Tilapia lake virus causes mitochondrial damage: a proposed mechanism that leads to extensive death in fish cells

Propagation of TiLV

The TiLV strain VETKU-TV01, previously isolated from the brain of infected red tilapia (Oreochromis sp.) (Tattiyapong, Dachavichitlead & Surachetpong, 2017), was used in the study. E-11 cells, a clone of SSN-1 cells isolated from snakehead fish (Ophicephalus striatus) (Iwamoto et al., 2000) were obtained from the European Collection of Authenticated Cell Cultures (ECACC), England (catalog number 01110916). E-11 cells were cultured in Leibovitz’s L-15 medium supplemented with 5% fetal bovine serum (Sigma, USA) and 2 mM L-glutamine at 25 °C in a CO₂-free environment until they reached 70–80% confluence. The culture medium was subsequently removed, and inoculated with a viral load of 0.1 MOI for 1 h at 25 °C. Following incubation, the virus suspension and culture medium were aspirated, and the cells were thoroughly rinsed. Cells were maintained in Leibovitz’s L-15 supplemented with 2% fetal bovine serum (Sigma, USA) and 2 mM L-glutamine at 25 °C in a CO₂-free environment. Daily microscopic observations were conducted until 80% of the cells exhibited CPE. The protocol for handling the virus was approved by the Institutional Biosafety Committee (IBC), Faculty of Veterinary Medicine, Kasetsart University under the protocol number IBC-63-V02.

Virus purification by glucose gradient centrifugation

Once 80–100% CPE formation was observed, infected E-11 cells were disrupted using three rounds of freeze-thaw cycles followed by centrifugation at 3,000× g for 10 min. The supernatant containing TiLV was collected and stored at −80 °C until further use. The supernatant was thawed and re-suspended in a 30% sucrose solution in 14 × 89 mm thin wall polypropylene centrifuge tubes (Beckman Coulter, USA). The suspension was then centrifuged at 40,000 rpm (10,000× g) for 1.5 h at 4 °C using an Optima L-90K Ultracentrifuge (Beckman Coulter, Brea, CA, USA). The pellet was collected and resuspended in one mL of TN buffer (0.1 M NaCl, 0.01 M Tris pH 7.4). The suspension was overlaid on top of a glucose gradient solution consisting of two mL layers of 30%, 40%, and 50% (w/v) sucrose in TNE buffer (0.1 M NaCl, 0.01 M Tris pH 7.4, 3 mM EDTA) and subjected to centrifugation at 40,000 rpm for 1.5 h at 4 °C. Two mL of each fraction was collected and mixed with 10 mL of phosphate buffer saline (PBS) buffer. To remove excess sucrose solution, the suspension was ultracentrifuged at 40,000 rpm for 30 min at 4 °C. The supernatant was discarded, and 500 µL of PBS solution was added to each fraction, which was then stored at 4 °C until further analysis.

Transmission electron microscopy (TEM) with positive staining

Uninfected and infected E-11 cells were collected at 0, 1, 3, and 6 dpi. Cells were trypsinized from the culture flasks and transferred to a 1.5 mL tube at room temperature (25 °C). The cell suspension was centrifuged at 2,000 rpm for 5 min at 4 °C (Centrifuge 5418R; Eppendorf, Hamburg, Germany). The supernatant was discarded, and the pellet was resuspended in 500 µL of 0.1 M PBS, followed by incubation in 2.5% glutaraldehyde in 0.1 M PBS at 4 °C overnight. The following day, the samples were thoroughly rinsed with 0.1 M PBS for 10 min, three times. The cell pellets were then incubated with 1% osmium tetroxide in dH₂O for 1 h according to a previous protocol (Barreto-Vieira & Barth, 2015). The pellets were rinsed with dH₂O for 10 min three times, dehydrated in acetone, and embedded in resin. Ultrathin sections were prepared by cutting samples at 90 nm thick using a glass knife. Samples were placed on a thin copper grid for 15 min and stained with 5% uranyl acetate for 15 min and lead citrate for 15 min. Samples were examined under a Hitachi HT7700 transmission electron microscope (Hitachi) at the Scientific Equipment and Research Division, Kasetsart University, Bangkok, Thailand. All micrographs were taken at 80 kV.

Transmission electron microscopy (TEM) with negative staining

The purified TiLV was suspended in PBS (4 °C) and then transferred to Formvar^® film-coated copper grids with 400 mesh sizes (Electron Microscopy Science, Hatfield, PA, USA) for 30 min. The grids were washed with dH₂O before being stained with 40 µL of 2% uranyl acetate or 1% phosphotungstic acid for 1 min using filtered paper to remove the staining solution. The samples were dried for 7 days and then observed with the TEM operating at 80 kV.

Cell viability assay

The cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, E-11 cells were seeded in a 96-well plate at a density of 4 × 10⁴ cells/mL per well and incubated overnight with L-15 medium supplemented with 5% FBS at 25 °C without CO₂. The cells were then infected with TiLV at 0.1 MOI for 1 h in a humidified incubator at 25 °C. Next, cells were incubated with MTT at a concentration of 5 mg/mL in L-15 media for 4 h in a 37 °C humidified incubator. The uninfected and infected E-11 cells were collected at 1 h (0 dpi), 1, 3, and 6 days after TiLV inoculation and used as control and experimental groups, respectively. The media containing MTT were then removed and replaced with 100% DMSO to solubilize formazan, and the absorbance of the solubilized formazan in each group was measured using a hybrid multi-mode microplate reader (Synergy™ H1; Agilent, Santa Clara, CA, USA) at a wavelength of 590 nm.

Measurement of ATP concentration

The ATP concentration of the cells was evaluated using the CellTiter-Glo^® luminescent cell viability assay (Promega, Madison, WI, USA). E-11 cells were incubated at 25 °C, 100% O₂ overnight. The cells were then treated with TiLV at 0.1 MOI (n = 3) for one hour before being replaced with Leibovitz’s medium containing 2 fetal bovine serum (2% FBS L-15). Cells treated with 2% FBS L-15 and blank were included as negative controls. The measurement of ATP concentration was performed on 0, 1, 3, and 6 dpi, by extrapolating from the ATP (Sigma, St Louis, MO, USA) standard curve (10–1,000 nM). Luminescence was detected using a luminometer (Synergy H1™, BioTek^® Instruments, Inc., Winooski, VT, USA) for 1 s at 37 °C, and the relative light units were read using Gen5™ software (BioTek^® Instruments, Inc., Winooski, VT, USA).

Detection of red-to-green ratio in JC-1-stained E-11 cells

The mitochondrial function of E-11 was evaluated using the fluorochrome 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) (Molecular probes Inc, USA). E-11 cells were plated in 24-well flat-bottom plates and allowed to reach 70–100% confluence. The cells were then infected with 0.1 MOI of TiLV for 1 h, followed by the replacement of 2% FBS L-15, and further incubation at 25 °C. Uninfected E-11 cells were used as a control. At 0, 1, 3, and 6 dpi, the cells were incubated with 5 µM of JC-1 for 30 min, washed with PBS twice, and visualized under an inverted fluorescence microscope (IX73, Olympus, Japan). Green and red images were captured and analyzed under the BW channel (bandpass 460–495 nm; barrier filter 510 nm; dichroic mirror 505 nm) and GW channel (bandpass 530–550 nm; barrier filter 575 nm; dichroic mirror 570 nm), respectively. All pictures were merged into a new image using cellSens dimension™ 2.3 software (Olympus, Tokyo, Japan). The intensity of green and red colors was randomly analyzed from three dispersed areas with more than 1,000,000 pixels, and the average red-to-green (R/G) ratios were calculated and compared between uninfected and TiLV-infected E-11 cells.

Determination of mitochondrial mass in E-11 cells

The number of mitochondria was investigated using MitoTracker™ Red CMXRos staining (Invitrogen™, Eugene, OR, USA). The E-11 cell line was cultured in Leibovitz’s L-15 media with 5% FBS at 25 °C without CO₂. After trypsinization and cell counting, the cells were seeded onto collagen-coated cover slips and allowed to incubate until they reached 80–90% confluence. Subsequently, the cells were inoculated with TiLV at 0.1 MOI and then stained with MitoTracker™ in a dark room. Following staining, the cells were fixed with methanol and permeabilized using Triton X-100 (Sigma-Aldrich, St Louis, MO, USA). To visualize the cell nuclei, the cells were further incubated with 4′,6-diamidino-2-phylindole (DAPI) at a concentration of 1:1,000. Representative images were acquired using confocal laser scanning microscope model FLUOVIEW FV3000 (Olympus, Tokyo, Japan) with specific filters for MitoTracker™ Red (579 nm excitation/599 nm emission), and DAPI (358 nm excitation/461 nm emission).

Statistical analysis

All data were statistically analyzed using GraphPad™ Prism software (San Diego, CA, USA). Results were shown as the mean ± standard error of the mean (S.E.M.). The data were tested for normal distribution using Kolgomorov-Smirnov test, and all data followed a Gaussian distribution. The cell viability, ATP measurement and JC-1 red-to-green ratios were compared between uninfected and TiLV-infected at 0, 1, 3, and 6 dpi using two-way ANOVA, followed by Tukey’s as a post-hoc test. Statistical significance was considered at p-value less than 0.05.

TiLV caused morphological changes and cytopathic effects in E-11 cells

The infection of E-11 cells with TiLV resulted in significant morphological changes and cytopathic effects (CPE). Within 24 h of infection, the infected cells showed altered morphology, with CPE progression from 10% to 90% between 1 to 6 days post-infection (dpi). At 1 dpi, uninfected cells remained normal (as seen in Fig. 1A), while infected cells exhibited vacuolation and pyknotic nuclei (as seen in Fig. 1B). By 3 dpi (as seen in Fig. 1C) and 6 dpi (as seen in Fig. 1D), extensive vacuolation, shrinkage, and distinct CPE formation were observed, with only a limited number of viable cells remaining and discoloration of the culture media at 6 dpi. These findings demonstrate that TiLV infection leads to substantial changes in the morphology and viability of E-11 cells.

Ultrastructural changes of E-11 cells during TiLV infection

The ultrastructure of uninfected and TiLV-infected E-11 cells was compared using TEM. Figures 2A and 2C depict the ultrastructure of uninfected cells, while Figs. 2B and 2D demonstrate the ultrastructure of TiLV-infected cells. Within 1 h post-infection (0 dpi), a viral particle was observed at the plasma membrane of the infected E-11 cells (Fig. 2B; inset). However, no significant changes in the cellular structure and organelles were noticed at this early time point. Both uninfected and TiLV-infected cells exhibited intact nuclear membranes, normal mitochondria (Figs. 2C and 2D), and typical rough endoplasmic reticulum (rER) at 0 dpi (Figs. 2A, 2B, and 2C).

At 1 dpi, the uninfected cell displayed both normal mitochondria and mitochondria with partial loss of cristae (Fig. 3A). In contrast, TiLV-infected cells exhibited initial changes, including swollen mitochondria, indistinct mitochondrial membrane structure, and cristae degeneration (Fig. 3B). At 3 dpi, cristae remained visible in the mitochondria of uninfected cells (Fig. 3C), while progressive mitochondrial degeneration was observed in TiLV-infected cells, characterized by extensive cristae loss. Furthermore, TiLV-infected cells displayed the formation of lamellar bodies and a large number of free TiLV particles (Fig. 3D). At 6 dpi, uninfected cells still maintained intact mitochondrial membranes and cristae (Fig. 3E). In contrast, TiLV-infected cells exhibited multiple cytoplasmic vacuolations. At this time point, the mitochondria showed progressive degeneration, including swelling, major structural distortion, delamination of the inner and outer mitochondrial membranes, and complete loss of cristae. Intracytoplasmic TiLV particles were also prominently present (Fig. 3F). Additionally, the ultrastructure of isolated TiLV particles was examined in Fig. S1. The particles were found to have a round or oval shape, with diameters ranging from 50 to 120 nm. The particles exhibited a central electron-dense core surrounded by a capsid-like bilaminar structure. Notably, the spike protein was not observed on the surface of the TiLV particles.

TiLV caused extensive mitochondrial damage and cell death

To evaluate the impact of TiLV infection on cellular viability and mitochondrial damage in E-11 cells, the MTT assay, ATP measurement, and JC-1 staining were employed (Fig. 4). The MTT assay revealed that TiLV infection resulted in significant cell death, with a progressive decline in the number of viable cells from 104.39 ± 5.85% at 0 dpi to 6.89 ± 7.21% at 6 dpi (Fig. 4A). Likewise, the amount of ATP concentration in E-11 cells following TiLV infection gradually reduced from 1.01 ± 0.01 µM at 0 dpi to 0.77 ± 0.03 µM at 6 dpi (Fig. 4B). Notably, the JC-1 staining demonstrates the alteration of mitochondrial membrane potential as indicated by red-to-green fluorescence (R/G) ratio in TiLV-infected cells (Figs. 4C & 4D). At 0 dpi, there was no significant difference in the R/G ratio (1.22 ± 0.06) in TiLV-infected cells compared to uninfected cells (1.50 ± 0.05). However, at 1 dpi, the R/G ratio decreased to 1.03 ± 0.06 and remained at a similar level at 3 dpi (1.012 ± 0.04) and 6 dpi (1.00 ± 0.11). Additionally, the MitoTracker™ Red staining revealed loss of mitochondrial mass following TiLV infection at 1 dpi (Fig. 5). Comparison of the MTT assay, ATP measurement and JC-1 staining between control and TiLV-infected cells revealed statistical significance at 1, 3, and 6 dpi (p < 0.05). These results indicated significant damage to the mitochondria and reduction in cellular viability in E-11 cells following TiLV infection.