Full-Genome Analysis Of Vero Cell-Adapted Porcine Epidemic Diarrhea Virus (PEDV) And Establishment Of Indirect Immunofluorescence Assay

Porcine epidemic diarrhea virus（PEDV）is one of the viruses that cause huge economic losses to the pig industry.The virus can infect pigs of all ages,especially for those newborn piglets under seven days without effect maternal antibody,whose the incidence and mortality range will reach from 80%to 100%.Since the re-outbreak of PED in 2010,the traditional vaccine has failed to provide effective protection.Therefore,studing the whole-genome changes of different generations strains which passaged in Vero cells can provide a theoretical basis for further understanding of the genetic variation and pathogenic mechanisms of PEDV.In this study,the genomes of the 200th and 6th generations of ZH02 strains were sequenced,and the entire genome sequences of ZH02-P0,ZH02-P6,ZH02-P65 and ZH02-P200 were compared.The results showed that compared with other generations of strains,the ZH02-P200 strain had the most mutations in the ORF1a/b gene and S gene,and the more conservative ones were ORF3,E,M,and N genes.In the ORF1a/b gene,there are 21nucleotide sense mutations and 469 nucleotide deletions.Among them,the nonstructural proteins Nsp3 and Nsp12～13 have the most obvious mutations.In S gene,there are 10nucleotide sense mutations and 36 nucleotide deletions.The mutation of the epitope COE is the most popular.The deletion of 2 nucleotides in the cytoplasmic region of the S gene caused the amino acid translation to terminate prematurely.In addition,there are 4nucleotide and 7 nucleotide sense mutations in the M and N genes.These results indicate that the decrease of PEDV pathogenicity results from mutations in multiple genes.Animal regression experiments were also performed in research.Two groups of 7-day-old piglets,A and B,were orally administered 2 mL of 1×10^6.0TCID₅₀/mL of 6th and 200th generation virus strains.160 hours after challenge,piglets in group B did not die,and no PEDV was detected in the grinding fluid of duodenum and intestinal lymph nodes.Piglets in group A had typical clinical symptoms of PEDV infection and died.The PEDV could be detected in the grinding fluid of duodenum and intestinal lymph nodes.This suggest that the ZH02-P200 strain has weakened during cell passaging and is potential to develop a safe oral vaccines.In this study,we screened hybridoma cell lines that secreted anti-PEDV N protein monoclonal antibodies using the limiting dilution method.Vero cells were infected with PEDV and stained with the candidate monoclonal antibody of anti PEDV N protein as the first antibody and Goat anti-mouse IgG labeled with fluorescein FITC as the second antibody in established indirect Iimmunofluorscence Assay（IFA）,the optimal conditions were that cells were fixed with 80%acetone at-20℃for 30 min,stained with monoclonal antibody of anti PEDV N protein diluted 1:1000 as primary antibodies and incubated at 4℃overningt,goat anti-mouse IgG labeled with fluorescein FITC as the second antibody diluted 1:100 and incubated at 37℃for 1 h.The method for detecting PEDV has good specificity.