Studies On The Isolation And Identification Of PEDV Endemic Strains,and The Neutralization Of Chimeric IgG/IgA Antibodies

In addition to the diseases of African swine fever and porcine reproductive and respiratory syndrome,porcine epidemic diarrhea(PED)is currently the third most serious disease affecting the pig industry.Infection of the sow will result in the illness or even death of the entire litter of piglets.The disease is caused by Porcine epidemic diarrhea virus(PEDV)and its clinical symptoms include diarrhea,vomiting,wasting and even death from dehydration in piglets.The symptoms are easily confused with those caused by Porcine transmissible gastroenteritis virus(TGEV),Porcine Rotavirus(PRo V),Porcine deltacoronavirus(PDCo V)and various bacterial diarrhea pathogens.Due to the rapid onset and spread of the disease,the application of therapeutic antibodies has the potential to treat piglets when the conventional vaccination has failed.For protection against enteroviruses,Ig A antibodies in sow mammary secretions are essential to assess the protection of piglets against PEDV,but the presence of proteases in the gastrointestinal tract results in only a limited amount of Ig A antibodies reaching the intestine to neutralize the virus.Therefore,it was the aim of this study to first construct and express an anti-PEDV chimeric Ig G/Ig A antibody with broad-spectrum neutralizing activity,and then to evaluate the therapeutic efficacy of the chimeric Ig G/Ig A antibody using the isolated PEDV prevalent strains.The detailed findings of the study are as follows:1.In this study,a total of 3,261 diarrhea samples from different pig farms in 16 provinces from 2018 to 2021 were tested,and the results showed that the positive detection rates were 62.47% for PEDV,11.41% for PRo V,3.31% for PDCo V,and no positive detection for TGEV,MRV and SADS-Co V.The S gene sequencing and analysis of the 81 samples showed that the nucleotide sequence identity between the strains ranged from 93.0% to 93.7% when compared to the G1 representative strain CV777;94.7% to 96.4% when compared to the S-INDEL representative strain ZL29;96.1% to 99.3% when compared to the G2 representative strain AJ1102 or LW-L.Among the 81 PEDV strains,the numbers of strains of type G2 a,G2b and S-INDEL were 59,17 and 5,respectively.2.Based on the results of the clinical diarrhea samples and the background information of the samples,fresh intestinal tissues sent from four provinces,including Shandong,Sichuan,Fujian and Zhejiang,were selected for PEDV isolation and identification.The results showed high purity levels of the isolated strains SD01,SC14,FJ70 and ZJ78,and the virus reached its peak proliferation at 24 h after inoculation,and typical virus particles could be observed by electron microscopy.The four PEDV strains were tested for pathogenicity in piglets,and the SD01 strain was selected as the test virus,and a challenge model of the SD01 strain on 15-day-old piglets was established.3.A CHO cell line expressing anti-PEDV chimeric antibody Ig G/Ig A was constructed based on the laboratory’s PEDV m Ab 8A3A10(Ig G)that with high neutralizing activity.The expression plasmid containing heavy chain region of the chimeric antibody was composed of the VH,Cγ1 and hinge regions of m Ab 8A3A10,and the Cα2 and Cα3 structural domains of the α-chain of murine immunoglobulin,which was subsequently ligated with the light chain of m Ab 8A3A10 by enzymatic digestion to obtain the plasmid p CHO-Ig G/Ig A-HC-LC carrying both heavy and light chain genes.p CHO-Ig G/Ig A-HC-LC was transfected into CHO cells and CHO cell lines stably expressing anti-PEDV chimeric Ig G/Ig A antibodies were obtained by stress screening.In vitro cell neutralization assays were performed using seven PEDV strains,including the original laboratory strains CV777(subtype G1),P014(subtype G2b)and HN1303(subtype G2a),as well as strains SD01(subtype G2a),SC14(subtype G2a),FJ70(subtype G2b)and ZJ78(subtype S-INDEL).The results showed that the chimeric Ig G/Ig A antibodies obtained were able to neutralize the different PEDV strains with antibodies ranging from 1:32 to 1:64.The results of in vivo trials also showed that oral treatment with chimeric Ig G/Ig A antibodies in piglets after the challenge of PEDV strains HN1303 and SD01 significantly alleviated clinical diarrhea and vomiting,and reduced intestinal pathological damage and pathogenic infection in piglets,in addition to reducing piglet detoxification and effectively delaying or preventing piglet mortality.In conclusion,based on the isolation and identification of the prevalent PEDV strains,the chimeric Ig G/Ig A antibodies were tested in cell neutralization and in vitro therapeutic efficacy tests,which proved that the chimeric antibodies have good clinical therapeutic effect against PEDV infection.