Immune Gene Expression Modulation and in Vitro Inhibitory Effect of TiO₂ Nanoparticles under UV irradiation on Viral Necrosis Virus

1. Introduction

Many fish farms use open systems, where water is taken from a natural pond and used in tanks before being returned to the main body of water. A large amount of water is consumed each day, and inorganic substances and organic nutrients are released, polluting the aquatic environment and affecting fish health status [1,2].

In recent years, photocatalyst has been widely studied in water treatment [3], including in recirculating aquaculture systems (RAS) for the off-flavours [4] or ammonia removal [5,6,7,8] with positive effects on water quality, in terms of ammonia and nitrogen compounds concentrations and animal health.

The photocatalytic treatment offers great potential as an industrial technology for the detoxification or remediation of wastewater due to several factors: the process uses natural oxygen and light, possibly also sunlight, thus it is effective under ambient conditions.

The most largely employed photocatalyst, i.e., TiO₂, is low cost, readily available, non-toxic, chemically and mechanically stable, and has a high turnover; moreover the formation of large amounts of intermediate potentially toxic species is largely avoided; this technology allows the oxidation of organic molecules to CO₂ in a complete way: all these beneficial actions of TiO₂ thin films formation or growth on the internal walls of photoreactors are reported by [9,10].

The industrial research on TiO₂ photocatalysis is mainly aimed at using its oxidation properties, for sterilization, sanitation, and anti-pollution applications in the aqueous phase [11]. Photocatalytic degradation of high ammonia concentration water solutions by TiO₂. Nowadays the photocatalytic destruction of organic matter is also exploited in photocatalytic antimicrobial coatings, typically thin films applied to furniture in hospitals and other surfaces susceptible to contamination with bacteria, fungi and viruses [12].

The most commonly used photocatalyst, i.e. TiO₂, is inexpensive, readily available, non-toxic, chemically and mechanically stable [13,14]. Under UV irradiation reactive oxygen species, namely hydroxyl radicals (^•OH⁻) and superoxide anions (^•O₂⁻), are produced on TiO₂ [15]. In the case of viruses, these reactive radicals can degrade the capsid and envelope proteins and phospholipids of non-enveloped and enveloped viruses, respectively, leading to leakage and subsequent degradation of genetic information [16,17,18,19].

TiO₂ NPs are frequently used in paint, skin care, and cosmetic products, among other applications [20]. Because of this broad use, TiO₂ NPs build up and release a lot of material into aquatic habitats [21]. Many NPs have been used as treatments against viruses, and the advantages of their use is the specific target action and increase the efficiency of treatment with minimum side effects in species of aquaculture interest as reviewed by [22]. For instance, silver nanoparticles (AgNPs) inhibit hepatitis B virus (HBV) [23], herpes simplex virus type 2 (HSV-2) [24] and human immunodeficiency virus 1 (HIV-1) [25,26] in vertebrate, as well as WSSV, Vibrio cholerae, V. harveyi, and V. parahaemolyticus infection in shrimp [27,28,29,30]. Gold nanoparticles (AuNPs) reduce the replication of foot-and-mouth disease virus (FMDV) in cloven-hoofed animals [39] and V. parahaemolyticus as shrimp infection [40]. Moreover, titanium dioxide can deactivate rotavirus, astrovirus, and feline calicivirus (FCV) [40]. In aquaculture, numerous viral organisms affect a significant portion of global fish production, resulting in significant economic impacts on aquaculture [42,43,44]. In particular, RNA viruses cause the most severe fish diseases, such as nervous necrosis virus, which is responsible for encephalopathy and retinopathy (VER) [45]. Fish nodavirus is a non-enveloped icosahedral virion containing two positive-sense single-stranded genomic RNAs and is classified as a betanodavirus of the Nodaviridae.

In this study, the effect of TiO₂, at different concentrations, was evaluated on Nodavirus in the dark and under UV irradiation, to determine TiO₂ mechanisms of virus inactivating, of the cytopathic effect on fish cells, and immune system interaction of TiO₂ NPs at different concentrations activated by UV irradiation.

2. Materials and Methods

2.1. TiO₂ Powder Characterization

Commercial Aeroxide TiO₂ P25 powder was purchased from Evonik and used as received. Scanning electron microscopy (SEM) images for morphology investigation were acquired with a Zeiss ΣIGMA field emission instrument. The X-ray diffraction (XRD) pattern for crystal phase determination was recorded on a Philips PW3020 powder diffractometer, using the Cu Kα radiation (λ = 1.54056 Å). The Rietveld refinement method was performed for quantitative phase analysis, using the “Quanto” software [39]. The average crystallite size was calculated by means of the Scherrer equation applied to the main reflections centered at 2θ = 25.425° for anatase and 2θ = 27.575° for rutile. The BET specific surface area (SSA) and BJH mesopore volume and size were measured by N₂ adsorption/desorption at liquid nitrogen temperature (77 K) in a Micromeritics ASAP 2020 apparatus, after out-gassing the sample at 300°C in vacuum for 1 h. The diffuse reflectance (R) spectrum was measured on a Jasco V-670 spectrophotometer equipped with a PIN-757 integrating sphere, using barium sulfate as a reference, and then converted into a Taucplot, (KM hν)^1/2 vs. hν, where KM is the Kubelka-Munk transform of the reflectance R, with KM = (1−R)²/2R, for bandgap energy determination.

2.2. Preparation of TiO₂ Suspensions

The P25 powder was dispersed in aqueous suspensions as follows: a suspension containing 5 g/L of TiO₂ was prepared first, which was diluted with distilled water, to obtain suspensions containing 2.5, 2.0, 1.25, 0.75, and 0.3 mg/mL of TiO₂.

2.3. Cells and virus

The cell line (E-11) of Japanese striped fish (Ophiocephalus striatus) [47] was maintained in L15 medium (Leibovitz L-15, Eurobio) supplemented with 10% fetal calf serum (Gibco) and 10% antibiotic (10,000 units/mL penicillin and 10,000 g/mL streptomycin, Gibco, Life Technologies). Betanodavirus isolate (PG105/D19) was obtained from clinically infected specimens of Dicentrarchus labrax from the isolate collection of the Virology Laboratory of the National Institute of Marine Sciences and Technologies (INSTM), Tunisia.

2.4. Investigation of TiO₂ Cytotoxic Properties on the E-11 Cell Line

The MTT assay was performed to measure the cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes that reduce MTT to formazan. The insoluble formazan crystals are dissolved with a solubilizing solution and the resulting-colored solution is analyzed by measuring the absorbance at 500-600 nm with a multiwall spectrophotometer. In this study, the protocol reported in ref. [29] was adopted with a small change in the incubation temperature (25°C instead of 37°C). The absorbance was measured at 590 nm. The viability threshold (%) was calculated as follows: Viability threshold (%) = (absorbance of different TiO₂ suspensions / absorbance of the control) × 100.

2.5. TiO₂ Inhibitory Properties on Nodaviruses Replication

An equal volume (50 µL) of Nodavirus solution and TiO₂ suspension were mixed and used for the described treatments. Six TiO₂ concentrations were tested (5.0, 2.5, 2.0, 1.25, 0.75 and 0.3 mg/mL). The UV irradiation was performed by exposing VNNV-TiO₂ mix Eppendorf tubes, at a distance of 20 cm, to a UV-Clamp source (15 W) with a peak at 253.7 nm. The irradiated mixtures were retrieved at 5 different time intervals (5 min, 15 min, 30 min, 1 h and 16 h), then inoculated on E-11 cells. The mock and positive controls were always added.

A total volume of the irradiated mixtures (100 µL) was withdrawn at 5 different time intervals. Simultaneously, the same assay was performed in the dark by mixing an equal volume of Nodavirus and TiO₂ (5 mg/mL) and incubating for 1h at 25°C prior to cell inoculation. The negative and positive controls were always added. Five 96-well plates with confluent E-11 cells (7x104 cells/well) were freshly prepared 24 h before the start of the experiment. Each plate corresponds to a sample interval. The assay was created in duplicate.

2.6. RNA Extraction and Real-Time qPCR Analysis

Total RNA was isolated from cell supernatants using the GF-1 kit (VIVANTIS) according to the manufacturer's instructions. Quantitative reverse transcription qPCR was performed using Luna One-Step Universal Probe RT-qPCR kit and with gene-specific primers related to Nodavirus and various immune genes such as antiviral protein genes (IFN-I, Mx); pro-inflammatory genes (IL-1β, IL-8); anti-inflammatory gene (TNF-α, TGF-β); genes involved in the cellular response (CD4, CMH1-β); genes involved in the humoral response (IgM) and stress gene (Hsp30) (Table 1). The reaction was processed in the 7500 Real-Time PCR System (Applied Biosystems). The β -actin gene was selected as the housekeeping gene. The CT2 method was used to calculate relative gene expression [30]. Validation of amplification efficiency was performed for each gene to use the amplification protocols in the current study.

3. Results

3.1. TiO₂ Powder Characterization

SEM micrograph (Figure1-A) reveals that commercial P25 TiO₂ consists of micro-aggregates of irregularly shaped, ca.10 to 50 nm sized NPs. According to the Rietveld refinement of the XRD pattern (Figure 1-B), the sample is composed of 82% anatase and 18% rutile with no trace of brookite. The Scherrer average crystallite size is 21 nm for anatase and 26 nm for rutile. The N₂ adsorption/desorption isotherm at 77 K (Figure 1-C) shows a relative narrow H1-type hysteresis loop typical of mesoporous materials with a BJH desorption cumulative volume of pores of 0.357 cm³/g and average pore width of 170 Å.

The measured BET specific surface area (SSA_BET) is 48 m²/g. If a spherical shape of the particles were assumed, the average particle diameter D_m can be calculated from the SSA, as:

D_m = 6000 / (SSA_BET × ρ_TiO2),

(1)

with SSA_BET in m²/g, ρ_TiO2, the TiO₂ density, in g/cm³ and D_m in nm. If ρ_TiO2 = 4 g/cm³ is used (the mean value between the density of anatase and rutile), then D_m = 30 nm, in line with the SEM micrograph and the Scherrer average crystallite size. Finally, the Tauc plot of the diffuse reflectance spectrum (Figure 1-D) clearly shows the presence of two slops. The extrapolation of these two linear regions to the abscissa yields an energy of the optical bandgap of 3 eV, characteristic of the rutile phase, and of 3.2 eV, typical of the anatase phase.

3.2. Investigation of TiO₂ Cytotoxic Properties on the E-11 Cell Line

Based on the MTT measurements, the wells treated with TiO₂ (5 mg/mL) showed a higher viability threshold compared to those treated with the other TiO₂ concentrations (Figure 2). In fact, TiO₂ concentrations ranging from 2.5 to 0.3 mg/mL significantly reduced the viability of E-11 cells.

3.3. In Vitro Investigation of the Inhibitory Properties of TiO₂ on Nodavirus Replication

Daily observations were performed to check the cellular cytopathic effect (CPE) after different treatment conditions (see Figure 3).

In general, the cell supernatants were very turbid and morphological changes were not visible under the reversed-phase microscope. Therefore, the wells content, which were already irradiated by UV at different time points after UV irradiation (5 min, 15 min, 30 min, 1 h and 16 h), regardless of the occurrence of CPE. It is worth noting that treatment of cells with a TiO₂-nodavirus mixture incubated in the dark (i.e., not-irradiated) did not significantly inhibit Nodavirus replication on E-11. This result highlights the synergistic effect of combined UV-irradiation and TiO₂ NPs inoculation. Continuous CPE was observed daily. The results obtained in darkness were then omitted in the rest of this study.

3.4. Relative Expression of the Viral Capsid Gene (CP) in E-11 Cells

E-11 cells were inoculated with several UV-irradiated TiO₂-Nodavirus mixtures. No clear dose-dependent inhibitory effect was obtained. However, a significant decrease in viral CP gene expression was observed when TiO₂ solution was used at concentrations of 2.5 mg/mL and 1.25 mg/mL (Figure 4). Interestingly, the inhibition of viral gene expression lasted up to 16 h after infection.

3.5. Immune Genes Expression

Immunogenes from E-11 fish cells exposed to TiO₂ co-incubated with Nodavirus showed dose-dependent up-regulation at 1 h. Cells exposed to 1.25 mg/mL of TiO₂ showed the highest level with increased expression of TGF-α, TGF-β and Hsp30 genes compared to virus control. In addition, there was an increase in the expression of IFN-1, Mx, Hsp30, Il-1β, CD4 and Il-8 genes for cells exposed to 2.0 and 2.5 mg/mL TiO₂ concentrations. With a TiO₂ concentration range of 2.0 to 0.3g/L MHC1-β gene expression increased, while cells exposed to low TiO₂ concentrations (0.75 and 0.3 mg/mL) showed an increase in IgM gene expression (Figure 5).

4. Discussion

Numerous viral organisms affect a significant portion of global fish production [48,49], resulting in significant economic impacts on aquaculture [50]. In particular, RNA viruses cause the most serious fish diseases [40] like the neural necrosis virus: betanodavirus responsible for encephalopathy and retinopath [41]. The current study is an in vitro evaluation of the application of TiO₂ powders as an antiviral treatment in aquaculture. TiO₂ powder characterization

A comparative study of the effect of TiO₂ at different concentrations on Nodavirus in the dark and under UV irradiation was performed to determine the mechanisms of the inactivating effect of TiO₂. The investigation of the cytopathic effect was also carried out. The MTT assay shows that the cell viability decreases after addition of TiO₂ at different concentrations, especially in the 2.5-0.3 mg/mL range. These results are consistent with those of previous studies [42], showing a significant reduction in cell viability upon co-exposure of the BF2 fish cell line to TiO₂ at 2.5 and 1.25 g/L. Despite the fact that the MTT assay either overestimates the cell viability or fails to detect a reduction in cell number and viability [44], this effect is most likely produced by the superoxide anions (^•O₂⁻) generated by TiO₂ NPs [40].

No clear cytopathic effect was observed under the reversed-phase microscope at different TiO₂ concentrations. Consistent with present results, a comparative analysis between TiO₂ NPs and ZnO NPs has shown that TiO₂ NPs are less cytotoxic than ZnO NPs [38], meaning that TiO₂ NPs are safer than ZnO NPs with lower cytotoxicity. Another study showed that TiO₂ NPs, when coated with glycated polyethylene, exhibit stronger antiviral activity and lower cytotoxicity compared to bare TiO₂ NPs [41].

To further investigate the antiviral activity of TiO₂, the relative expression of the viral capsid gene (CP) in E-11 cells was examined. The results showed a significant decrease in viral CP gene expression using a TiO₂ suspension at a concentration of 2.5 g/L or 1.25 g/L (Figure 4). This result is in line with those reported by others [47], who showed that the UV-activated TiO₂ surface is able to degrade the capsid and envelope proteins as well as the phospholipids of the non-enveloped virus. Consistent with the present results, previous studies have shown that an astrovirus, a non-enveloped virus that causes diarrheal diseases, was inactivated, but in this case the TiO₂ had been irradiated with visible light [40].

Consistent results were obtained in previous studies on the impact of the use of different NPs on the immune system. Studies on Mytilus galloprovincialis showed that such particles induce pro-apoptotic processes. Their uptake by hemocytes was rapid and affected the phagocytic function [52,53,54]. Similarly, studies of earthworms exposed to TiO₂ revealed bioaccumulation and significantly reduced phagocytosis from 0.1 mg/mL of TiO₂ NPs. However, TiO₂ did not cause cytotoxicity on coelomocytes [52].

In the present study, co-incubation of Nodavirus with different concentrations of TiO₂ exposed to UV light for 1 h differentially regulated the relative expression of genes associated with immunity and stress in cells, such as antiviral protein genes (IFN-I, Mx); pro-inflammatory genes (IL-1, IL-8), anti-inflammatory gene (TGF-, TGF-), genes involved in the cellular response (CD4, CMH1-), gene involved in the humoral response (IgM) and stress gene (Hsp30). Remarkably, the TiO₂ concentration of 1.25 mg/mL increased expression of all target genes and induced an inflammatory response to Nodavirus, which has been never reported in the literature. Studies have now been conducted to examine the interaction of TiO₂ NPs with the immune system: human macrophages were exposed in vitro to non-toxic concentrations of various nanoparticles including TiO₂, which were shown [53] to significantly affect the expression of several Toll-Like Receptors (TLR), molecules. These latter receptors represent a critical class involved in the initiation of the inflammatory response and innate immune responses. Particularly intriguing is that NPs like TiO₂ could increase the expression of TLR chains required for virus-dependent stimulation [53]. TLR7 is another receptor that is crucial for detecting viruses. It binds to viral ssRNA and activates the signaling pathway that depends on IRF-3 to produce IFN-a/b [54,55]. According to [53], macrophages exposed to nanoparticles such as TiO₂ have increased susceptibility to viral infections. Our in vitro research has shown that the different doses of TiO₂-NPs can possibly boost the fish's immune system to fight viral infections, although the doses are much higher compared to the estimated environmental concentration of 0.025 mg/mL [55,56].

Several research results suggest that nanoparticles can replace antibiotics in the treatment of infections. TiO₂ NPs proved to be the safest of all NPs, cheap and easy to manufacture, compared to other NPs.

Authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.

5. Conclusions

This in vitro study shows that TiO₂ NPs reduce viral infection, while boosting the fish's innate immunity markers, and might be able to suppress more pathogens, but further experiments, such as in vivo tests, are required. In particular, further investigations should clarify whether the inactivation of TiO₂ NPs depends on the virus titer, the incubation time and the TiO₂ concentration. However, this work has shown that the use of TiO₂ NPs to control Nodaviruses in fish farming has great potential.