Establishment and Utility of a Multi-Envelope Pseudovirus Assay to Identify Lead Antiviral Compounds.

Abstract

Background: Viral glycoproteins, attach the virus onto host cell surface and this causes the envelope of virus to fuse with the cell membrane of host. This is necessary in the case for most, if not all, of enveloped viruses to enter the host cell. Despite significant breakthroughs in this sector, identifying and developing effective antiviral medications remains challenging. To tackle newly developing and re-emerging viruses, it is essential to develop a reliable and fast assay that can identify broad-spectrum entry inhibitors that can stop viral entry into host cells. Methods: To determine which transfection method was best to produce the specific pseudovirus; Lipofectamine, polyethyleneimine (PEI), and Calcium phosphate transfection were employed in producing Marburg, Ebola, and SARS-CoV-2 pseudoviruses. Cytotoxicity assay of the compounds obtained from Bacillus and Streptomyces spp. was performed on the permissive cell lines (293T and ACE-293 cells) using resazurin (Alamar blue) assay. An inhibition assay was done by using one-tenth of the cytotoxic concentration (CC 50) of the compounds against the various pseudoviruses. Results: ACE2 HEK293 cell line was produced for SARS-CoV-2 pseudovirus infection. Polyethyleneimine (PEI) was effective in producing Marburg pseudovirus. However, the lipofectamine method was the most effective in producing high pseudovirus titers for SARS CoV-2 and EBOV-Makona. Resazurin was used in determining the cell toxicity of all 32 compounds against Human Embryonic Kidney (HEK293T) and ACE2 HEK293 cells. The CC 50 of the 32 compounds was determined using a dose-response curve. For the inhibition out of the 32 compounds screened on the 3 different pseudoviruses: 2 compounds inhibited MARV, 5 inhibited EBOV and 5 inhibited SARS-CoV-2 pseudoviral entry. Conclusion: The plasmids used to produce the pseudoviruses are reliable for pseudovirus production and can be used for viral infectivity and inhibition assays. PEI is effective in producing MARV pseudovirus and lipofectamine is also effective for producing SARS-CoV2 and EBOV pseudoviruses. Five compounds showed inhibition against SARS-CoV-2. Compound (30) showed active pan-inhibitory effect against Marburg and Ebola pseudoviruses. The assay was reliable in showing compounds that had some inhibitory activity against all the three pseudoviruses. Significance: This assay is good for finding formal hit compounds against viral entry of the three viruses used and can also be optimized for other enveloped viruses of concern to screen for entry inhibitors.