Study On The Nano Drug Delivery System Targeting White Spot Syndrome Virus

White spot disease（WSD）,caused by white spot syndrome virus（WSSV）,is an acute and highly lethal disease to shrimp.As invertebrates do not have an adaptive immune system and rely mainly on innate immunity for defense,drug treatment and the production of specific pathogen free（SPF）are effective measures for the prevention and treatment of WSD.At present,there are no effective commercial drugs to control WSD,and the researches on drug prevention and control of WSD are still scarce.Thus,it is necessary to develop anti-WSSV drugs.The high production costs and long production cycle have limited the development of SPF shrimp.Medicinal plants have the advantages of rich resources,environmental friendliness,low toxicity,and easy degradation,which is an ideal raw materials source of safe and harmless aquatic drug.Nano-targeted drug delivery systems have the advantages of easy penetration of tissue barriers,targeted drug delivery and improvement of drug efficacy,and have become research hotspots in the medical field.In the study,medicinal plants were screened against WSSV in red swamp crayfish Procambarus clarkii,and then the main compounds of medicinal plant were identified for anti-WSSV activity and mechanism research.The anti-WSSV compound would be used for the construction of nano-targeted drug delivery systems.Additionally,single-walled carbon nanotubes（SWCNTs）,single-chain antibody P75E8 specifically against WSSV and the anti-WSSV compounds were selected for construction of nano-targeted drug delivery systems.The anti-WSSV activity of nano-targeted drug delivery systems were evaluated in vivo.The results obtained in this study were as follows:1. The screening of medicine plants against anti-WSSV activityReal-time quantitative PCR and standard curve capable of absolutely quantifying the copy numbers of WSSV were used to detect the dynamic changes of the virus copy numbers in different tissues of WSSV-infected crayfish.The results showed that WSSV could infect gills,hemolymph,hindgut,muscle and hepatopancreas of crayfish.Among them,the copy numbers of the gills are the highest,and the hepatopancreas is the lowest.The proliferation curve of WSSV in crayfish is a typical one-step growth curve.The expression trends of WSSV immediate early（IE）genes ie1,early genes dnapol and late genes vp28 were consistent with the genomic DNA replication.The anti-WSSV activities of 30 medicinal plant extracts were investigated in WSSV-infected crayfish.Among these,Gardenia jasminoides,Eucommia ulmoides,Morinda officinalis,Acanthopanax senticosus,Polygonatum sibiricum,Evodia rutaecarpa and Pulsatilla chinensisand had significant antiviral activity,and the inhibition rates were 92.31%,78.65%,69.00%,64.50%,62.01,57.87%and 46.58%,respectively.2. Study on anti-WSSV activity of the crude extracts of G.jasminoidesThe anti-WSSV activity of the crude extracts of G.jasminoides were evaluated in WSSV-infected crayfish.The results showed that:G.jasminoides extracts inhibited WSSV genome replication in a concentration-dependent manner;The inhibition rate was more than 90%when the treatment concentration was 100 mg/kg;The extracts could significantly inhibit the expression of IE genes ie1,early genes dnapol and late genes vp28;After treatment with extracts（100 mg/kg）for 7 d,the cumulative survival rate of WSSV-infected crayfish increased by 60%.In addition,the extracts could increase the expression of antioxidant-related genes（c Mnsod,m Mnsod,cat,and gst）and apoptosis-related genes（bax and bax inhibitor-1）in the gills of WSSV-infected crayfish.Quantitative analysis of geniposide（GP）,geniposidic acid（GPA）,and genipin（GN）in G.jasminoides extracts by LC/MS showed that the contents of GP,GPA and GN were 171.93 mg/g,1.25 mg/g and 0.46 mg/g,respectively.3. Study on anti-WSSV activity of three active ingredients of G.jasminoidesThe anti-WSSV activities of GP,GPA,and GN were evaluated in WSSV-infected crayfish.The results showed that:GP,GPA,and GN could significantly inhibit WSSV replication in crayfish;The inhibition rate was more than 90%when the concentration was 50 mg/kg;Following GP,GPA,and GN（50 mg/kg）treatment for 10 d,the cumulative survival rates of the WSSV-infected crayfish increased by 70.0%,43.33%,and 50.0%,respectively.The anti-WSSV activities of GP,GPA,and GN were further evaluated in WSSV-infected shrimp Litopenaeus vannamei.The results showed that the inhibition rates of GP,GPA,and GN on WSSV replication were all over 90%when the treatment concentration was 50 mg/kg.In addition,GP,GPA and GN could protect crayfish from oxidative damage and inflammatory responses.The pharmacokinetics of crayfish and shrimp showed that the pharmacokinetic curves of GP and GPA were similar,and the highest drug contents were in hepatopancreas,and the lowest contents were in gill tissue.4. The effect of GP,GPA and GN on function of WSSV proteinsThe effects of GP,GPA,and GN on the proliferation of WSSV were studied,and the results showed that these three compounds mainly affect the early stage of WSSV replication.The eukaryotic plasmids of WSSV envelope proteins VP19,VP24,VP26,and VP28 were constructed.Then these plasmids were transfected into Drosophila S2 cells,and S2 cells were treated with GP,GPA,and GN,respectively.The results showed that GP,GPA,and GN effectively inhibited the expression of proteins VP19,VP24,VP26,and VP28,of which GN had the most significant inhibitory effect on VP28,indicating that these three compounds can target WSSV proteins to block WSSV infection.In addition,GP,GPA,and GN could inhibit the expression of the STAT gene,thereby suppressing the transcription of the early gene ie1,the early gene dnapol,and ultimately inhibit the replication of WSSV genome DNA and the expression of late gene vp28.5. Study on the nano-targeted drug delivery system against WSSVSingle-chain antibody P75E8 specifically against WSSV were selected as the template to build P75E8 prokaryotic expression system by genetic recombination technology,and prepare P75E8 protein by fermentation and purification techniques.Elisa test showed that P75E8protein has higher affinity with WSSV.Cell co-transfection experiments revealed that PE5E8migrated from the nucleus to the cytoplasm and bound to the WSSV envelope protein VP28.Yeast two-hybrid and Western blot results showed that P75E8 bound to the VP28_460-612 region of the C-terminus of VP28.GPA,P75E8 and SWCNTs were selected as anti-WSSV drugs,targeting ligands and nanocarriers to construct a nano-targeted drug delivery system（SWCNTs-GPA-P75E8）.The antiviral effects of GPA,SWCNTs-GPA and SWCNTs-GPA-P75E8 were evaluated in WSSV-infected crayfish,and the results showed that SWCNTs-GPA-P75E8 had the strongest anti-WSSV activity.When the GPA concentration was 25 mg/kg,the survival rates of WSSV-infected crayfish increased to 23.33%,36.67%and 46.67%within 10d after treatment with GPA,SWCNTs-GPA and SWCNTs-GPA-P75E8.The pharmacokinetic study showed that the GPA contents in the gills of WSSV-infected crayfish were significantly higher in the SWCNTs-GPA-P75E8 treatment group than that in the GPA and SWCNTs-GPA treatment groups.Medicated bath treating larvae shrimp showed that:GPA,SWCNTs-GPA and SWCNTs-GPA-P75E8 could kill WSSV virus in larvae,and the antiviral effect of SWCNTs-GPA-P75E8 was significantly higher than that that in the GPA and SWCNTs-GPA treatment groups.In summary,G.jasminoides can inhibit the replication of WSSV and reduce the mortality of WSSV-infected crayfish;The active ingredients GP,GPA and GN of G.jasminoides can effectively control WSSV replication by host antiviral response and directly acting on viral envelope proteins;The nano-targeted drug delivery system can target WSSV protein VP28,achieve targeted treatment of WSSV,and improve the anti-WSSV activity of GPA,provide theoretical foundations for highly efficient anti-WSSV and production of SPF shrimp.