Pathogen Molecular Characteristics And Immune Control Of Reemergenced Porcine Epidemic Diarrhea

Since late2010, severe outbreaks of porcine epidemic diarrhea virus (PEDV)infection have reemerged in China Mainland. In order to study the reason of there-emergence, a whole length of S gene, each of12PEDV strains isolated fromdifferent districts, central China from2010to2012, was amplified and cloned byRT-PCR, and then sequenced and analyzed. An indirect ELISA was developed todetection anti-PEDV antibody, which would be suitable for monitoring anti-PEDVantibody and epidemiologic survey of PED. In addition, a product of IgY againstPEDV was developed from egg yolk of chickens immunized with field strain ofPEDV and utilized to successifully control the disease in the pig herds.1. Molecular Characterization and Phylogenetic Analysis of Porcine EpidemicDiarrhea Virus Field Strains in Central China during2010～2012OutbreaksSince late2010, porcine epidemic diarrhea virus (PEDV) has been re-emerging incentral China. To explore the possible reason of the PEDV outbreaks, twelve PEDVfield strains were isolated from different swine breeding farms in central Chinaduring2010～2012, and molecular diversity, phylogenetic relationships of thesestrains with other PEDV reference strains were investigated. Sequence analysis of Sgenes revealed that the central China PEDV isolates had several specific nucleotidesand amino acids which were different from PEDV reference strains. In addition, theentire S genes of eleven central China PEDV isolates were found to be ninenucleotides longer in length than CV777and large number of amino acid variationswas accumulated in the N-terminal region of S gene. Phylogenetic analysis showedthat the central China PEDV isolates had close relationship with Korea strains(2007～2009), Thailand strains (2007～2008), Vietnam strains (2009～2010), Japanstrains (2010), and other prevailing strains from other parts of China (2010～2012).However, they differed genetically from European strains (CV777, Br1/87), Chinastrains (2003～2007) and the vaccine strains (CV777) used in China. These resultsimply that a rapid variation and evolution of central China PEDV strains hasoccurred in recent years, and a more efficient vaccine strain should be selected toprevent and control outbreaks of PEDV in China.2. Prokaryotic Expression of N Gene Major Antigen Region of PEDV andDevelopment of an Indirect ELISA Based on the Expressed ProteinThe complete N genes of PEDV isolated from Henan province were cloned,sequenced and analyzed. Two major antigen Region of N gene was amplified by RT-PCR and inserted into the expression vector pET-32a, and the recombinantplasmid was named pET-32a-N. A fusion protein was expressed in BL21(DE3) withtransfected pET-32a-N and induced by IPTG. The molecular weight of therecombinant protein was about41.3kD analyzed by SDS-PAGE, and the immuno-reaction activity of the recombinant protein was confirmed by Western-blotting whenmouse positive serum against PEDV was used. An indirect ELISA for detectingantibody against PEDV was developed by using expressed protein N as coatingantigen. Results showed that the optimal coating concentration for N protein is0.226μg/well, and the optimal dilution of the serum is1:100and that of the HRP-SPAis1:2000.The positive criterion for this ELISA assay is OD450≥0.28. When fieldserum samples were tested with the ELISA developed above, the positive rate was84.26%(166/197) for sows serum, and27.93%(31/111) for piglets serum. The resultsindicated that this ELISA developed was specific, sensitive, reproducible and suitableas a rapid serology detection method for monitoring anti-PEDV antibody andepidemiologic survey of PED.3. Study and Application the Hyperimmunized Yolk Antibodies of PEDVAccording to the variation of virus and ineffective prevention of existing vaccine,the hyperimmunized yolk antibodies against PED was made by PEDV epidemicstrains to control the occurrence and epidemic of PED, and the control effect wasstudied. The hens were immunized with the inactivated vaccine of PED made byPEDV epidemic strains. When AGP titer of yolk antibody reached to1:128, thehyperimmunized yolk antibodies against PEDV were made by eggs collected,artificially infection and treatment test and clinical therapeutic test were conducted toobserve the effect of hyperimmunized yolk antibody. Artificially infection andtreatment test showed that the mortality rate was100%in the control group, and only10%in the antibody treatment group. The result of clinical therapeutic test indicatedthat artificial prevention efficiency was91%, and the treatment efficiency was90%(1343pigs from5large-scale pig farms in different cities). The results suggest thatyolk antibody made by PEDV epidemic strains has significant control effect againstthe occurrence and epidemic of PED.