Isolation,Identification Of Porcine Epidemic Diarrhea Virus Epidemic Strains And Effects Of Autophagy On PEDV Replication

Porcine epidemic diarrhea(porcine epidemic diarrhea)is an infectious intestinal diseases caused by porcine epidemic diarrhea virus(PEDV)with the clinical features of mental disorders,vomiting,watery diarrhea.Fresh piglets have occurred 3 to 4 days after diarrhea usually died due to dehydration death,with mortality rate of up to 100%.Since the winter of 2010,PED in most provinces of China increased significantly the incidence for worse vaccine immunization protection,causing serious harm.And PED is one of the major diseases that caused the death of piglets in domestic farms.In May 2013,then the disease first occurred in the United States,and then spread popularly in the North American continent.The controlling means to this disease mostly dependented on immunization prevention,but in recent years,the prevalence of PEDV hypervariable strains have made the market usage of vaccine protection is limited.In this paper,considering the genetic characteristics of the mutant strains,after isolation and purification,the stable cell-adapted strains was obtained.The molecular genetics evolution was analyzed and the effects of autophagy on PEDV replication were investigated.The research results are of great significance to elucidate the pathogenesis of PEDV and the development of new vaccines.1.Isolation and Identification of Porcine Epidemic Diarrhea Virus YC2014 StrainIn 2014,a certain scale pig farm in Jiangsu occurred diarrhea epidemic,collecting the incidence of piglets positive disease(small intestine mucosa and intestinal contents)after treated with Vero cell culture and trypsin incubation method,blind passed 3 generations after a significant cell syncytial lesions-cell vacuolization,finally occurrence of cleavage.The harvested samples were RT-PCR tested for only PEDV positive.Further indirect immunofluorescence assay confirmed that the diseased cells could react specifically with anti-PEDV Npro antiserum.The titer of the isolated virus was determined by plaque assay at 105.7TCID50/100 uL.Virus-infected Vero preparation of ultra-thin slices by transmission electron microscopy to further confirm can be observed with about 90nm diameter of the virus particles inclusion body structure,the virus core dense,with a clear capsule structure.The isolated virus was named YC2014 strain.2.Sequence analysis of PEDV whole genome sequence and genetic evolution analysisAccording to the whole genome sequence of CV777 classical strain,seven pairs of primers were designed,and seven overlapping fragments were amplified and then the whole genome sequence was obtained.The results showed that the whole genome of YC2014 strain was 28077nt(Accession No.KU252649)and the genome structure was 5,UTR-ORF1a-ORF1b-S-ORF3-E-M-N-3'UTR.YC2014 strain and the current domestic and foreign epidemic strains and vaccine strains were compared,and utilize MEGA6 software to develop phylogenetic tree.The results showed that the sequence of S gene of YC2014 strain shares only 93.9%?94.4%similarity of DR13,JS2008 or CV777 strain,and there were 15 insertion and multiple base deletions in the S1 region which as the virulence gene of PEDV.Base phylogenetic analysis,PEDV can be divided into two subgroups:a group consists of the domestic epidemic strains since 2010,and the United States/Europe isolated strains since 2013.Another subgroup contains traditional vaccine strains and early isolations.The YC2014 strain is differently related to the traditional strain and vaccine strain,and located in different branches,but more similarity to the epidemic strains isolated in recent years.That indicating that YC2014 is the current prevalent strain,can developed as candidate vaccine strains for the PEDV controlling currently.3.The effect of cell autophagy on PEDV replicationAfter the Vero cells infected with YC2014,the autophagosomes were observed by transmission electron microscopy.And the distribution of fluorescent spotting location was observed by laser confocal microscopy.Besides the autophagy-related gene protein LC3B was detected by western blot.The results show that PEDV infected Vero cells can induce the formation of autophagy bilayer structure in host cells,and make LC3-GFP-mRFP in the cell spotting localization,up-regulation of LC3B-? expression level.The similar result s were obtained in IPEC-J2 cells,indicating that PEDV infection induced cell autophagy.Transfection of GFP-mRFP-LC3 double fluorescent system showed that PEDV infection induced complete autophagy.UV-inactivated viruses can not induce autophagy that autophagy relies on PEDV replication.To investigate the effect of autophagy on viral replication,it was found that the autophagic inducing drugs Rapamycin treated Vero cells with PEDV infection decreased the PEDV titer.Autophagy inhibitor 3-MA treated cells after infection with PEDV cells increased the titer of PEDV.The studies have shown that autophagy inhibits PEDV replication.These studies provide a new perspective for exploring the interaction between PEDV and host cells and the pathogenesis of PEDV.