Study On The Variation Of S Gene And Its Antigenic Difference In The New Strain Of PEDV In Liaoning Province

ObjectiveThe disease characteristics,genetic variation and antigen difference were studied in order to clarify the genetic variation and antigen variation of Liaoning swine epidemic diarrhea strain in the research,which also laid the foundation for establishing the corresponding diagnostic methods,screening and development of the corresponding vaccine and provided the basis for improving the specificity of the vaccine and the formulation of targeted control measures.Method In Dalian,Yingkou,Jinzhou and Huludao cities of Liaoning Province,cases of diarrhea in piglets at the age of 3~20 days were collected and diagnosed by pathology,etiology and PCR diagnosis.The cases diagnosed as PED and other diseases were excluded.On the basis of observing and recording the changes of dissection,the histopathological and immunohistochemical characteristics of PED were observed by collecting the sections of gastrointestinal tract,lymph nodes,heart,liver,kidney and other organs.The PED positive samples were collected and the virus was isolated and cultured by Vero cells.The virus was identified by electron microscope observation and gene sequence analysis.The S gene of Liaoning PEDVLN-2015-1 new strainwascloned and sequenced which analyzed by software of DNAMAN,Vector NTI and MEGA6.06 at the same time.The nucleotide and amino acid sequences of S gene were homologous and mutated with those of CV777,AJ1102 and PEDV,and the antigenic epitopes of S gene were compared with those of CV777.The genetic variation and antigen change of the new PEDVLN-2015-1 strain were analyzed.In order to confirm whether the variation of S protein sequence of new strain of PEDV caused the change of its antigenicity,we constructed S gene expression protein of new strain of PEDV and traditional strain of virus by using E.coli prokaryotic expression system,and prepared polyclonal antibody against S protein.Through indirect ELISA test,Western blot and cross neutralization test,the antigenic differences between the new strain S protein and the traditional CV777 strain S protein were compared and analyzed.ResultThe observation of disease characteristics showed that the epidemic diarrhea of piglets in Liaoning mainly concentrated at age of 3~20 days,and the incidence and mortality of piglets were over 90%.The main characteristic pathological changes were intestinal dilatation and inflation,thinning and transparency of intestinal wall,accumulation of milky clot in stomach,redness of gastric fundus mucosa with mucus,mesenteric hyperemia,hyperemia and swelling of mesentery and other lymph nodes,degeneration of other solid organs.The main histopathological changes were atrophy,necrosis and exfoliation of villus epithelium,hyperemia of lamina propria,proliferation of lymphocytes,structural disorder of glands and incomplete glandular epithelium.Other organs such as gastric mucosal epithelium atrophy,shedding,kidney,liver,lung,lymph nodes have metamorphosis and exudative changes.Immunohistochemical assay showed that the newly isolated PEDV protein was expressed in the whole small intestine and cecum,and was strongly expressed in the villus epithelial cells and small intestinal glands of duodenum.The results of pathological changes and immunohistochemistry showed that the new strain of Liaoning was more extensive than the traditional strain,which was consistent with the high mortality rate of disease in clinic.This indicates that the PEDV epidemic strains in this area have higher pathogenicity,and that the pigs immunized with traditional vaccines are still infected,indicating that their antigenicity may have changed,which needs further study and confirmation.A new PEDV epidemic strain named PEDVLN-2015-1 was successfully isolated from Vero cells in Liaoning Province.The results of genetic variation and epitope analysis of the full-length 4158 bpS gene obtained by sequencing showed that the mutation was mainly concentrated in S1 region compared with the traditional strain CV777 gene.There were only point mutations in the S2 region of insertion,deletion and point mutation.There were 3 BP,9BP and 3 BP insertions at 173,186 and 437,respectively.There was a 6 BP deletion at 489 and a 13 bp continuous mutation was found at 212-224.At the 59 and 140 sites,there were 3AA and 1 AA deletion at 163 and 3 amino acid mutations at 55,68,159 and 199,respectively.Homology analysis showed that PEDVLN-2015-1 strain and traditional new strains and in2010 the former low homology between 93.2%-95.6%,and in recent years at home and abroad variant strain homology,and the homology of HB-HA2015 strain recently,99.2%sequence similarity,and the domestic listed mutant AJ1102 97.6% homology and phylogenetic analysis showed that the strain and strain variation in recent years at home and abroad popular belong to the same group of G2 subgroup,but the formation of a small branch and HB-HA2015,and the distance is far from traditional strain evolution.Antigenic epitope analysis showed that 16 amino acid mutations were found in the S gene of the new strain in 5epitope regions.The comparative analysis of antigenic difference showed that the titer of cross-reaction between S protein of PEDV LN-2015-1 strain and traditional CV777 was low,which indicated that there was obvious antigenicity difference between the two strains,although they could form a certain cross-protection ability.Conclusion(1)A new epidemic strain of PEDV,PEDVLN-2015-1,was successfully isolated from Liaoning Province.The disease was characterized by high morbidity and mortality of suckling piglets,invasion of various organs such as gastrointestinal tract,mesenteric lymph nodes,etc.The pathogenicity is stronger;(2)Compared with the traditional strain CV777,the new strain PEDVLN-2015-1 had obvious variation in S gene which had low homology with the traditional strain before 2010 and high homology with the domestic and foreign variant strains in recent years.The homology of AJ1102 was higher than that of the mutant strains listed in China,but there were some differences.(3)Antigenic epitope analysis showed that 16 amino acids were mutated in 5 epitope regions between the new strain PEDVLN-2015-1S gene and the traditional strain CV777.The antigenicity difference between the new strain S protein and the traditional strain CV777 was further confirmed by antigen cross reaction test.This study confirmed that the pathogenicity and antigenicity of the new isolates of PEDV in Liaoning Province have changed greatly,so it is necessary to screen or develop a new strain vaccine to prevent and control the disease.