Non-Replicating SARS-CoV-2 for Safer Research
University of Pittsburgh researchers have produced SARS-CoV-2 viruses that are infectious but replication-defective. These single-cycle viruses can encode reporter genes and could be a safe research tool requiring only biosafety level 2 (BSL-2) precautions. Recombinant DNA versions of the SARS-CoV-2 genome have been produced which lack the genes needed for structural proteins required for replication, and are encoded in a bacterial artificial chromosome (BAC). These infectious, replication-defective, reporter viruses may be vital research tools in the understanding of SARS-CoV-2 and the development of new treatments and future vaccines.
Description
SARS-CoV-2 is a deadly human coronavirus that emerged in 2019, resulting in the COVID-19 pandemic. To date, millions have died, and many more have been left disabled through post-viral complications (i.e., long COVID). While widespread vaccinations began in early 2021, new variants continue to emerge that can escape immune response resulting in illness, hospitalizations and deaths. Effective treatments and universal vaccines are required to prevent these deaths and other health complications associated with COVID-19 infection. A replication-defective virus would allow the study of SARS-CoV-2 pathology and treatments in a lower security BSL-2 (or ABSL-2) laboratory making research safer and more accessible, particularly in early career laboratories and in developing nations where BSL-3 laboratory access is limited.Applications
• SARS-CoV-2 research• SARS-Cov-2 vaccines
Advantages
Currently SARS-CoV-2 must be handled in a BSL-3 laboratory due to the infectiousness and lethality of SARS-CoV-2. This BSL-3 requirement increases the cost and limits research, particularly in low resource settings. SARS-CoV-2 has four structural proteins, spike (S), envelope (E), membrane (M) and nucleocapsid (N). E and M are necessary for virus assembly, release and host pathogenesis. Recombinant DNA lacking the genes for M (M), or both E and M (EM) proteins, can be cloned into a BAC enabling direct infection and expression in cells. Replication of viruses only occurs following trans-complementation of the missing viral genes (i.e., M separately expressed in cells). M and EM SARS-CoV-2 viruses only infect cells for a single cycle meaning these viruses would not require BSL-3 precautions and may be handled in more accessible BSL-2 settings. Finally, the lack of infectivity means these M and EM viruses may be suitable for use as vaccines.Invention Readiness
DNA sequences for M and EM SARS-CoV-2 with fluorescent reporter genes have been produced and cloned into a BAC. Compared to WT SARS-CoV-2, viral production was nearly 100-fold less after passage 1, undetectable after passage 3, and were not infectious. Expression of the missing proteins restored infectivity. There is no evidence of codon-optimized M RNA (used for trans-complementation) with the M SARS-CoV-2 genome.IP Status
Patent PendingRelated Publication(s)
Fischer, H. L., Kline, C., Duprex, W. P., McCarthy, K. R., Watkins, S. C., Conway, J. F., & Ambrose, Z. (2025). Deletion of the Envelope gene attenuates SARS-CoV-2 infection by altered Spike localization and increased cell-to-cell transmission. Cold Spring Harbor Laboratory. https://doi.org/10.1101/2025.05.20.655126
Zhang, H., Fischer, D. K., Shuda, M., Moore, P. S., Gao, S., Ambrose, Z., & Guo, H. (2022). Construction and characterization of two SARS‐CoV‐2 minigenome replicon systems. Journal of Medical Virology, 94(6), 2438–2452. https://doi.org/10.1002/jmv.27650