title: Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain.
abstract: The porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replication in vitro, it enhanced virus replication in vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses.
  
  
  OBJECTIVE
  The tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro but enhanced virus replication and induced higher-level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural, & Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Zhongyan
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Yokoyama
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural, & Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA Boehringer Ingelheim (China) Investment Co., Ltd., Shanghai, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Chen
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural, & Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA Department of Virology II, National Institute of Infectious Disease, Tokyo, Japan.
authors.forename: Tomoichiro
authors.initials: T
authors.lastname: Oka
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural, & Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Kwonil
authors.initials: K
authors.lastname: Jung
authors.affiliation: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Kyeong-Ok
authors.initials: KO
authors.lastname: Chang
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural, & Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Thavamathi
authors.initials: T
authors.lastname: Annamalai
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural, & Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA wang.655@osu.edu saif.2@osu.edu.
authors.forename: Qiuhong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural, & Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA wang.655@osu.edu saif.2@osu.edu.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Saif
mesh_terms: D000220:Adaptation, Biological; D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D059386:Reverse Genetics; D029341:Sapovirus / Q000235:genetics / Q000254:growth & development*; D012692:Serial Passage; D013552:Swine; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics; D014776:Virus Cultivation
chemical_list: D015678:Viral Structural Proteins; D012324:RNA-Dependent RNA Polymerase