In vitro susceptibility of eighteen clinical isolates of human monkeypox virus to tecovirimat

Abstract

Background: In 2022, an outbreak of mpox that started in European countries spread worldwide through human-to-human transmission. Cases have been mostly mild, but severe clinical presentations have been reported. In these cases, tecovirimat has been the drug of choice to treat patients with aggravated disease.

Objectives: Here we investigated the tecovirimat susceptibility of 18 clinical isolates of monkeypox virus (MPXV) obtained from different regions of Brazil.

Methods: Different concentrations of tecovirimat were added to cell monolayers infected with each MPXV isolate. After 72 hours, cells were fixed and stained for plaque visualization, counting, and measurement. The ortholog of F13L gene from each MPXV isolate was polymerase chain reaction (PCR)-amplified, sequenced, and the predicted protein sequences were analyzed.

Findings: The eighteen MPXV isolates generated plaques of different sizes. Although all isolates were highly sensitive to the drug, two showed different response curves and IC50 values. However, the target protein of tecovirimat, F13 (VP37), was 100% conserved in all MPXV isolates and therefore does not explain the difference in sensitivity.

Main conclusions: Our results support screening different MPXV isolates for tecovirimat susceptibility as an important tool to better use of the restricted number of tecovirimat doses available in low-income countries to treat patients with mpox.

Fig. 1:. diversity of monkeypox virus (MPXV) plaque phenotype and effect of tecovirimat on virus replication: BSC-40 cells were infected with 300 plaque-forming unit (PFU) of each MPXV clinical isolate for 72 hours when cells were fixed and stained. (A) Box and Whiskers (10-90 percentile) plot showing the size diversity of nearly 1,500 viral plaques (minimum of 550 plaques) measured for each isolate in three independent experiments. Median ± standard deviation (SD) is shown. (B, C) Infection proceeded in the presence of different concentrations of tecovirimat for 72 hours when cells were fixed and stained. Data are represented as mean ± SD. (C) Half maximal inhibitory concentration (IC₅₀) was calculated based on the linear regression equation formula for each dose-response curve. All experiments were performed in triplicate.

Fig. 2:. effect of tecovirimat on plaque size of monkeypox virus (MPXV) isolates 450 and 221: BSC-40 cells were infected with 300 plaque-forming unit (PFU) of each of the 18 clinical isolates of MPXV for 72 hours in the presence of different concentrations of tecovirimat when cells were fixed, stained, and photographed. (A) Representative images of wells with viral plaques generated in the cell monolayers (upper row) and size-representative plaques photographed at 4x magnification. (B, C) Box and Whiskers (10-90 percentile) plot showing the size diversity of at least 590 plaques (maximum of 830 plaques) measured for each concentration of tecovirimat in three independent assays. Median ± standard deviation (SD) is shown. (D) Percent of control plot values obtained in B and C expressed as mean ± SD. m indicates the slope values for each linear regression line and the p-value indicates that the difference between the slopes is not significant (ns). All experiments were performed in triplicate.

Fig. 3:. multi-alignment of F13 amino acid sequences: (A) The multi-alignment was screened for single nucleotide polymorphisms (SNPs shown in blue) using Base-by-base, opting for monkeypox virus (MPXV) isolate 08 as the reference sequence. Preserved residues are shown in white. The red dotted box indicates sequences from the 2022 mpox outbreak. Asterisk (and blue residue in B) indicates the lysine that replaced glutamic acid in all 2022 sequences. (B) Representative F13 protein sequence from Brazilian MPXV isolates, highlighting key motifs critical to VP37 functionality and known tecovirimat resistance residues.