Preliminary Establishment Of A Cas13a Strip Test For Detection Of Encephalomyocarditis Virus And Screening DHEA Derivatives Of Anti-EMCV Drugs

Encephalomyocarditis virus(EMCV)is a micronucleus virus of the genus Cardioviruses.The virus mainly causes myocarditis,diabetes,nervous system and reproductive system diseases,can infect a variety of mammals including pigs,is a zoonotic pathogen.At present,there is no standard nucleic acid detection method for EMCV in China,and there is no specific therapeutic drug for clinical infection of EMCV.Therefore,it is of great significance to establish a rapid detection method and screen antiviral drugs for this virus.This study mainly established a transverse flow strip detection method for EMCV based on CRISPR-Cas13a technology and selected antiviral drug screening of Dehydroepiandrosterone(DHEA)derivatives.The main research contents and results are as follows:1.Preliminary establishment of CRISPR-Cas13a strip detection method for EMCVFirstly,VP1 gene of EMCV structural protein was selected as the target gene to establish a SYBR Green I fluorescent quantitative RT-PCR method for detection of EMCV.The lower limit of detection was 1×10~1copies/?L,which showed good reproducibility and high specificity.Secondly,the Recombinase aided amplification(RAA)primers which could stably amplify the VP1 gene of EMCV were screened,and then the CRISPR RNA sequences targeting VP1 gene were designed based on a series of RAA primers.The EMCV VP1 gene was specifically detected by using cr RNA binding and Cas13a protein to recognize and process RNA from various sources.On this basis,combined with the detection method of Cas13a strip test kit,the CRISPR-Cas13a strip test method for rapid visualization of VP1 gene was established.The lower limit of sensitivity was 1×10~1 copies/?L,which was similar to the traditional fluorescence quantitative method.The detection procedure only needs 85min,which is obviously shorter than the traditional fluorescence quantitative method.The method can specifically detect EMCV and has good specificity.The method was used to detect clinical samples.Comparing with the traditional fluorescence quantitative PCR method,the results showed that the coincidence rate of the two methods was 100%.The above results indicated that the CRISPR-Cas13a nucleic acid strip detection method for EMCV was preliminarily established in this study,laying a foundation for the development of clinical rapid detection kit for EMCV.2.Molecular screening of DHEA derivatives inhibiting EMCV replicationFirst,nine DHEA derivatives were diluted to 10?M,and the antiviral molecules were screened on BHK-21 cells susceptible to EMCV.The results showed that three derivatives of AV4001,AV4005 and AV4006 could significantly inhibit EMCV propagation.Secondly,AV4001 and AV4006 were selected to determine the cytotoxicity and antiviral effect.The results showed that the maximum concentration of AV4001 and AV4006 was 10?M and 200?M respectively.Both showed dose-dependent tolerance to EMCV with IC50 of 9.324?M and 325.8?M,and SI of 22.40 and 1.87,respectively.On this basis,the mechanism of AV4001 and AV4006 inhibiting EMCV replication was further studied.The results showed that when AV4001 or AV4006 were added after EMCV infected cells,the antiviral effect was the most significant,and the two derivatives had no effect on the absorption and invasion of EMCV cells.This suggests that the antiviral effect of DHEA derivatives may mainly occur in the revirus replication stage.3.In vivo validation of antiviral effect of DHEA derivative AV4001AV4001 derivative molecule with the strongest antiviral effect was selected,and the antiviral effect of the molecule was further verified in vivo through mouse infection model.A total of 48 BALB/C mice aged 6 weeks were randomly divided into three groups:EMCV+DMSO,EMCV+AV4001,and DMEM+AV4001.16 mice in each group were intraperitoneally injected with 10~4TCID50 EMCV or isovolumetric DMEM on day0,and continuous administration of DMSO(50mg/kg AV4001)or isovolumetric DMSO from day 0 to day 3.The symptoms of the mice were observed continuously until day 14.On day 3,5 tissue samples were collected from each group,and the remaining 11 mice were used for clinical symptom observation and survival rate calculation.Mice infection experiments showed that the mortality rate of mice in EMCV+DMSO group was 100%,and the infected mice developed severe neurological symptoms and died quickly.The mortality rate of mice in EMCV+AV4001 group was 72.73%.Except for sudden death,other dead mice showed no obvious neurological symptoms.DMEM+AV4001 group had no death and no symptoms.Clinical symptom evaluation and tissue TCID50 viral load detection results showed that AV4001 treatment can reduce the mortality of mice,alleviate the clinical symptoms of mice,and reduce the viral load of EMCV in the brain tissue of mice.H&E staining results showed that vascular cuff and microglial nodules were obvious in the brain tissue of mice in the EMCV+DMSO group,while there was no obvious inflammatory reaction in the brain tissue of mice in the EMCV+AV4001 group.This study shows that AV4001 is effective in the treatment of EMCV infection in vivo.In this study,a CRISPR-Cas13a EMCV strip test assay for rapid clinical detection was established,and a DHEA derivative molecule with inhibition of EMCV replication was screened,These results lay the foundation for the development of rapid detection kits and therapeutic drugs for EMCV.