title: Attenuation of an original US porcine epidemic diarrhea virus strain PC22A via serial cell culture passage.
abstract: Although porcine epidemic diarrhea (PED) has caused huge economic losses in the pork industry worldwide, an effective live, attenuated vaccine is lacking. In this study, an original US, highly virulent PED virus (PEDV) strain PC22A was serially passaged in Vero CCL81 and Vero BI cells. The virus growth kinetics in cell culture, virulence in neonatal pigs and the whole genomic sequences of selected passages were examined. Increased virus titers and sizes of syncytia were observed at the 65th passage level (P65) and P120, respectively. Based on the severity of clinical signs, histopathological lesions and the distribution of PEDV antigens in the gut, the virulence of P100 and above, but not P95C13 (CCL81), was markedly reduced in 4-day-old, caesarian-derived, colostrum-deprived piglets. Subsequently, the attenuation of P120 and P160 was confirmed in 4-day-old, conventional suckling piglets. Compared with P120, P160 replicated less efficiently in the intestine of pigs and induced a lower rate of protection after challenge. Sequence analysis revealed that the virulent viruses [P3 and P95C13 (CCL81)] had one, one, sixteen (including an early termination of nine amino acids) and two amino acid differences in non-structure protein 1 (nsp1), nsp4, spike and membrane proteins, respectively, from the fully attenuated P160. However, the overall pattern of attenuation-related genetic changes in PC22A differed from those of the other four pairs of PEDV wild type strains and their attenuated derivatives. These results suggest that PEDV attenuation can occur through multiple molecular mechanisms. The knowledge provides insights into potential molecular mechanisms of PEDV attenuation.
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, USA.
authors.forename: Chun-Ming
authors.initials: CM
authors.lastname: Lin
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, USA.
authors.forename: Yixuan
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Department of Veterinary Population Medicine and Veterinary Diagnostic Laboratory, University of Minnesota,1333 Gortner Avenue, St. Paul, MN 55108, United States.
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Marthaler
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Gao
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, USA; State Key Laboratory of Veterinary Etiological Biology, OIE/National Foot-and-Mouth Disease Reference Laboratory of China, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
authors.forename: Xinsheng
authors.initials: X
authors.lastname: Liu
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, USA; College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, China.
authors.forename: Lanlan
authors.initials: L
authors.lastname: Zheng
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, USA. Electronic address: saif.2@osu.edu.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Saif
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, USA. Electronic address: wang.655@osu.edu.
authors.forename: Qiuhong
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000276:immunology*; D018929:Cell Culture Techniques / Q000662:veterinary; D002522:Chlorocebus aethiops; D018352:Coronavirus Infections / Q000662:veterinary* / Q000821:virology; D003967:Diarrhea / Q000662:veterinary* / Q000821:virology; D023281:Genomics; D000071497:Immunogenicity, Vaccine; D053485:Porcine epidemic diarrhea virus / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity*; D012692:Serial Passage / Q000662:veterinary; D013552:Swine; D013553:Swine Diseases / Q000821:virology*; D014613:Vaccines, Attenuated; D014709:Vero Cells; D014765:Viral Vaccines; D014774:Virulence
chemical_list: D000956:Antigens, Viral; D014613:Vaccines, Attenuated; D014765:Viral Vaccines
keywords: Attenuation; Cell culture adaptation; Genome; Porcine epidemic diarrhea virus; Sequence analysis