Antiviral Effects of Tecovirimat and Cellular Ultrastructural Changes in Human Bronchial Epithelial Cell Line Following Monkeypox Virus Infection

Abstract

The mpox virus (MPXV) Clade IIb outbreak in 2022 was the biggest one ever to occur outside Africa, causing different types of clinical symptoms and levels of disease severity. There is no currently approved treatment for mpox, but Tecovirimat has proven effective against known orthopoxviruses in several animal models and Vero cell cultures. Since serious complications, including lung involvement, have been reported, especially in immunocompromised people, we investigated the effects of MPXV infection on the in vitro model of lung airway epithelium (Calu-3 cell line) and examined MPXV replication kinetic and related ultrastructural changes, also performing dose-response studies to measure Tecovirimat antiviral activity. Our results highlighted an active replication of MPXV in Calu-3 cells linked to mitochondrial structural modifications with perinuclear relocation and the formation of cytoplasmic vacuoles. Treatment with Tecovirimat consistently reduced viral replication both in supernatants (81%) and inside cells (77%) and ultimately stopped viral infectivity (92% of cytopathic effect reduction) after 48 h of infection. Drug administration inhibited the final wrapping of mature viral particles, causing extensive cytoplasmic vacuolation. Our results demonstrated Tecovirimat's in vitro effectiveness against MPXV at the nanomolar concentration on Calu-3 cells. This suggests a potential rationale for using this drug for patients with mpox severe disease and lung involvement.

Keywords: Calu-3 cells; MPXV; antiviral activity; electron microscopy; mitochondria; mpox disease; tecovirimat.

Figure 1

Quantification of MPXV DNA in Calu-3 cultures and transmission electron microscopy images of infected cells. (A) Calu-3 cells infected with MPXV at MOI 0.1 and cultured for 72 h at 37 °C. Kinetic of viral yield in cell supernatants quantified by qRT-PCR CPE and expressed as Log copies/mL.(represented as circle) (B) Kinetic of intracellular level of MPXV DNA quantified by qRT-PC and expressed as Log copies/ng (represented as square). (C–F) Representative electron microscopy images of infected cells: (C) numerous mature viral particles are visible in the cytoplasm of Calu-3 cells at 48 h.p.i.; (D) morphological features of wrapped virions (WV) are shown; (E) different steps of MPXV maturation in a cell at 24 h.p.i., crescent (arrow), immature virion (IV), immature particles with eccentric DNA condensation (IVN), and mature virus (MV) displaying typical dumbbell shape of the viral core; (F) cytopathic changes induced by MPXV at 72 h.p.i., abundant presence of vesicular structures (arrows) with electron-dense lipidic content, and numerous mature viral particles (MV) are visible. Scale bars: (C,E) = 500 nm; (D,F) = 200 nm.

Figure 2

Mitochondria modification in MPXV-infected Calu-3 cells. (A) Radial distribution of mitochondria (m) around the granular site (G) of the viral factory. (B) Close association of mitochondria with viral factories (f) in the perinuclear area. (C) High magnification of large mitochondria near the nucleus. (D) Densely packed mitochondria (m), with altered structure, in areas of viral assembly. N = nucleus. Scale bars: (A,B) = 1 um; (C,D) = 500 nm.

Figure 3

Mitochondria redistribution in MPXV-infected Calu-3 cells. Confocal microscopy representative images of uninfected cells (A) and infected cells at different times post-infection (B–D). Green staining shows localization of mitochondria. Random dots of fluorescence are visible in control cells (A) and at 7 h.p.i. (B). The mitochondrial network appears strongly modified at 24 h.p.i. (C) as highlighted by strong fluorescence localization around cell nuclei (blue). Cells at 48 h.p.i. display more scattered staining (D). Staining: Nucleus Blue (DAPI); mitochondria Green (anti-Tim23/Alexafluor488). Scale bars: (A–D) = 5 μm.

Figure 4

Effect of Tecovirimat on Calu-3 cells. Calu-3 cells were cultured at 37 °C up to 72 h in the absence or presence of scalar doses of Tecovirimat. (A) 20× bright-field images Calu-3 cells at 48 h; (B) CellTiter-Glo was used to measure the antimetabolic effect of Tecovirimat at 48 h and (C) at 72 h.

Figure 5

Tecovirimat antiviral activity on Calu-3 cells. Calu-3 cells were infected with MPXV at MOI 0.1 and cultured for 72 h at 37 °C in the absence or presence of scalar doses of Tecovirimat. (A) Kinetic of viral yield in cell supernatants quantified by qPCR and results expressed as Log copies/mL. (B) Kinetic of MPXV DNA at the intracellular level by qPCR and results expressed as Log copies/ng. (C) Viral titer in cell supernatants evaluated as TCID₅₀/mL. Experiments were performed as three independent replicates; mean and SEM are shown in the picture. (D) 20× bright-field images at 48 h.p.i. (E) Half-maximal inhibitory concentration (IC₅₀) measured as CPE percentage of reduction at 48 h.p.i.

Figure 6

Transmission electron microscopy images of MPXV infection and antiviral treatment with 10 nM. (A) Control uninfected and untreated cells after 48 h of culture. (B) Uninfected cells cultured in the presence of Tecovirimat for 48 h display normal morphological features. (C) Untreated cells 24 h.p.i.; in the insert higher magnification of virions show the presence of envelope (arrowheads). (D) Viral factories (f) and few mature virions are visible in Tecovirimat-treated cells after 24 h of MPXV infection; absence of envelope is visible in higher magnification of virions showed in the insert. (E) Untreated cells 48 h.p.i. (F) Extensive vacuolation in Tecovirimat-treated cells after 48 h of MPXV infection. N = nucleus; m = mitochondria; IVN = immature particles with eccentric DNA; MV = mature virions; f = viral factories. Scale bars: (A–E) = 1 μm; (F) = 500 nm.