Antiviral Mechanism Of Two-dimensional Graphene Analogues Nanomaterials

Infectious diseases caused by viruses and bacteria constantly threaten the health of both humans and animals. Although some viral diseases have been well controlled or even eradicated by vaccination, some are not controlled. Low cross-protection among different serotypes and variation between numerous strains hamper prophylaxis of these viral infections, therefore, studies of pathogenesis and potent viricide are very important for the control of these diseases.In this study, we developed a Vero cell line that stably expresses PEDV ORF3 gene to evaluate the influence of PEDV ORF3 on host cells and the virus proliferation. These results provided information on the function of PEDV ORF3 and were helpful in understanding the mechanisms of PEDV replication. Then we identifed the antiviral activity of two-dimensional nanomaterials against PEDV and PRV for the first time, and proposed a possible antiviral mechanism. This study shed lights on novel virucide development. The main contents are as follows: 1. Construction of Vero cell line that stably expressing PEDV ORF3 to analysis itsinfluence on cell cycle, vesicles formation and virus replicationVero cell lines that stably express PEDV ORF3 were obtained using lentivirus system. The expression of ORF3 was identified by RT-PCR, qPCR and western blot. Vero-ORF3 cell line did not lose the expression of ORF3 during passages. Vero cells and Vero-ORF3 cells were analysed for cell cycle progression by flow cytometry. Over 40% of the Vero-ORF3 cells accumulated in the S-phase whereas only 28.03% Vero cells accumulated in this phase, indicating that ORF3 expression in Vero cells could prolong the S-phase, which was crucial for virus replication. On the other hand, there were significantly more Golgi-related vesicles in Vero-ORF3 cells than host Vero cells and Vero cells transfected with truncated ORF3. Moreover, double membrane vesicles（DMVs） which were involved in coronavirus replication were observed in Vero-ORF3 cells. These results indicated that PEDV ORF3 prolongs S-phase and facilitates formation of vesicles, which were closely related to virus replication. Vero and Vero-ORF3 were infected with isolated fiels virulent PEDV（YN strain） and attenuated PEDV（AH-M and CV777 vaccine strain） to further confirm the influence of ORF3 on PEDV proliferation. The results of TCID50 and qPCR demonstrated that attenuated PEDV virus strain AH-M and CV777 were significantly higher in Vero-ORF3 cells than in Vero cells, while the virulent YN strain showed no significant difference. The expression of PEDV N protein on Vero-ORF3 was higher than on Vero cells. Based on these results, we inferred that PEDV ORF3 exerted a positive regulatory effect on the attenuated PEDV proliferation. 2. Mechanism of the antiviral activity of GOWe found that GO exhibit excellent antiviral activity on both porcine epidemic diarrhea virus（PEDV, RNA virus） and pseudorabies virus（PRV, DNA virus） in a concentration and time dependent manner. GO was added to the cell culture at different stages of viral infection to determine at which stage of viral replication GO works. GO exhibits antiviral activity only when it was incubated with virus prior to viral adsorption but does not work after or even simultaneously with the onset of viral infection, indicating that the antiviral activity of GO was most probably due to the physical interaction by direct contact. To elucidate the antiviral activity of GO, sevral GO-based materials were used. Both GO and rGO show similar antiviral activity, indicating that the oxygen-containing group is not essential for the antiviral activity. The polylaminate GtO shows much weaker antiviral activity than single-layered GO and rGO, whereas the non-nanosheet Gt showed no inhibition, suggesting that the nanosheet structure is important for the antiviral activity. Furthermore, GO combined with the cationic PDDA（GO-PDDA） has no antiviral activity, but the nonionic GO-PVP shows similar antiviral activity as GO, indicating that the negative charge is required for the antiviral mechanism of GO and rGO. In conclusion, we found that the negatively charged GO has more chances to interact with viruses through electrostatic interactions prior to viral entry, resulting in virus damage and inactivation due to its single-layer structure and sharp edge. 3. Antiviral activity of two-dimensional graphene analogue MoS₂ and WS₂ nanosheetWe found that other two-dimensional nanosheet materials, like MoS₂ and WS₂ also exhibit good antiviral activity. However, their antiviral efficiency was lower than GO, accompany with lower cytotoxicity compared to GO, which may associated with their antiviral mechanism. Antiviral activity of MoS₂ and GO agaisnt PRV in vivo was evaluated on mice. Four-week old Balb/c mice were challenged with 103TCID50 PRV. PRV-infected mice all died 4 dpi, while 60% mices were survival in MoS₂ incubation group and GO incubation group. Moreover, when the mice were intravenously injected with MoS₂ 1 h post challenge, 40% mice were protected, while the treatment with GO intravenous injection showed no protection. In GO incubation group, MoS₂ incubation group and MoS₂ injection group, the mortality of mice and the virus distribution in mice brain tissue were decreased, and the mice postponed symptom for 1d. These results indicated that MoS₂ and GO could inhibit PRV replication in vivo, and MoS₂ showed therapeutic effect. 4. Toxicity of MoS₂ and GO on miceTo evaluate the toxicity of MoS₂ and GO on mice, 20 fold dose（2 mg/kg body weight for GO and 10 mg/kg body weight for MoS₂） were intravenous injected in mice. Pathological examinations were performed in 2, 14 and 60 d post injection. Results showed that there were no damage in lung, liver, kidney and brain. The liver and kideny function parameters were all in the normal reference range. These results demonstrated that MoS₂ and GO showed no acute toxicity in our experimental condition.In conclusion, we found that PEDV ORF3 prolonged S phase in cell cycle and facilitated vesicles formation, thus promoted the proliferation of attenuated PEDV. These results provided information on the function of PEDV ORF3 and were helpful in understanding the mechanisms of PEDV pathogenesis. We also reported on the broad-spectrum antiviral of two-dimensional nanomaterials with potential antiviral mechanism, which might shed some lights on novel virucide development.