Screening Of Compounds Against Pseudorabies Virus And The Study Of Their Inhibitory Mechanisms

Pseudorabies(PR)is an acute viral neurological infection of pigs caused by Pseudorabies virus(PRV),which manifests clinically as neurological symptoms in piglets,respiratory symptoms in large and medium-sized pigs,abortion or stillbirths and mummified foetuses in pregnant sows.Vaccination is a major commonly used method for the control of PR,but in addition to vaccination,pharmacological control holds some promise for research as a potential strategy for PR control.There are no specific drugs for PRV infection,so the search for drugs to inhibit PRV infection is of great importance for the treatment of PR.1.Construction of recombinant PRV tracer virus containing EGFPIn this study,the left and right homologous arms of the UL23(TK)gene of PRV JSY13 were linked to the EGFP-containing pBeloBAC11 vector using the Bacterial artificial chromosome(BAC)technique and seamless cloning method.The pBAC-FR-EGFP was cotransfected with the PRV JS Y13 genome into African green monkey kidney cells(Vero),which were homologously recombined at both ends of the JSY13 UL23 gene,and the UL23 gene was replaced by the BAC-EGFP sequence.Subsequently,the recombinant PRV was purified by plaque formation assay and the growth characteristics of the recombinant virus were characterized.The results showed that the left and right homologous arms of the UL23 gene of PRV JSY13 were successfully linked to the EGFP-containing pBeloBAC 11 vector,and the transfer vector pBAC-FR-EGFP was successfully constructed.pBAC-FR-EGFP was co-transfected with the PRV JSY13 genome using the calcium phosphate transfection method,and the green fluorescent virus could be observed under the fluorescence microscope after 72 hours.The green fluorescent virus could be observed under fluorescence microscope after 72 hours,indicating that the EGFP-containing recombinant rJSY13-△TK-EGFP was successfully constructed.The recombinant virus was purified using the plaque formation assay,and the recombinant virus rJSY13-△TK-EGFP was finally purified.The results of the viral proliferation curve showed that the proliferation efficiency of the constructed rJSY13-△TKEGFP strain was slight lower than its parental strain.2.Screening of anti-PRV drugs and their inhibitory mechanismsThe constructed PRV rJSY13-△TK-EGFP was used as a tracer virus to screen 240 natural compounds for anti-PRV drugs according to the intensity of virus fluorescence after drug treatment.The toxicity of the screened drugs to cells was then assayed using the CCK-8 cellular activity kit.In addition,the concentrations of drugs required to inhibit 50%of the virus(IC50)were measured using a Cell-based-ELISA method.Subsequently,PK15 cells were pretreated with different concentrations of the drug and inoculated with JSY13(0.15 MOI),and the effect of the drug on PRV proliferation was further verified by Western blot and indirect immunofluorescence assay(IFA).The effect of the drug on the different replication stages of PRV,including attachment,entry,and release,were determined by quantitative realtime PCR(qRT-PCR)and plaque formation assay.Finally,the effects of the drugs on PRV proliferation in vivo were investigated on a mouse model by observation of clinical symptoms,anatomical lesions,viral load and survival numbers.The results showed that the fluorescence intensity of PRV was significantly reduced after treatment with bufalin,gallocatechin gallate(GCG),and Saikosaponin D(SSD),indicating that the three drugs could inhibit PRV proliferation.The results of cytotoxicity assay showed that the cellular activity of bufalin and GCG remained above 80%at drug concentrations of 120 μM and 160 μM,and the drug concentration(CC50)required for SSD to kill 50%of host cells was 14.430 μM.Cell-based-ELISA results showed that the IC50 of bufalin,GCG,and SSD to PRV proliferation was 0.033 μM,0.410 μM,and 1.692 μM,respectively.The results of Western blot and IFA showed that as the concentrations of bufalin,GCG and SSD increased,the drugs inhibited PRV proliferation more significantly.The investigation of the effect of the three drugs on the replication cycles of PRV showed that bufalin inhibited the proliferation of PRV mainly by affecting the entry of PRV;GCG inhibited the proliferation of PRV mainly by affecting the entry and release phases of PRV;SSD inhibited the attachment,entry and release phases of PRV significantly.Since bufalin can inhibit PRV at 0.033 μM and it is not toxic to cells at 120 μM,this suggests that bufalin may be a relatively safe and effective PRV inhibitor.So,the bufalin was chosen to perform an animal experiment on PRV proliferation in vivo.The results of animal experiment with bufalin showed that the intraperitoneal injection of bufalin delayed the onset of neurological symptoms caused by PRV JSY13.The results of autopsy showed that bufalin significantly reduced the congestion and haemorrhage in all tissues of mice.The results of qRT-PCR showed that the PRV load in the liver,spleen,lung and brain of bufalin-treated mice was significantly reduced compared with that of PRV-attacked mice.The results showed that the PRV load in the liver,spleen,lung and brain tissues of the bufalin-treated mice was significantly lower than that of the control group.When mouse mortality was recorded and plotted,it was found that bufalin increased the survival rate of mice by 20%(2/10).In conclusion,this study found that bufalin,GCG,and SSD significantly inhibited the replication of PRV in PK15 cells.Bufalin also inhibited the replication of PRV in BALB/c mice to a certain extent and reduced mouse mortality.This study provides a basis for the research of antiviral drugs for PRV and also provides a reference for the development of antiviral drugs for other herpesviruses.