Effect Of(+)-Usniacin On Replication Of Procine Epidemic Diarrhea Virus And The Passage Of PEDV SH Strain

Porcine epidemic diarrhea（PED）is an acute gastrointestinal disease of pigs caused by porcine epidemic diarrhea virus（PEDV）,which can cause vomiting,watery diarrhoea and other symptoms,with mortality rate of up to 100%in newborn piglets.Before 2010,the disease was sporadic in China.A massive outbreak at the end of 2010 brought huge economic losses to China’s pig industry.Due to the emergence of mutant strains,the traditional vaccines can not effectively prevent the disease,and there are no effective antiviral drugs for clinical treatment,so it is great significance to strengthen the screening of vaccine strains and the study of antiviral drugs for the prevention and control of the disease.In this study,the drug（+）-Usniacin,which effectively inhibits the proliferation of PEDV,was screened through in vitro experiments to prove that it plays an antiviral role in the process of viral replication.It was preliminarily proved that its antiviral effect may be related to cell autophagy.Meanwhile,in order to cultivate attenuated strains,SH strain with partial deletion of N gene isolated from laboratory was successively passaged to 100 generations on Vero cells,and whole genome sequencing and analysis were performed in this study.The result shown that the virus gradually adapted to cells during cell passage and maintained certain genetic stability.1（+）-Usniacin inhibits PEDV replication and its machanismIn order to study the effect of（+）-Usniacin on PEDV replication and its mechanism,the non-toxic concentration of（+）-Usniacin on Vero cells was first determined by CCK-8.The results showed the concentration of（+）-Usniacin below 15 μM had no toxicity to cells.Then the inhibitory effect of（+）-Usniacin on PEDV was determined by dose-dependent method.The inhibitory effect on PEDV replication was enhanced with the increase of（+）-Usniacin concentration by western blotting,qRT-PCR and indirect immunofluorescence assay.To explore whether（+）-Usniacin has good inhibitory effect on different strains of PEDV,the classical strain CV777,the variant strain YZ and the deleted strain SH of PEDV were used in the study.The results showed that（+）-Usniacin had good inhibitory effects on different strains of PEDV.By adding（+）-Usniacin at different stages of PEDV replication,it was found that the drug exerted viral inhibition mainly at the replication stage of PEDV.To study the possible antiviral mechanism of this drug,the expression of LC3 protein was detected by western blotting,and it was found that PEDV could promote the autophagy process of Vero cells after infection with PEDV.（+）-Usniacin can inhibit the autophagy of Vero cells,which indicates that the antiviral effect of（+）-Usniacin may be related to autophagy.This study reveals for the first time the anti-PEDV effect of（+）-Usniacin and finds that its anti-PEDV mechanism may be related to the autophagic process of cells,which laying a foundation for the therapeutic research of PEDV.2 The passage of PEDV SH strain on Vero cells and its analysis of genetic stabilityIn order to obtain the attenuated strain of PEDV,the PEDV SH strain isolated and preserved in the laboratory was passaged from 81 to 100 generations on Vero cells in this study,and its gene sequence and TCID₅₀ were determined.The gene sequence was compared with the original viral gene sequence,and its TCID₅₀ was compared with that of 20,40,60 and 80 generations of viruses to analyze the genetic stability and cell adaptability.The results showed that the TCID₅₀ of SH strain gradually increased and the proliferation rate became faster during continuous passage.The genome of SH strain is 28002 bp in length,and there are no insertions or deletions during successive passages,but there are 18 base mutations.It includes 1 mutation in 5’UTR,6 mutations in ORF1a,5 mutations in ORF 1b,4 mutations in S gene,1 mutation in E gene,and 1 mutation in N gene,no mutation in the rest of the genes.The results declared that the SH was conservatively inherited.And the consecutive 36 nt deletion of the N gene found in the initial isolation of SH strains is highly conserved during successive passages.This study laid a foundation for the screening of PEDV vaccine strains.