Study On The Effect Of Hypericin-loaded Graphene Oxide Against A Novel Duck Reovirus

Novel duck reovirus?NDRV?has a wider host spectrum than the classical duck reovirus?DRV?.There are no biological products for the prevention and treatment of the virus disease at present,which brings great difficulties to disease prevention and control.Hypericin?HY?is a traditional Chinese medicine monomer extracted from Hypericum perforatum.It has broad-spectrum antiviral activity,including various retroviruses,but its application is limited by its poor water solubility and cytotoxicity.With the development of nanotechnology,graphene oxide?GO?has been used in biomedical field.GO has high drug loading capacity because of its unique structure,and has become the most promising biomaterial in biomedical applications.In this study,the cytotoxicity of HY on DF-1 cells was reduced by loading HY into GO.The characterization and loading efficiency of the complex were evaluated.The antiviral effect of the complex on NDRV-TH11 strain of duck reovirus in vivo and in vitro was studied,which laid a foundation for the development of antiviral drugs against duck reovirus.This study is divided into the following parts:1 Study on loading,characterization and cytotoxicity of hypericin by graphene oxideGraphite oxide/hypericin complex?GO/HY?was prepared by blending method,and its loading efficiency was calculated to be 72.5%.The morphology of hypericin was observed by atomic force microscopy?AFM?.The effective loading of hypericin on graphene oxide was determined by lamellar analysis.MTT assay was used to detect the cytotoxicity.The results were as follows:When GO concentration reached 100?g/mL,the cell survival rate was 93.16%.Compared with the single HY,the toxicity of GO-loaded HY to DF-1 cells was lower.When the concentration of HY reached 72.5?g/mL,the cell survival rate was 91.3%,while the DF-1 cell survival rate under GO/HY was still 93.83%.2 Study on the anti-NDRV activity of graphene oxide/hypericin complex?GO/HY?in vitroThe anti-NDRV activity of GO/HY in vitro was detected by CPE and titer method with TH11 strain of duck reovirus as the research object.The results were as follows:The inhibition of GO/HY on DNRV-induced DF-1 cell lesion was observed by CPE method.The results showed that when GO/HY concentration was 72.5?g/mL,the cytopathic effect could be completely inhibited.DF-1 cells infected with NDRV were treated with GO/HY at different concentrations.The results showed that the viral titer of TCID₅₀0 in GO/HY treatment group decreased gradually with the increase of GO/HY concentration compared with that in virus control group.The above results showed that GO/HY had good anti-NDRV activity in vitro.3 The effects of GO/HY on the replication of NDRV in vitroThe effects of GO/HY on the replication of NDRV in vitro were observed by virus growth curve,plaque test,Western blot and indirect immunofluorescence?IFA?.The results were as follows:The growth curve of NDRV in virus group and GO/HY treatment group?72.5?g/mL?was determined.The results showed that the viral titer of virus control group could reach 10⁷.8.8 after 48 hours of exposure,and that of virus treatment group could only reach 10⁴.2.2 after 48 hours of exposure,which indicated that hypericin could significantly inhibit the proliferation of NDRV in DF-1.The results of plaque test showed that the number of empty plates in each dose of GO/HY treatment group was significantly less than that in virus control group,and there was no empty plate in negative control group.This indicated that GO/HY could significantly inhibit the virulence of DNRV.The expression of?C protein was detected by Western blot.The results showed that when GO/HY concentration was 18.125?g/mL,specific bands appeared at 35 kDa,but the band size was obvious decreased,GO/HY medium and high concentration treatment group no objective band appeared.IFA analysis showed that the viral group showed obvious green fluorescence,while the GO/HY treatment group?72.5?g/mL?showed a significant decrease in green fluorescence intensity.From the above results,GO/HY can significantly inhibit the replication ability of DNRV in vitro,and the inhibition effect is dose-dependent.4 Study on the action characteristics of GO/HY on NDRVThe antiviral effects of GO/HY on NDRV infected cells at different stages were compared by SYBR Green II fluorescence quantitative PCR.The results were as follows:The anti-NDRV activity of GO/HY in vitro was investigated by three ways:cell protection by drugs,virus penetration into cells and intracellular reproduction.The relative expression of indolent?C gene was detected by SYBR Green II quantitative fluorescence PCR.The results showed that the relative expression of indolent?C gene was significantly decreased after infection compared with that in the early dosing group.It can be seen that GO/HY plays an important role in the process of NDRV infection with DF-1,which is the adsorption stage of virus invading cells.5 Anti-NDRV activity of GO/HY in vivoA duckling model of Cherry Valley duck with NDRV-TH11 virulent strain was established by intramuscular injection.The results were as follows:GO/HY in each dose group could prolong the survival time of the ducks.The virus infection group began to die on the 3rd day,and all died after 5 days,the survival rate was 0.The survival time of drug treatment group was prolonged,and the survival rate of high dose group was up to 40%.This indicated that hypericin increased the survival rate of poisoned ducks.The viral load in liver and lung tissues of ducks in each treatment group was detected by SYBR Green II fluorescent quantitative PCR method.The results showed that the higher the dose of treatment,the lower the transcription level of?C gene,indicating that hypericin had a significant inhibitory effect on viral load in tissues.Histopathological sections showed that GO/HY could significantly improve the pathological changes of liver and lung tissues of ducks exposed to GO/HY,and the results of immunohistochemistry showed that the distribution of virus in liver and lung decreased with the increase of drug dosage.In this study,hypericin was loaded with graphene oxide,and the anti-NDRV effect of GO/HY complex in vivo and in vitro was investigated.It was found that GO/HY could inhibit the replication of NDRV by inhibiting the transcription of NDRV target gene and the expression of target protein at an early stage.At the same time,GO/HY can delay the time of death by inhibiting the replication of NDRV in infected ducks and alleviate the damage of target organs.The results of this study lay a foundation for the development of anti-duck reovirus drugs.