HIGHLY VIRULENT PORCINE EPIDEMIC DIARRHEA VIRUS (PEDV), CAUSING UP TO 100% MORTALITY IN NEONATAL PIGLETS, EMERGED IN CHINA IN LATE 2010. IT SPREAD TO THE US IN SPRING 2013. DURING THE FIRST YEAR OF PEDV OUTBREAKS IN THE US, PEDV KILLED ABOUT 7 MILLION PIGLETS, ACCOUNTING FOR 10% PIG POPULATION AND $900 MILLION TO $1.8 BILLION ECONOMIC LOSSES. CURRENTLY, PEDV HAS SPREAD TO 39 STATES IN THE US. IT HAS BECOME THE MOST ECONOMICALLY IMPORTANT PORCINE ENTERIC VIRAL PATHOGEN AND HAS CAUSED IMMENSE ECONOMIC LOSSES AMONG THE PORK INDUSTRIES IN MANY COUNTRIES. EFFECTIVE AND SAFE VACCINES ARE DESPERATELY DESIRED BUT STILL NOT AVAILABLE. KNOWLEDGE ON VACCINES FOR OTHER SWINE ENTERIC VIRUSES (SUCH AS ROTAVIRUS AND TRANSMISSIBLE GASTROENTERITIS VIRUS) SUGGESTS THAT ORAL IMMUNIZATION OF SOWS WITH LIVE ATTENUATED VACCINES (LAVS) IS THE MOST EFFECTIVE APPROACH TO PROTECT NEWBORN PIGLETS AGAINST ENTERIC VIRUSES, INCLUDING THE HIGHLY VIRULENT PEDV. HOWEVER, A MAJOR CONCERN FOR USING LAVS IN THE FIELD IS THAT A LAV STRAIN MAY REVERT TO VIRULENCE VIA MUTATION AND RECOMBINATION. WE HYPOTHESIZE THAT SAFE AND EFFICACIOUS LAVS CAN BE ENGINEERED USING NEW STRATEGY AND REVERSE GENETICS TECHNOLOGY: INACTIVATION OF CONSERVED VIRAL GENES ENCODING MODULATORS OF INNATE IMMUNITY AND VIRUS REPLICATION AND ADDITIONAL MUTATIONS WITHIN THE NON-ESSENTIAL SEQUENCES OF STRUCTURAL OR ACCESSORY PROTEINS WILL ATTENUATE AND STABILIZE PEDV; ADDITIONAL REWIRING TRANSCRIPTIONAL REGULATION SEQUENCE (TRS) OF PEDV MAKES IT HIGHLY RESISTANT TO RECOMBINATION. WE HAVE ESTABLISHED A ROBUST REVERSE GENETICS SYSTEM FOR PEDV THAT CAN BE USED AS A PLATFORM TO DESIGN VACCINE CANDIDATES. THREE AIMS ARE PROPOSED: 1) DESIGN PEDV VACCINE CANDIDATES WITH THE MULTIPLE TARGETED MUTATIONS THAT ARE RESISTANT TO REVERSION AND RECOMBINATION USING REVERSE GENETICS; EVALUATE VIRAL PROPAGATION AND GENETIC STABILITY IN VITRO; 2) EVALUATE ATTENUATION AND GENETIC STABILITY OF THE ATTENUATED PEDV VACCINE CANDIDATES IN NEONATAL PIGS; AND 3) EVALUATE THE IMMUNOGENICITY AND PROTECTION OF THE OPTIMAL TWO VACCINE CANDIDATES IN WEANED PIGS. THIS PROJECT WILL DEVELOP SAFE, ATTENUATED PEDV VACCINE CANDIDATES FOR FUTURE EVALUATION AS MATERNAL VACCINES IN SOWS TO INDUCE LACTOGENIC IMMUNITY IN PIGLETS. ADDITIONALLY, THE INFECTIOUS CLONE OF SUCH AN ATTENUATED VIRUS WILL BE A GOOD PLATFORM TO QUICKLY GENERATE TIMELY VACCINE CANDIDATES FOR NOVEL EMERGING PEDV STRAINS THAT DISPLAY SIGNIFICANT ANTIGENIC VARIATION. KNOWLEDGE OBTAINED FROM THIS PROJECT WILL ALSO AID IN INNOVATIVE VACCINE DESIGN AGAINST OTHER ANIMAL (SUCH AS PORCINE DELTACORONAVIRUS AND SWINE ACUTE DIARRHEA SYNDROME (SADS)-CORONAVIRUS, WHICH CAUSE SIMILAR CLINICAL SIGNS TO PEDV BUT INDUCE NO CROSS-PROTECTION) AND HUMAN CORONAVIRUS DISEASES, SUCH AS SEVERE ACUTE RESPIRATORY SYNDROME (SARS) AND MIDDLE EAST RESPIRATORY SYNDROME (MERS).