| Porcine Epidemic Diarrhea(PED)is a highly pathogenic intestinal infectious disease that causes acute watery diarrhea,vomiting and even dehydration in piglets caused by Porcine Epidemic Diarrhea Virus(PEDV).Since 2010,PED has been widespread in China,causing huge economic losses to the pig industry in my country.The main reason is that its S gene mutations have caused the vaccine developed by the PEDV CV777 strain to fail to form a fully effective protection for pigs.Therefore,it is of great significance to develop the molecular biological characteristics and pathogenic mechanism of PEDV epidemic strains for the development of new vaccines.This study carried out the genetic evolution and sequence alignment analysis of the full-length sequence of the S1 gene and ORF3 gene of 5 PEDV isolates.Phylogenetic analysis of the S1 gene showed that the PEDV YJH strain belongs to the G2 b type,and the remaining 4 isolates belong to the G2 a type.The amino acid comparison analysis with the classic strain CV777 showed that the S1 gene of the 5strains had amino acid mutations,5 amino acid insertions and 2 amino acid deletions at56-60 different positions: 5 isolates were all in the first There was an insertion of 4amino acids(QGVN)at position 59-62,aspartic acid or lysine at position 139,and a deletion of 2 amino acids(DI)at position 163-164.The comparison analysis of ORF3 gene revealed that 5 strains had the same amino acid changes at 6 positions(V21A;V54I;V79I;L92F;A101T;N166S),among which the three isolates of PEDV HH,PEDV SQ and PEDV TQ The same amino acid changes(L25S,I70 V,C107F,N168S)occurred at 4 positions,and the two strains of PEDV YJH and PEDV HY had the same amino acid changes at 2 positions(F80V;H182Q).The evolution analysis of the ORF3 gene showed that the 5 PEDV isolates isolated in 2017 belonged to the G2 type,while the vaccine strain belonged to the G1 type.The results of animal experiments showed that the 5 isolates were highly pathogenic to piglets,among which the PEDV YJH strain was the most virulent,and the PEDV HY strain was weaker than other strains.In this study,PEDV YJH strain and PEDV HH strain were successively subcultured in vitro to 90 and 140 generations.Evolutionary analysis showed that PEDV YJH strain after serial passage still belongs to G2 b type,and PEDV HH strain still belongs to G2 a type.The amino acid comparison results of the S1 gene showed that the 27 th and 57 th amino acid changes(S27L;E57K)and the 55 th and 56 th amino acid deletions existed at the same time when the two strains were passed to the 140 th generation.Cell adaptability studies have found that the 25 th generation(F25)of PEDV YJH strain requires a trypsin concentration of 5 μg/μL,but when it is passed to the140 th generation(F140),it is found that the virus can infect without adding trypsin.The cells proliferate.S protein is the key protein that determines PEDV pancreatin dependence.The comparison of S protein between F25 and F140 viruses shows that there are two amino acid deletions and 12 amino acid mutations.Animal experiment results show that PEDV YJH strain has weakened virulence after continuous passage,is not pathogenic to piglets,and has good safety.Serum antibody test results prove that this attenuated strain can induce immune response in piglets and can be used as a PEDV attenuated live Candidate strains for vaccines.In this study,by analyzing the genetic variation of PEDV isolates,using passaging methods to weaken the PEDV epidemic strain,and analyzing its biological characteristics such as pancreatin tolerance,it provides basic materials for the further establishment of PED prevention and control technology. |
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