therefore, antibody neutralization assays based on the S protein have been extensively studied for SARS-CoV-2 pseudoviruses. The reported pseudovirus-based assays correlate well with WT virus-based assays, while also generally having higher throughput and requiring less sample serum compared to traditional assays10.
3. Inhibitor screening
Screening of small‐molecule inhibitors against SARS-CoV-2 has also been performed using SARS-CoV-2 pseudoviruses. Yang et al. used pseudotyped SARS-CoV-2 to screen an approved drug library of 1,800 small molecular drugs for SARS-CoV-2 virus entry inhibitors. Fifteen active drugs were identified as specific SARS-CoV-2 S pseudovirus entry inhibitors, and further antiviral tests using native SARS-CoV-2 virus in Vero E6 cells confirmed that seven of these drugs significantly inhibited SARS-CoV-2 replication, reducing supernatant viral RNA load with a promising level of activity.
Figure 6 shows the different application scenarios of the SARS-CoV-2 pseudovirus, as well as the choice of different packaging systems and reporter genes.
Figure 6: The application of SARS-CoV-2 pseudovirus8
Limitations of pseudoviruses
Despite the advantages of pseudoviruses listed above, the system still has many limitations.
First, pseudoviruses can only be used to study viruses with specific envelope proteins, such as influenza viruses, coronaviruses, retroviruses, and Ebola viruses. However, for viruses without envelope proteins, such as rotavirus and poliovirus, the pseudovirus system is difficult to function.
Second, pseudovirus mimics are also very limited in characteristics compared to live viruses. In most cases, pseudoviruses can only mimic the role of the envelope proteins of live viruses in mediating virus entry into cells in vitro, but cannot mimic the process of proliferation and release after entering cells. Therefore, results from assays using pseudotyped viruses should be compared and validated against the live virus‐based assay, which remains the gold standard11.
Nonetheless, pseudovirus systems have their unique advantages. Since the pseudovirus cannot self-replicate and can only carry out a single cycle of infection, it reduces the virus mutation rate and has high biological safety. In addition, it can reduce the risk of laboratory operations and replace live virus for vaccine development and immunogenic neutralizing antibody level detection, reducing the difficulty of the operation and the cost of testing.
At present, the epidemic caused by SARS-CoV-2 infection is still severe, and the continuous maturity of pseudovirus technology can better play an important role in the research and development of diagnosis, treatment and preventive prevention and control products.
1. Pal, M., Berhanu, G., Desalegn, C., & Kandi, V. (2020). Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2): an update. Cureus, 12(3).
2. Chen, M., & Zhang, X. E. (2021). Construction and applications of SARS-CoV-2 pseudoviruses: a mini review. International Journal of Biological Sciences, 17(6), 1574.
3. Kishko M G. Molecular and Functional Properties of Transmitted HIV-1 Envelope Variants: A Dissertation[J]. 2011.
4. Chan, J. F. W., Kok, K. H., Zhu, Z., Chu, H., To, K. K. W., Yuan, S., & Yuen, K. Y. (2020). Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerging microbes & infections, 9(1), 221-236.
5. Ge, S., Lu, J., Hou, Y., Lv, Y., Wang, C., & He, H. (2021). Azelastine inhibits viropexis of SARS-CoV-2 spike pseudovirus by binding to SARS-CoV-2 entry receptor ACE2. Virology, 560, 110-115.
6. Ansarah-Sobrinho, C., Nelson, S., Jost, C. A., Whitehead, S. S., & Pierson, T. C. (2008). Temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation. Virology, 381(1), 67-74.
7. Huang, W., & Wang, Y. (2020). Application in the evaluation of fake virus technology in the prevention and control of new sudden viral infectious diseases.
8. Xiang Q, Li L, Wu J, et al. Application of pseudovirus system in the development of vaccine, antiviral-drugs, and neutralizing antibodies[J]. Microbiological Research, 2022: 126993.
9. Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 181(2), 281-292.
10. Bentley, E. M., Mather, S. T., & Temperton, N. J. (2015). The use of pseudotypes to study viruses, virus sero-epidemiology and vaccination. Vaccine, 33(26), 2955-2962.
11. Tamin, Azaibi, et al. “Development of a neutralization assay for Nipah virus using pseudotype particles.” Journal of virological methods 160.1-2 (2009): 1-6.