title: Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A.
abstract: Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus. In the United States, highly virulent PEDV strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Using reverse genetics, we removed open reading frame 3 (ORF3) and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-ΔORF3-RFP. icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild-type-virus- or icPEDV-infected pigs, and the virus formed smaller plaques than those of PC22A. Together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting.
  
  
  OBJECTIVE
  Porcine epidemic diarrhea virus (PEDV) emerged in the United States in 2013 and has since killed 10% of U.S. farm pigs. Though the disease has been circulating internationally for decades, the lack of a rapid reverse-genetics platform for manipulating PEDV and identifying genetic factors that impact transmission and virulence has hindered the study of this important agricultural disease. Here, we present a DNA-based infectious-clone system that replicates the pathogenesis of circulating U.S. strain PC22A both in vitro and in piglets. This infectious clone can be used both to study the genetics, virulence, and transmission of PEDV coronavirus and to inform the creation of a live attenuated PEDV vaccine.
authors.affiliation: University of North Carolina Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Beall
authors.affiliation: University of North Carolina Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Boyd
authors.initials: B
authors.lastname: Yount
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Chun-Ming
authors.initials: CM
authors.lastname: Lin
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Yixuan
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Qiuhong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Linda
authors.initials: L
authors.lastname: Saif
authors.affiliation: University of North Carolina Chapel Hill, Chapel Hill, North Carolina, USA rbaric@email.unc.edu.
authors.forename: Ralph
authors.initials: R
authors.lastname: Baric
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D018352:Coronavirus Infections / Q000473:pathology / Q000635:transmission / Q000662:veterinary* / Q000821:virology; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D003967:Diarrhea / Q000473:pathology / Q000821:virology; D017353:Gene Deletion; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D053485:Porcine epidemic diarrhea virus / Q000235:genetics* / Q000472:pathogenicity*; D011995:Recombination, Genetic; D059386:Reverse Genetics / Q000379:methods*; D013194:Staining and Labeling; D016019:Survival Analysis; D013552:Swine; D013553:Swine Diseases / Q000473:pathology / Q000635:transmission* / Q000821:virology*; D014481:United States; D014774:Virulence; D000097573:Red Fluorescent Protein
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein