The Porcine Epidemic Diarrhea Virus Isolate Complete Genomic Sequence Analysis And Pathological Research

Porcine epidemic diarrhea virus(PEDV)has been causing enormous losses on pig farming industry.In 2010,on account of the variant strains,porcine epidemic diarrhea became one of the worldwide porcine pandemic diseases.To preliminarily explore the mechanism of PEDV mutation and prevalence,in 2017,a PEDV isolate,BJsy13/2017 collected at one pig farm in Beijing was sequenced for complete genome analysis.As well as,histopathological and cytopathologic research were then conducted on SHpd/2012 strain.Complete genome sequencing and analysis was conducted for Bjsy/13/2017 isolate.The complet genome was divided into 22 segments,which varied from 800 to 3000 nt,then were amplified by PCR and inserted into cloning vectot PED-T,finally sequencing was conducted,as well as the primers were designed by Oligo 7.0.The analysis showed that compared with the classical CV777 isolate,gene recombination was found in S gene of the new isolate,which was similar to other recent PEDV isolates in China.And the new isolate has a closer genetic relationship with eastern China isolates,meanwhile bioinformatic analysis was done on S protein.Histopathological study was conducted for SHpd/2012.Twelve five-day-old piglets were picked up and divided into three group A,B,and C,with 4 pigs in each group.Group A was for blank control,Group B(gavage)and Group C(intramuscular injection)were for PEDV challenge.When severe diarrhea and vomit were observed in Group B and C,tissue samples were collected and made into tissue slices.As a result,lesion organs included mainly intestines,liver and kidneys.Intestine lesions appeared as intestinal villus atrophia,severe epitheliums defluvium,meanwhile,intestinal wall cell edema,appearance of eosirophilic particles inner enterocyte,lumen stenosis and the inflammatory cell infiltration all came up.Compared with other lesion organs,intestines suffered a more significant lesion,and gained a larger damaged area.In liver,lesion came up as mainly cellular edema and congestion.In kidneys,steatosis and necrosis emerged in renal tubular epithelial cells.Some lumens contained fibrin exudation,but inflammatory cell infiltration was not captured.However,apparent pathological changes of tissues weren't observed in lymph nodes,spleen and lungs.Ultrastructure observation of Vero cells infected by SHpd/2012 isolate was conducted.Vero cells inoculated SHpd/2012 strain was cultured for different time and ultrathin sections were produced for ultrastructure observation by transmission electron microscope,TEM.Result showed that at the incipient phase of the infection(6 h),though with intact cellular structure,part of mitochondria and rough endoplasmic retiulums expanded,cytoplasm vacuolization emerged;with the infection lasting(12 h),the virus particles gradually increased,more vacuolar structures appeared in cytoplasm,and virus particles successively appeared in cytoplasm and nuclei;as infection lasting for 24 hours,with an observably increasing viron amount,the mitochondria vacuolization and cell fusion became more prominent,even nucleus lysis was observed;at the final phase of infection(48 h),with severe vacuolization,nuclear condensation and cell infusion,the neonatal PEDV virons were budding from the infected Vero cells.All the results above laid a scientific foundation for furture study on PEDV prevalence and pathogenic mechanism.