title: Construction and in vitro evaluation of a recombinant live attenuated PRRSV expressing GM-CSF.
abstract: BACKGROUND
  Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be an important problem for the swine industry. Inactivated vaccines and modified-live virus vaccines are widely used in the field; however, the efficacy of these PRRSV vaccines is suboptimal due to poor immunogenicity. Granulocyte-macrophage colony stimulating factor (GM-CSF) has been extensively used as an effective genetic and protein adjuvant to enhance the efficiencies vaccines expressing tumor or pathogen antigens. The purpose of this study was to determine if GM-CSF could increase the efficiency of PRRSV vaccine.
  
  
  METHODS
  The GM-CSF gene was inserted in the HuN4-F112 vaccine strain by overlap PCR. The expression of GM-CSF by the recombinant virus was confirmed with methods of indirect immunofluorescent assay (IFA) and Western blotting. The stability of recombinant virus was assessed by cDNA sequence and IFA after 20 passages. To detect the biological activity of GM-CSF expressed by the recombinant virus, bone marrow-derived dendritic cells (BMDCs) were isolated and co-cultured with the recombinant virus or parental virus and the surface phenotypes of BMDCs were examined by flow cytometric analysis. The cytokines secreted by BMDCs infected with PRRSV, or treated with LPS, GM-CSF or medium alone were evaluated by ProcartaPlexTM Multiplex Immunoassays and qRT-PCR.
  
  
  RESULTS
  A novel modified-live PRRSV vaccine strain expressing GM-CSF (rHuN4-GM-CSF) was successfully constructed and rescued. The GM-CSF protein was stable expressed in recombinant virus-infected cells after 20 passages. Analysis of virus replication kinetics showed that the novel vaccine strain expressing GM-CSF had a similar replication rate as the parental virus. In vitro studies showed that infection of porcine BMDCs with rHuN4-GM-CSF resulted in increased surface expression of MHCI+, MHCII + and CD80/86+ that was dependent on virus expressed GM-CSF. The expression of representative cytokines was significantly up-regulated when BMDCs were incubated with the recombinant GM-CSF expressing virus.
  
  
  CONCLUSIONS
  Our results indicated that the expression of GM-CSF during infection with a vaccine strain could enhance the activation of BMDCs and increase cytokine response, which is expected to result in higher immune responses and may improve vaccine efficacy against PRRSV infection.
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. yulingxue1984@126.com.
authors.forename: Lingxue
authors.initials: L
authors.lastname: Yu
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. yanjunzhou406@163.com.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China. yanjunzhou406@163.com.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. jyfabc1983@sohu.com.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China. jyfabc1983@sohu.com.
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. 402518438@qq.com.
authors.forename: Wu
authors.initials: W
authors.lastname: Tong
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. 307766180@qq.com.
authors.forename: Shen
authors.initials: S
authors.lastname: Yang
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. 381163550@qq.com.
authors.forename: Fei
authors.initials: F
authors.lastname: Gao
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. 1134845535@qq.com.
authors.forename: Kang
authors.initials: K
authors.lastname: Wang
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. 272955728@qq.com.
authors.forename: Liwei
authors.initials: L
authors.lastname: Li
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. 240582640@qq.com.
authors.forename: Tianqi
authors.initials: T
authors.lastname: Xia
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. 879335043@qq.com.
authors.forename: Qun
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. gztong@shvri.ac.cn.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China. gztong@shvri.ac.cn.
authors.forename: Guangzhi
authors.initials: G
authors.lastname: Tong
mesh_terms: D000276:Adjuvants, Immunologic / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000276:immunology / Q000821:virology; D015870:Gene Expression; D042822:Genomic Instability; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D008969:Molecular Sequence Data; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D014776:Virus Cultivation; D014779:Virus Replication
chemical_list: D000276:Adjuvants, Immunologic; D016207:Cytokines; D012367:RNA, Viral; D016178:Granulocyte-Macrophage Colony-Stimulating Factor