Inhibitory Effect of ZnO-NPs and NF-CS-ZnO Nano-Hybrid Against Herpes Simplex Virus Type 1 Infection

Background: Since the viruses are intracellular parasite and their replication depends on the host cells, to develop a medication against the viral infections, some substances should be provided having no damage on the host’s cells and tissue. Although the antiviral effects of zinc ion against the viral pathogens has been conspicuous, undesirably it shows a severe toxicity on the cells in cell culture and host tissues in animal models. Methods: To optimize, zinc oxide nanoparticles with antiviral property at the lowest cell toxicity, nano-brillar chitosan-ZnO nano-hybrid was synthesized and then, its inhibitory effect against the HSV replication was investigated in comparison to ZnO nanoparticles using plaque reduction and Real time PCR method. Results: Although the infectivity of HSV-1 was reduced in initial steps of virus entry, the signicant reduction of viral titer was observed in post exposure of Nano particles on HSV-1 infected cells. In contrary, no changes of viral load were identied in pre-incubation experiment, co-incubation of virus and nanoparticles prior to virus inoculation. Conclusion: Nano-brillar CS could dramatically decrease the cell cytotoxicity of ZnO, even though the antiviral effect of ZnO nanoparticles did not change. Nano hybrids appears to penetrate into the host cells and intracellularly inhibits the virus multiplication.


Background
Viruses are one of the main pathogens, which can cause severe diseases in human being (1). Although annually a wide range of drugs are offered to eradicate the viral microorganisms, due to the unique properties of viruses such as mutation's capability in generating new strains and particularly the emergence of drug resistant strains, most of available drugs are becoming useless (2,3). Furthermore, the usage of some viral medications has been restricted as they cause devastating side effects on the individuals (4,5). Therefore, there is a growing need to provide new, safe and effective drugs against viral infections.
In the recent years, by the growth of nanotechnology, a numerous variety of nanoparticles especially metal oxide nanoparticles have been developed to inhibit the infectivity of aboard range of human pathogens (6, 7). Amongst all, the application of zinc oxide nanoparticles (ZnO-NPs) as an antimicrobial agent has been more appealed as they represent several unique antimicrobial mechanisms such as the induction of oxidative stress and disruption of the bacterial membrane due to the accumulation of ZnO-NPs (8, 9). Moreover, recent studies showed ZnO-NP has an e cient inhibitory activity against the several viral infections such as in uenza, herpes simplex, rhinoviruses and corona viruses. It seems zinc ions can inhibit the virus's multiplication via interference with viral genome replication (10)(11)(12).
In spite of high antiviral effect of Zno-NPs, scienti c evidence shows that the ZnO-NP has a signi cant cytotoxicity on host cells (13,14), thus the optimum concentration of this material in inhibition of viral replication must be accurately controlled. At the same time, some reports have shown that ZnO-NPs modi cation by natural polysaccharide materials such as cellulous, alginate, hyaluronic acid, starch and chitosan, can reduce ZnO-NPs cytotoxicity (15)(16)(17). Chitosan (CS) is known as a natural polysaccharide with remarkable properties such as biocompatibility and biodegradability. Furthermore, in some studies, antimicrobial activities of CS against the bacterial and fungal strains have been demonstrated (18). It has also been shown that the hybridization of nano-sized metal oxides and CS can improve the antibacterial properties (19,20). Besides, it seems that CS can increase the permeability of nanoparticles into cells (21), which can be considered as an e cient option for developing antiviral drugs to inhibit the viral replication into cells. In the previous studies, hybridization of CS nanoparticles with antiretroviral drugs has improved the anti-HIV therapy as cell targeting e ciency increased by 92% compared to the conventional drug control (22). There is a possibility that CS not only can be utilized as a drug delivery candidate, but also has intrinsic antiviral effects (23).
It is established that CS can be produced in various morphologies by using different preparation processes, including natural powder, spherical and nano-structure (24). Among these morphologies, nanobrillar's form can exhibit prominent properties in biomedical application due to the higher aspect ratio (25). Since viruses replicate into cells, this characteristic might increase the antiviral activity of ZnO-NPs.
In the current study, antiviral effect of prepared ZnO-NPs and nano-brillar CS-ZnO (NF-CS-ZnO) nanohybrid was evaluated in the cell culture system. To this aim, Herpes simplex virus type 1(HSV1) replication, one of the members of herpesviridae family which is known as the leading cause of a broad spectrum of human disease including herpetic stomatitis, recurrent herpes labials, keratitis and encephalitis (26, 27) was targeted by synthetics nanoparticles.

Synthesis of ZnO-NPs
ZnO-NPs were synthesized within a simple co-precipitation method. Firstly, the 0.5 M zinc acetate dehydrate solution was prepared in an Erlenmeyer ask at room temperature. Secondly, 0.5 M solution of sodium hydroxide was treated to the zinc acetate dehydrate solution (PH of the solution reached 7) under ultrasonic homogenizer condition (200 watts, 100% amplitude, UP400St, Hielscher, Germany). The achieved light milky suspension was named ZnO-NPs. Finally, to remove the excess of zinc acetate and other impurities, the above suspension was washed several times using deionized water and ethanol and then dried using a vacuum oven at 60 °C for 24 h.  For the AO uorescent dye assay, Vero cell monolayer were seeded in 6-well tissue culture plates and incubated ZnO-NPs and NF-CS-ZnO at 50 µgr/ml concentration. According to manufacturer's protocol AO orescent dye assay was performed 24h after incubation. Brie y, cells were washed twice by PBS and then, 100 μg/ml AO uorescent dye was added to each well. Finally, an inverted uorescent microscope (Jeol JSM 6490) was used for the observation of alive cells within 30 s.

Assessment of NPs antiviral activity
To experiment the effect of NPs in HSV replication's inhibition, three main stages of viral life cycle on Vero cells cultured in 24 well plate was evaluated. To explain brie y, in the pretreatment stage, Vero cell was exposed at the lowest toxic concentration of NPs for 4 h. Then, cell monolayer was washed by PBS and infected by HSV1 strain (MOI: 0.01) for merely 1 hour. Unbound viruses were removed by washing and then fresh DMEM with 2% FBS was added to each well. In another stage named post infection, at rst, the Vero cells was inoculated by virus strain and then, after 1 hour, cells were washed by PBS and nally cells were treated by different NPs. To evaluate, the direct virucidal activity of NPs, the pre incubation experiment was performed as a mixture of virus and nanoparticles was provided in a micro tube. The microtube was incubated on a shaker at room temperature for roughly 4 hours. The microtube was then centrifuged for 10 min at 4000 g and infection of cells was performed by 500ul of supernatant.
Eventually, after 48 h incubation of the cells at 37° C, the supernatant of each well was collected and tittered for infectivity of HSV1 on Vero cells by plaque reduction assay.

Plaque reduction assay
To determine the inhibitory effects of nanoparticles on HSV-1, plaque assays were performed on Vero cells. The cells were seeded onto 6-well culture plates in DMEM with 10% FBS to achieve nearly 95% of con uence. Serial dilution of supernatants collected in previous experiment were provided and then 500 ml of each dilution were added to each well. To allow virus attachment to cells, the plate was incubated at 37° C for 1 h. Then, after washing cells by PBS, 3 ml of 1.5% DMEM/agar was overlaid to each well.
The plates were incubated for 72 h until the formation of plaques. To observe plaques, 1.5% DMEM/agar with Neutral red 0.01% was added to each well and plates incubated for 24 h. Finally, the number of plaques in plate was counted and viral titer was calculated according to the following formula. A reduction in the number of plaques in each experiment was determined by comparison with a positive control.
*Viral titer formula: No. of Plaques / (Dilution factor x Volume of diluted virus/well) = pfu/ml

Quantitative Real-Time PCR assay
To determine HSV viral load, Real-Time PCR assay was carried out in the following. Total RNA was extracted from supernatants collected in the interference experiments using the viral nucleic acid kit (Roche, Germany). The extracted RNA was then subjected to reverse transcription using the cDNA Synthesis Kit (Bio fact, Korea). The late gene UL27 (encoding glycoprotein gB) were determined by using Rotor-Gene Q instrument in a thermal pro le of 95 °C for 2 min (1 cycle), 95 °C for 30 s, and 60 °C for 15 s (35 cycles, respectively) (29). The transcription levels of total RNA in each sample were normalized against the GAPDH gene. Relative quanti cation was carried out based on the 2 −ΔΔCT threshold cycle method.

Statistical analysis
ANOVA and Least Signi cant Difference (LSD) test was done to compare viral titer reduction in each experiment. The P value of <0.05 was considered statistically signi cant. All the data are given as the mean ± SD. SPSS software, version 22 was run to do all calculations.

Characterization of prepared ZnO-NPs and NF-CS-ZnO nano-hybrid
FE-SEM micrographs of the synthesized ZnO-NPs and NF-CS-ZnO nano-hybrid are shown in Fig.1 (a, b). According to Fig.1a, the successful formation of ZnO-NPs with the spherical morphology at around 50-70 nm was illustrated. Moreover, it can be clearly seen that in the specimen of prepared NF-CS-ZnO nanohybrid (Fig. 1b), the spherical ZnO-NPs have favorably covered the surface of NF-CS. Importantly, it seems that NF-CS act as a steric hindrance to prevent aggregation of ZnO-NPs. Figure 1c

Nanoparticles and Nano hybrids 's toxicity on cell survival
NPs cytotoxicity against the Vero cells was analyzed by MTT assay and AO uorescent staining. The results of MTT assay showed that the viability of cells signi cantly decreased to 40% when Vero cells was treated by 50 μg/ml of ZnO-NPs. However, Vero cells exposed with 25, 50 and 100 μg/mL of ZnO-CS-NPs showed the cell survival roughly 90, 80 and 65% respectively (Fig.2 a). The microscopic assay on normal cells in comparison with Vero cells treated by nanoparticles also indicated that toxic concentration of ZnO-NPs has lead cells becoming tiny, round and detached from the bottom plate ( Fig.2.b). Accordingly, AO orescent dye results showed that the population of viable cells (green cells) incubated with ZnO-CS-NPs was similar to the control group while the population of viable cells in ZnO NPs group was signi cantly reduced (Fig. 2.c). Therefore, based on the obtained results, the optimum concentration of 25 μg/ml of ZnO-NPs and 50 μg/ml of ZnO-CS-NPs was applied for the all experiments.

Antiviral activity of the ZnO-NPs and ZnO-CS-NPs
To evaluate antiviral effect of nanoparticles in vitro, three main experiments were designed as explained in the method section and then viral load was assayed by plaque reduction method. As illustrated in Fig.3a, b, both ZnO nanoparticles and ZnO-CS nano-hybrids could signi cantly inhibit the viral replication in the post and pretreatment stages as the viral titer substantially decreased in comparison with the control group. The reduction of cytopathic effects of infected cells in different experiments can also be observed in Fig. 4. Amongst three stages, no remarkable changes were observed in viral infectivity in pre incubation stage.

Real time PCR results
To con rm, the viral titer reduction in the presence of nanoparticles, the relative quantity of HSV genome in Vero cells exposed by nanoparticles (treat group) compared with HSV infected cells (control group) using real time PCR. As shown in Fig.5, the copy number of HSV gene B in the pretreatment and post infection groups was signi cantly declined (P = 0.02), while no change was observed on pre incubation experiment.

Discussion
Although the effect of zinc ion against a wide range of viral pathogens has been proven since 1967, the utilization of it as a viral medication has been restricted as it is quite toxic for host cells (29).
In the current study, to optimize, Zno nanoparticles with antiviral property and the least toxicity on host cells, Zno NPs coated with nano bers chitosan was offered to investigate their inhibitory effect against HSV replication in Vero cells. As gure three and ve shown, both Zno and Zno-CS nanoparticles could signi cantly inhibit viral replication in the post and pretreatment stages as the viral titer substantially decreased in compared with group control. Interestingly though, Zno-Cs nanoparticles has shown a lower toxicity on Vero cells con rming that surface chitosan of nanoparticles plays a key role in reduction of cell cytotoxicity on Vero cells (Fig. 2).
As the results shows (Fig. 3,5), it seems that nanoparticles are not able to directly trap and destroy the structure of viral particles, as the declining of viral titer was no signi cant in the pre incubation assay. In fact, in this stage, nanoparticles were treated by HSV virus in a free cell system at four hours and then the exposed HSV particles was inoculated into Vero cell line. This experiment might obviously support this idea that zinc oxide nanoparticles has no physical interaction by HSV particles and as a result, they do not have e cient direct virucidal activity. Nevertheless, in the pretreatment experiment and, particularly, in the post infection stage, the reduction of viral infectivity was vividly observable (Fig. 3,5). To explain this nding, the antiviral activity of zno nanoparticles are more likely to be intracellularly exerted. As the dimension of nanoparticles are very small nearly 100nm, they can effectively penetrate into the cells and interfere with viral replication. In consistent with our ndings, a few previous studies also showed that zinc ions could block viral polymerase activity or hinder viral polyproteins processing (10-12). Although, the viral titer also has declined in the pretreatment experiment, the reduction was more signi cant in post infection experiment. The aim of pretreatment assay was showing the nanoparticles capability in blocking cellular receptors in term of hindrance of virus entry. However, it seems nanoparticles are more capable to enter cells and intracellularly inhibit viral replication instead of involving in the virus attachment stage to cell receptors.

Conclusions
In conclusion, although zinc oxide has a considerable cytotoxicity on host cells, we could optimize it by creating zno nanoparticles coated by chitosan, which have shown notable antiviral activity against HSV virus. Furthermore, the present results indicate that the inhibitory mechanism exerted by nanoparticles is more probably in the result of interference of NPs with one of the viral replication stages into cells.
Although it can be promising to use zinc oxide-cs nanoparticle in treatment of viral diseases, more experiments need to investigate the antiviral activity of such nanoparticles against different types of viral pathogens in vitro and in vivo as well.

Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request

Competing Interests
The authors have declared that there are no con ict of interest.    Microscopy assay of infected cells, which were treated by NF-CS-ZnO nano-hybrid in three different experiment.