Breeding Of High Proliferation Vero Cells Of Porcine Epidemic Diarrhea Virus And Its Cultivation,Identification And Intestinal Pathogenesis On Target Cell SIEC

Porcine epidemic diarrhea（PED）is an acute contagious enteric disease that caused by Porcine epidemic diarrhea virus（PEDV）,which can infect all ages of swine,characterized by severe enteritis,vomiting,and watery diarrhea,finally death,caused great economic losses to the global swine industries.PEDV transmission in pigs mainly occurs through oral-fecal route,but recently it was reported that long distance spreading of the virus by air may be possible,which means PEDV will pose a serious threat to the swine-producing industry across the world.Although a series of biological methods have been developed for detection of PEDV infection,there is no effective vaccine and drug against this disease,mainly due to the rapid virus mutation,unclear of pathogenesis,the low infection rate of the current available cells and the epidemic situation in some areas are not available to PEDV infection.In order to improve these issues,present study aimed to harvest higher sensitive and higher yield Vero cells of PEDV infection by limited dilution technology and then used these cell lines to explore PEDV epidemic situation in northwest China.At the same time,used target cells SIECs to study the pathogenesis of isolated PEDV,compared the differences of PEDV proliferation on SIEC cells and Vero cells.Details are as follows:1.Single-cell Vero cloning was conducted via limiting dilution.Via limiting dilution technology and expanded culture,the infection rate of PEDV was detected by IFA,and the cells showed the highest infection rate was chosen as parent cells to do the second selection.After three rounds selection,we obtained four single-clone cells,named Vero/G2A8C4,Vero/G2A8D4,Vero/G2A8E7,and Vero/G2A8G6 to support higher propagation and yield of PEDV compared with parental Vero cells.When infected with 1 MOI PEDV at 96 hpi,the infection rate of PEDV in Vero/G6 clone can reach up to 100%while only 15%in parental Vero cells.At the same time,Vero/G6 cells showed better cellular status than parental cells after 120 hpi.Infection cases at 0.1 MOI PEDV,Vero/G6 showed positive cells after 24hours while the maternal cell showed positive cells until 72 hours,thus Vero/G6 cells is more conducive to timely diagnosis under low virus infection cases.Therefore,Vero/G6 cells will provide a beneficial tool for the study of PEDV diagnosis,propagation and the development of vaccines.2.Since 2015,piggeries in northwest China outbroke a severe diarrhea,vomiting,dehydrationand death disease,death rate was as high as 90%¹00%,caused great economic losses to Chinese pig industries.In order to expose pathogens of the disease,27 intestinal samples were collected from death piglets breeding in Shaanxi,Gansu,Qinghai province and Ningxia Hui Autonomous Region.RT-PCR assay indicated PEDV infection rate was 92.6%and TGEV infection rate was only 18.5%.Sequence analysis showed that among the six isolated PEDV strains,five strains belonged to classical strains,and one was new mutant strains.Two style strains were used to infected piglets of 10 days old,and the clinical symptoms and pathological changes indicated that both of two strains were virulent to nursing piglets.qRT-PCR was used to detected the expression of pro-apoptotic factor BCL-G and JAB1 gene at the transcription level in piglet’s mesenteric lymph nodes,and the result showed the expression of apoptosis factor BCL-G and JAB1 was significantly enhanced by the isolated strains.Therefore,PEDV can significantly enchanced the apoptosis of small intestinal villus epithelial cells by increasing apoptosis factor BCL-G and JAB1 in endogenous cell apoptosis pathway.3.Since pig small intestinal epithelial cells（SIECs）is the target cells of PEDV infection,SIECs was selected as the cell model to study the change of cell activity after PEDV infection.The present studies showed PEDV infection depressed the activity of Na⁺-K⁺-ATPases and Ca²⁺-Mg²⁺-ATPases,decreased cells proliferation,and increased cells apoptosis clearly.Animal regression test showed PEDV proliferated in SIEC cells was more virulent than that in Vero cells,shown as the small intestine tissue damaged more serious,mesenteric lymph node swelling degree increased,peripheral blood capillary congested severity,the expression of apoptosis factor BCL-G and JAB1 of mesenteric lymph nodes also improved remarkably.These experiments revealed the pathogenesis of PEDV infection by infecting target cells,providing basic information for PEDV related research,as well as providing theoretical basis for the prevention and control of the disease.