The KM155C25 human cell line, referred to as C25 throughout this paper, was kindly provided by MyoLine, the platform for immortalization of human cells of the Institute of Myology of Paris, France, coordinated by Dr. Anne Bigot. The myoblasts were isolated from the semitendinosus muscle of a healthy 25-year-old male donor and immortalized by the overexpression of telomerase hTERT and cyclin-dependent kinase 4, thereby extending their proliferative capacity (Thorley et al., 2016). Cells were maintained as myoblasts in proliferation medium (PM) composed of four parts DMEM High glucose (Gibco, #10566016) and one part of medium 199 (Gibco, #41150020), supplemented with 20% FBS (fetal Bovine Serum #SH3008403 Cytiva) and growth factors: dexamethasone (0.2 µg/mL Sigma #D4902), epidermal growth factor (EGF 5 ng/mL Gibco #PHG311), Fetuin (25 µg/mL Sigma Aldrich #341506), basic fibroblast growth factor (bFGF 0.5 ng/mL Gibco #PHG0026) and gentamicin (50 µg/mL Gibco #15750060). To induce myoblast differentiation, cells were seeded in PM until they reached high confluence (90%). Then, the medium was replaced with differentiation medium (DM), which consisted of DMEM high glucose supplemented with insulin (10 μg/mL, Sigma #91077C). The cells were cultivated at 37 °C and 5% CO².

The ZIKV strain RIO-U1 (GenBank Accession number: KU926309) was isolated in 2016 from a urine sample of a patient from Rio de Janeiro, Brazil (Bonaldo et al., 2016). In this work, all experiments were performed at MOI 0.1, the same MOI used in our previous paper, where we compared gene expression profiling between myoblasts and myotubes (Riederer et al., 2022). Myoblasts were seeded in 100mm Petri dish at a density of 3.5x10³ cells/cm² (Corning #430167) in PM. Aliquots of the virus were diluted at an MOI of 0.1 in 2 mL of PM without growth factors, supplemented with 5% FBS, and added to the cells for 2 hours (2 h) at 37 °C, 5% CO₂. Non-absorbed viruses were removed, and 10 mL of PM supplemented with 20% FBS was added to the cells and cultured for 3 days. As a control, uninfected cells (MOCK) were grown under the same conditions described above.

Plaque assays were performed to determine viral titters (plaque-forming units, PFU). Vero E6 cell monolayers (5x10⁴/cm²) cultivated in 24-well plates (Corning) were incubated with 200μL of 10-fold serial dilutions of ZIKV for 2 h at 37 °C. After 1 h of incubation at 37 °C, the inoculum was removed and replaced with 1 mL of 2.4% carboxymethyl cellulose (CMC #C5013, Sigma-Aldrich) in Earle’s 199 medium (M0650, Merck). After 7 days of incubation at 37 °C, cells were fixed with 10% formaldehyde (P1005040000081, Dinamica) overnight, washed, and stained with 0.4% crystal violet (#C0775, Sigma-Aldrich) diluted in 20% methanol (34885, Sigma-Aldrich) to visualize plaques.

To determine the antiviral activity of the NITD008 inhibitor (SML2409, Sigma) in our model, 6x10³/cm² human myoblasts were grown on coverslips (13 mm) in 24-well tissue culture and infected with MOI 0.1 as described. After 2 h of ZIKV incubation, the cultures were treated with NITD008 at the following concentrations: 1, 2, and 5 μM (diluted in PM). After 72 h post-infection (72 hpi), supernatants were collected, and viral titters were quantified by plaque assay in VERO cells in triplicate for each concentration. A vehicle-only with a 5 μM dimethyl sulfoxide (DMSO #20688, ThermoFisher Scientific) concentration was set as the control treatment. In addition, cytotoxicity was analyzed in the supernatants of ZIKV-infected myoblasts and MOCK, and an LDH cytotoxicity assay Kit (Lactate Dehydrogenase (LDH) Colorimetric Activity #EEA013, Invitrogen) was performed according to the manufacturer’s instructions.

The effects of ZIKV on myoblast differentiation were analyzed using two protocols. First, myoblasts were grown at a density of 1.2 × 10⁴ cells/cm² in Lab-Tek 8-well Permanox cell culture slides (#177445, Thermo Scientific) in PM (20% FBS and growth factors) and infected with ZIKV at MOI 0.1 (100 µL/well of medium DMEM and 199 (1:5) supplemented with 5% FBS), after 2 h incubation, the cultures were then treated with NITD008 5 µM in PM for 72 h. And then, the PM was changed to DM for an additional 72 h to induce cell differentiation. In the second protocol, myoblasts were seeded at a high confluence, 7.5x10⁴ cells/cm² in Lab-Tek 8 well cell culture slides and infected with ZIKV. After a 2 h incubation and washing, DM was added to the cells, which were then cultured for an additional 72 h. A fusion index (FI) was calculated to evaluate the ability of myoblasts to differentiate into myotubes. The percentage of nuclei inside myotubes (cells with at least two nuclei) was determined over the total number of nuclei. Myocytes were defined as mononuclear and differentiated cells (positive for MF20). Cell cultures were stained with the anti-myosin heavy chain (MyHC) antibody (MF20 clone) by immunofluorescence to identify differentiated and fused myoblasts, as described below.

After 72 hpi, myoblasts were fixed with 4% PFA (Paraformaldehyde, Sigma Aldrich #158127) for 10 minutes (min), then permeabilized with 0.3% Triton X-100 (#42K0217 Sigma) in PBS for 15 min at room temperature. Fixed cells were blocked in buffer containing 1% bovine serum albumin (BSA #A0296), 2% FBS (Gibco # 12657029), and 0.1% Triton X-100 in 1x phosphate buffer saline (PBS Gibco#15374875) for 40 min. The cells were incubated for 1 h with anti-E Flavivirus protein antibody 4G2 (140719S0004G2P-Bio-Manguinhos, 1:50) or anti-desmin rabbit (AB907 Sigma-Aldrich, 1:30), followed by incubation with a fluorophore-conjugated secondary antibody (1:200) diluted in blocking buffer: anti-mouse Alexa Fluor 546 (A- 11030 Invitrogen) or anti-rabbit Alexa Fluor 488 (A-21206 Invitrogen) for 30 min in the dark. For myoblast differentiation and fusion staining, cells were incubated for 1 h with an anti-MyHc antibody (AB 2147781 Hybridoma Bank, MF20 clone 1:30), followed by incubation with anti-mouse Alexa Fluor 546. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) diluted in H2O at 1:20,000 (Life Technologies, D1306) for 10 min in the dark. The slides were mounted using Prolong Gold Antifade Mounting Media (P36930 Invitrogen). All incubations were performed at room temperature in the dark. Images were acquired using an AxioImager A2 fluorescence microscope with AxioVision Rel4.8 software (Zeiss, Oberkochen, Germany) or a Leica TSC SP8 Confocal device and LAS-X Software (Leica Microsystems, Wetzlar, Germany).

To quantify the number of nuclei and morphological changes, image analysis was performed using the software ImageJ FIJI (https://imagej.net/tutorials/). The “set scale” tool was used to measure distances in micrometers (µm). For initial image preparation, the “Image, Color, Split channels” function was applied, and channel AF488 was selected, which defines the structure being studied (desmin, a cytoskeletal protein in muscle cells). Threshold tool to reduce noise and “Binary, fill holes” to fill empty spaces, which improves the visibility of structures. “Analyze > Analyze Particles” function allows quantification of parameters such as cell area, circularity, roundness, and perimeter to analyze structure. Cells at the edge were defined as an exclusion criterion. For that, images were acquired using an Axio Imager A2 fluorescence microscope (Zeiss).

Cells in proliferation were detected using EdU (5-ethynyl-2′- deoxyuridine), a thymidine analog (EdU- Click 488 #FG07008), according to the manufacturer’s instructions. Infected cells were grown on coverslips (13 mm) for 72 h in PM. Over the last 2 h, cells were incubated with EdU at a final concentration of 5 µM in 300 µL of PM without growth factors at 37 °C and 5% CO². 72 hpi, cells were washed with PBS, fixed with 4% PFA for 10 min, and permeabilized with 0.5% Triton X-100 (42K0217, Sigma) in PBS for 20 min at room temperature. After a washing step with 3% BSA in PBS, cells were stained with EdU buffer detection for 30 min in the dark, using the components diluted according to the manufacturer’s instructions. After EdU staining, immunofluorescence was performed to detect ZIKV E protein (protocol described above). Images were acquired using an AxioImager A2 fluorescence microscope with Axio Vision Rel 4.8 software (Zeiss, Oberkochen, Germany). Quantification was performed using the FIJI ImageJ software.

To analyze the expression of adhesion molecules and the presence of viral proteins by flow cytometry, 3,5x10³/cm² myoblasts were seeded in 100 mm Petri dishes, infected with ZIKV, and cultured for 72 h. Myoblasts were trypsinized for 5 min at 37 °C (Trypsin/EDTA 0.25% Invitrogen) to remove adherent cells, then washed with PBS and centrifuged at 400g at 4 °C for 5 min. To detect intracellular molecules, cells were fixed with 4% PFA for 20 min at room temperature. After washing with PBS and centrifugation (400g, 5 min at 4 °C), cells were permeabilized using a Perm Buffer permeabilization/blocker buffer containing 0,3% Triton, 8% human serum (H4522 Sigma) in PBS for 1 h at 4 °C. After centrifugation at (400g, 5 min 4 °C) to remove the excess of the perm buffer, cells were incubated with the primary antibody 4G2 (Bio- Manguinhos, Fiocruz 1:200) for 1 h at 4 °C followed by incubation with an anti-mouse AF546-conjugated secondary antibody (#A11003 Invitrogen 1:300) for 30 min or anti-ki67 BV421 (#562899 Clone B56 BD Biosciences) at 4 °C. Cells were washed twice in PBS, and samples were acquired.

After 72 hpi, ZIKV-myoblasts and MOCK were washed with PBS, centrifuged at 400G, 4 °C, 5min, and resuspended in 300µL of 1x annexin-V binding Buffer (#556454, BD Bioscience) diluted in H₂O. Then, cells were incubated with 5 µL of Annexin-V APC (550475, BD Biosciences) for 15 min at room temperature. PI (6 µg/mL) was added to the cell suspension at room temperature for 2 min prior to acquiring samples in flow cytometry Experiments were performed using a FACS Celesta II cytometer (BD Biosciences) and analyzed with FlowJo software (V10).

To determine the viability of myoblasts, cells removed by trypsinization after 72 and 120 hpi were incubated with FVS520 cell viability dye (564407, BD Biosciences) diluted in PBS (1:4000) for 15 min at 37 °C. Positive controls (dead cells) were obtained by incubation with 70% ethanol for 3 min. For this analysis, live and dead cells were acquired using a FACS Celesta II cytometer (BD Biosciences) and analyzed with FlowJo software (V10).

DNA content was analyzed using flow cytometry to estimate the percentage of the cell population within each cell cycle phase. 72 hpi, cells were removed by trypsinization, washed with PBS, centrifuged at 400g, 4 °C for 5 min, and fixed with ice- cold 70% ethanol (diluted in distilled water) at 4 °C for 30 min. Cells were then washed with PBS and incubated with a hypotonic solution containing 50 µg/mL Propidium Iodide (#P3566–1 mg/mL Invitrogen), 100 µg/mL Ribonuclease A (RNAse A # 12091021), 2 mM MgCl2 (#208337 Sigma Aldrich), diluted in PBS for 40 min at room temperature in the dark. The samples were acquired using the FACS Celesta II cytometer (BD Biosciences) and analyzed using FlowJo software (V10).

Myoblasts were infected under the conditions described above. At 72 hpi, ZIKV-myoblasts and MOCK were submitted for counting the number of cell divisions. The formula used for calculating the number of cell divisions is derived from the basic principle of exponential growth (Wang et al., 2015). They enable us to estimate the number of cell divisions that occurred during the observed growth period or the average number of complete mitoses per cell during that period. n is the division number, n0 represents the initial number of cells (plated cells number), nt represents the final number of cells (counting day), as described below:

The adhesion capacity of ZIKV-infected myoblasts was estimated after 72 hpi culture period in PM. 4x10³ cells/cm² were seeded (in 100 µL of DMEM without serum) on coverslips (18 mm), pre-coated with ECM (extracellular matrix) protein: human recombinant Laminin isoform 111 (LM-111) 5 µg/mL (#LN111–02 BioLamina) or without coating for 2 h. LM-111 was diluted in PBS with calcium and Magnesium (#14040141 Gibco) and incubated for 1 h after adhesion at 37 °C, 5% CO2. LM-111 excess was removed, and myoblasts were allowed to adhere for 2 h at 37 °C. Non-adherent cells were gently removed by two washes with PBS, and the adherent ones were fixed with 4% PFA for 10 min and permeabilized with 0.3% Triton X-100 for 15 min. A blocking buffer containing 1% BSA, 2% SFB, and 0.3% Triton X-100 in PBS was added to the cells for 40 min. The desmin and ZIKV protein E detection protocol was performed using the immunofluorescence protocol above.

A Transwell migration assay was performed to evaluate the migratory capacity of human myoblasts that had been grown in 100 mm Petri dishes for 72 hpi. A cell suspension was seeded in membrane inserts on a 24-well plate using membranes with 8-μm pore sizes (Corning #3422), which were pre-coated with LM-111 at 5 μg/mL for 1 h at 37 °C and 5% CO². Infected and MOCK myoblasts (72 hpi) were seeded (5x10⁴/cm²) in the upper chamber in 100 μl of DMEM without serum, and 600 μl of the migration medium containing DMEM supplemented 1% FBS was put in the lower chamber as a chemoattractant. Cells were allowed to migrate for 4 h. Inserts were washed with PBS, and cells that did not migrate through the porous membrane were gently removed from the upper side of the insert with a cotton swab. Cells that migrated to the lower chamber were fixed with 3.7% formaldehyde for 15 min, washed with PBS, and stained with 0.4% crystal violet (42555 Sigma) diluted in 20% methanol for 10 min. Inserts were air-dried and counted under an optical microscope at 200x magnification. At least ten random microscopic fields were counted in each insert.

Data are presented as mean ± SD, the p values represent significant differences detected, and p<0.05 were considered statistically significant (p<0.05). Normality tests were performed on all data, including the Shapiro-Wilk test, and the T-test, followed by the nonparametric Mann-Whitney test correction test, which was used for pairwise comparisons depending on the data distribution. Two way-ANOVA was used to compare more than three groups, or Kruskal-Wallis test, depending on the data distribution. The reported sample size for a given dependent measure represents the number of replicates per group in 3 or 4 separate experiments. The specific statistical tests used for each experiment are provided in the figure legends. Statistical analyses were performed using GraphPad Prism 10 software.

We first evaluated the potential cytopathic effect of ZIKV infection in progenitor muscle cells. Viral particle production was quantified by collecting supernatants from ZIKV- infected myoblasts (MOI 0.1) at 24, 48, and 72 hpi, and viral titters were determined using a plaque assay. Figure 1 shows a progressive increase in viral titters up to 72 h (Figure 1A). Flow cytometry detection of the viral E envelope protein (using 4G2 antibody) indicated that approximately 50% of myoblasts were infected (Figure 1B). Supplementary Figure 1 outlines the gating strategies. These results were confirmed by immunofluorescence (Figure 1C). At 72 hpi, lower cell density was observed in ZIKV myoblasts, and no morphological changes were observed in the cultures analyzed under phase contrast optical microscopy (Figure 1D). Infected cultures had a reduced number of myoblasts compared to MOCK controls (Figure 1E) and were not related to loss of cell viability at this time point; however, this reduction occurred later, at 120 hpi (Figure 1G). Moreover, no apoptosis was observed in human ZIKV-infected myoblasts compared to MOCK controls at 72 hpi (Figure 1H). Therefore, at 72 hpi, a reduction in the number of cell divisions was observed in ZIKV-infected myoblasts (Figure 1F), which could explain the decreased number of myoblasts in the infected cultures.

Upon activation, muscle progenitor cells undergo multiple rounds of division, generating a high number of myoblasts, necessary for muscle regeneration (Dayanidhi and Lieber, 2014; Hindi and Millay, 2022). We evaluated whether ZIKV infection might impact myoblast proliferation using a nucleoside analog, EdU, which incorporates EdU during DNA synthesis. We observed no difference in the number of EdU-positive cells in ZIKV-infected cultures at 72 hpi compared to MOCK controls (Figure 2A). However, we found that infected myoblasts (4G2+ cells, immunolabeled for the E viral envelope protein) were almost all EdU negative (Figure 2A). We also observed a significant reduction in Ki67high infected myoblasts (Figure 2B). Ki67 is a nuclear protein expressed in cells during the cell cycle, most intensely in the S phase and mitosis (Dzulkifli et al., 2018). ZIKV-induced cell cycle arrest was confirmed in infected myoblasts 72 hpi by quantifying DNA content using PI, a DNA intercalator, via flow cytometry. Myoblasts decreased within the G2-M phase in the infected cultures, while an increase in the G0-G1 phases was observed (Figure 2C). These results may explain the lower number of cell divisions and consequently reduced cell numbers in ZIKV-infected myoblast cultures compared to MOCK controls (Figures 1E, F). However, our data does not answer whether ZIKV reduces, retains, or halts myoblast proliferation, and a more detailed evaluation of cell cycle pathways is needed to elucidate how ZIKV prevents myoblast proliferation.

Cell adhesion stimulates signals that regulate differentiation, migration, and survival. The first step of myoblast fusion is cell-to-cell recognition and contact, which will involve adhesion (Taylor et al., 2022). We investigated the adhesion capacity of ZIKV-infected myoblasts, and after 72hpi, the cells were detached from the cultures, seeded onto coverslips, and analyzed 2h after adhesion (Figure 3A). We observed that infected myoblasts exhibited lower adhesion than controls (Figure 3B). The cell area was significantly smaller, with less spread out in ZIKV-infected myoblasts than in controls, while circularity remained unchanged between groups (Figure 3C). These morphological changes were observed in all infected cultures, including those cells that were negative for ZIKV E viral protein labeling within the population (Figure 3C). Moreover, no significant correlation was observed between morphological alterations and the ZIKV viral E protein in infected (positive for ZIKV viral E protein) and non-infected cells (Figure 3D). The same effect was observed in wells were pre-coated with LM-111, an isoform known to stimulate myoblast adhesion and migration (González et al., 2017; Taylor et al., 2022; Dohi et al., 2025) (Supplementary Figure 4). Together, these results suggest that ZIKV infection reduces the ability of myoblasts to adhere and spread, an effect observed in both infected and non-infected cells within the same culture, likely due to a bystander effect.

Given that myoblasts migrate towards neighboring myoblasts or damaged fibers before fusing to form or repair myofibers (Hindi et al., 2017; Gao et al., 2024), we investigated the migratory capacity of myoblasts after ZIKV infection. To this end, Transwell migration assays were performed. Figure 4A shows the scheme of the transwell culture system. Infected myoblasts cultured for 72 hours were allowed to migrate from the upper chamber to the lower chamber through an 8 µm pore size membrane filter, pre-coated with LM-111 or without coating. Corroborating our previous results (Silva-Barbosa et al., 2008; González et al., 2017), LM-111 increased myoblast migration (MOCK LM) compared to the control without coating (MOCK no coating). However, ZIKV infection strongly reduced the migration capacity of myoblasts towards LM-111 compared to the LM-111 MOCK (Figure 4B). Taken together, these results suggest that ZIKV disrupts myoblast migration.

Myoblasts fuse into multinucleated syncytia, forming myotubes (Demonbreun et al., 2015). Myoblasts were maintained in PM for 72 hpi, then PM was replaced by DM to induce differentiation for an additional 72 h (Figure 5A). We observed that after 3 days of differentiation, ZIKV infection reduced the MyHC-positive population and myotube formation (Figure 5B). In addition, ZIKV infection reduced the total number of nuclei in myotubes, i.e., those containing at least 2 myonuclei formed from infected myoblasts, and increased the number of myocytes, defined as mononuclear cells expressing differentiation markers such as MyHC (Figure 5C). In brief, ZIKV infection decreases the total number of cells expressing MyHC. Actually, it reduces the number of fused cells, suggesting that ZIKV has a more substantial inhibitory effect on cell fusion than on cell differentiation. To further investigate the mechanisms underlying the impact of ZIKV on myoblast fusion, the replication inhibitor NITD008 was added to the cultures after incubation with ZIKV. NITD008 is a potent antiviral against ZIKV and other flaviviruses (Deng et al., 2016). Antiviral activity of NITD008 was confirmed in myoblasts infected with ZIKV (Supplementary Figure 5). Interestingly, we observed that inhibiting ZIKV replication restored myotube differentiation and fusion, with no alteration in cell numbers (Figures 5B,C). Alternatively, myoblasts seeded in high confluence (ready to differentiate) were infected, and the differentiation medium was added to the cells. With this protocol, differentiation was also affected, but to a lesser extent, compared to the differentiation of 3-day-infected myoblasts (Supplementary Figure 6). The viral protein was mainly detected in myocytes and was less detected in myotubes (Figure 5D). Conjointly, these results suggest that the effects of ZIKV infection on cell fusion may depend on viral replication, rather than just the presence of the virus.