title: Pathogenicity and genetic characteristics associated with cell adaptation of a virulent porcine reproductive and respiratory syndrome virus nsp2 DEL strain CA-2.
abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is the most common and world-widespread viral pathogen of swine. We previously reported genomic sequences and pathogenicity of type 2 Korean PRRSV strains belonging to the virulent lineage 1 family, which contain remarkable amino acid deletions in nonstructural protein 2 (nsp2 DEL) compared to VR-2332. Here, a virulent type 2 Korean PRRSV nsp2 DEL strain, CA-2, was serially propagated in MARC-145 cells for up to 100 passages (CA-2-P100). As the passage number increased, the phenotypic characteristics of cell-adapted CA-2 strains were altered, in terms of higher viral titers and larger plaque sizes compared to the parental virus. Pro-inflammatory cytokine genes, including TNF-α, IL-8, MCP-1, and MCP-2, were found to be significantly down-regulated in PAM cells with the CA-2-P100 strain compared to its parental nsp2 DEL virus. Animal inoculation studies demonstrated that the virulence of CA-2-P100 was reduced significantly, with showing normal weight gain, body temperatures, and lung lesions comparable to the control group. Furthermore, high-passage CA-2-P100 showed declined and transient viremia kinetics, as well as delayed and low PRRSV-specific antibody responses in infected pigs. In addition, we determined whole genome sequences of low to high-passage derivatives of CA-2. The nsp2 DEL pattern was conserved for 100 passages, whereas no other deletions or insertions arose during the cell adaptation process. However, CA-2-P100 possessed 54 random nucleotide substitutions that resulted in 27 amino acid changes distributed throughout the genome, suggesting that these genetic drifts provide a possible molecular basis correlated with the cell-adapted features in vitro and the attenuated phenotype in vivo. Taken together, our data indicate that the cell-attenuated CA-2-P100 strain is a promising candidate for developing a safe and effective live PRRSV vaccine.
authors.affiliation: Choongang Vaccine Laboratory, Daejeon 34055, Republic of Korea; College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea.
authors.forename: Seung-Chul
authors.initials: SC
authors.lastname: Lee
authors.affiliation: Choongang Vaccine Laboratory, Daejeon 34055, Republic of Korea.
authors.forename: Hwan-Won
authors.initials: HW
authors.lastname: Choi
authors.affiliation: Choongang Vaccine Laboratory, Daejeon 34055, Republic of Korea.
authors.forename: Eeuri
authors.initials: E
authors.lastname: Nam
authors.affiliation: Choongang Vaccine Laboratory, Daejeon 34055, Republic of Korea.
authors.forename: Yun-Hee
authors.initials: YH
authors.lastname: Noh
authors.affiliation: Animal Virology Laboratory, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea.
authors.forename: Sunhee
authors.initials: S
authors.lastname: Lee
authors.affiliation: Animal Virology Laboratory, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea.
authors.forename: Yoo Jin
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea.
authors.forename: Gun-Seok
authors.initials: GS
authors.lastname: Park
authors.affiliation: School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea.
authors.forename: Jae-Ho
authors.initials: JH
authors.lastname: Shin
authors.affiliation: Choongang Vaccine Laboratory, Daejeon 34055, Republic of Korea.
authors.forename: In-Joong
authors.initials: IJ
authors.lastname: Yoon
authors.affiliation: College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea. Electronic address: sykang@cbu.ac.kr.
authors.forename: Shien-Young
authors.initials: SY
authors.lastname: Kang
authors.affiliation: Animal Virology Laboratory, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea. Electronic address: changhee@knu.ac.kr.
authors.forename: Changhee
authors.initials: C
authors.lastname: Lee
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002460:Cell Line; D015536:Down-Regulation; D016679:Genome, Viral / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics; D008168:Lung / Q000473:pathology; D009154:Mutation; D019318:Porcine Reproductive and Respiratory Syndrome / Q000276:immunology / Q000473:pathology* / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000302:isolation & purification / Q000472:pathogenicity*; D011897:Random Allocation; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014774:Virulence / Q000235:genetics*
chemical_list: D000914:Antibodies, Viral; D016209:Interleukin-8; D014409:Tumor Necrosis Factor-alpha; D017361:Viral Nonstructural Proteins
keywords: Attenuation; Cell adaptation; Genome-wide sequencing; Immune response; Nsp2 deletion; PRRSV; Pathogenicity; Type 2 lineage 1