Fluorescent Clade IIb Lineage B.1 Mpox Viruses for Antiviral Screening

Abstract

The ongoing global outbreak of mpox caused by clade IIb viruses has led to more than 100,000 confirmed cases around the world, highlighting the urgent need for antiviral research to combat current and future mpox outbreaks. Reporter viruses expressing fluorescent proteins to monitor viral replication and virus spreading in cell culture provide a powerful tool for antiviral drug screening. In this work, we engineered two recombinant mpox clade IIb viruses by inserting, under the control of the vaccinia early/late promoter 7.5, the coding sequence of two different fluorescent proteins (EGFP and TurboFP635) in a previously unreported location within the viral genome. These recombinant viruses replicate in BSC-1 cells at rates similar to those of the parental virus. We show how these reporter mpox viruses allow the discrimination of infected cells by cell flow cytometry and facilitate the quantification of viral spread in cell culture. Finally, we validated these reporter viruses with two previously known inhibitors of poxvirus replication, cytosine arabinoside (AraC) and bisbenzimide.

Keywords: antiviral; bisbenzimide; clade IIb; fluorescent reporter; mpox; poxvirus.

Figure 1

Fluorescent clade IIb lineage B.1 MPXVs. (A) Schematic representation of the recombination process for the generation of MPXV2b-EGFP and MPXV2b-FP635. (B) BSC-1 cells were infected at 0.1 pfu/cell with MPXV2b-EGFP or MPXV2b-FP635 and screened under the fluorescence microscope at 48 hpi. FL: flanking left, FR: flanking right.

Figure 2

In vitro replication of MPXV2b-EGFP and MPXV2b-FP635. (A) BSC-1 cells were infected with the indicated viruses at 10 pfu/cell for the one-step growth curve. Virus production was determined by plaque assay at 24 hpi from fractions containing cell-associated viruses (CAV or extracellular viruses (EV). (B) BSC-1 cells were infected with the indicated viruses at 0.05 pfu/cell for the multi-step growth curve. Total virus production was determined by plaque assay at the indicated times.

Figure 3

Detection of infected cells by viral-driven fluorescence. (A) BSC-1 cells were infected at different MOIs with MPXV2b-EGFP or MPXV2b-FP635. Viral-driven fluorescence, relative to the mean fluorescence intensity at 48 hpi MOI 1 pfu/cell, was measured at the indicated times. Minimum of 5 replicates for each condition. (B) BSC-1 cells were co-infected with MPXV2b-EGFP and MPXV2b-FP635 at MOI 0.1 and 0.01 pfu/cell, respectively. At 24 hpi, cells were fixed, and infected cell populations were discriminated by flow cytometry.

Figure 4

Fluorescent MPXVs can ease the detection of antiviral activity. (A) BSC-1 cells were infected with MPXV2b-EGFP (MOI 0.1 pfu/cell) and treated with serial dilutions of H42 or AraC. Viral-driven fluorescence at 48 hpi, relative to the mean fluorescence of untreated control, is shown. (B) BSC-1 cells were infected with MPXV2b-FP635 (MOI 0.1 pfu/cell) and treated with serial dilutions of H42 or AraC. Viral-driven fluorescence at 48 hpi, relative to the mean fluorescence of untreated control, is shown. (C) BSC-1 cells were infected with MPXV2b (MOI 0.1 pfu/cell) and treated with the previously assayed concentrations of H42 or AraC. Viral titers at 48 hpi, measured by plaque assay, are shown. All the experiments were performed using at least triplicates.