title: Rescue and evaluation of a recombinant PRRSV expressing porcine Interleukin-4.
abstract: BACKGROUND
  The current vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) have failed to provide broad protection against infection by various strains of PRRSV. Porcine Interleukin-4 (pIL-4) plays an important role in the regulation of the immune response and has been used previously as an immunological adjuvant. The objective of this study was to construct a recombinant PRRSV expressing pIL-4 and to evaluate the immune response of the recombinant virus in piglets.
  
  
  METHODS
  The pIL-4 gene was inserted in the PRRSV (CH-1R strain) infectious clone by overlap PCR. Indirect immunofluorescence assay (IFA) and Western blotting were used to confirm the recombinant virus. The stability of the recombinant virus was assessed by DNA sequencing and IFA after 15 passages in vitro. Recombinant virus was injected into pigs and efficacy of immune protection was evaluated in comparison with the parental virus.
  
  
  RESULTS
  The recombinant virus (CH-1R/pIL-4) was successfully rescued and shown to have similar growth kinetics as the parental virus. The recombinant virus was stable for 15 passages in cell culture. Pigs vaccinated with CH-1R/pIL-4 produced a similar humoral response to the response elicited by parental virus, but IL-4 level in the supernatant of PBMCs from pigs vaccinated with CH-1R/pIL-4 was significantly higher than the parent virus at 28 days post-immunization (DPI). Flow cytometric (FCM) analysis showed that the percentage of CD4(+)CD8(+) double positive T (DPT) cells in the CH-1R/pIL-4 vaccinated group was significantly higher than the parental virus at 3 and 7 Days Post-Challenge (DPC), and the IL-4 level in the blood significantly increased at 7 DPC. However, the viral load and histopathology did not show significant difference between the two groups.
  
  
  CONCLUSIONS
  A recombinant PRRSV expressing porcine IL-4 was rescued and it remained genetically stable in vitro. The recombinant virus induced higher DPT ratios and IL-4 levels in the blood after HP-PRRSV challenge compared to the parental virus in piglets. However, it did not significantly improve protection efficacy of PRRSV vaccine.
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. zjlzlj@yahoo.com.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. zjlzlj@yahoo.com.
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agriculture Science, Harbin, 150001, China. wg0381@163.com.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. wangyanfushi@163.com.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. wangyanfushi@163.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agriculture Science, Harbin, 150001, China. leofighting@163.com.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. wxlong@nwsuaf.edu.cn.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. wxlong@nwsuaf.edu.cn.
authors.forename: Xinglong
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. hbch228@163.com.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. hbch228@163.com.
authors.forename: Baicheng
authors.initials: B
authors.lastname: Huang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. jean_lqy@sina.com.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. jean_lqy@sina.com.
authors.forename: Qiongyi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. lifeiyang2007@126.com.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. lifeiyang2007@126.com.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. 18717368270@163.com.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. 18717368270@163.com.
authors.forename: Biyun
authors.initials: B
authors.lastname: Xue
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. Dingpeiyang1990@163.com.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. Dingpeiyang1990@163.com.
authors.forename: Peiyang
authors.initials: P
authors.lastname: Ding
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. farazvet@yahoo.com.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. farazvet@yahoo.com.
authors.forename: Shahid Faraz
authors.initials: SF
authors.lastname: Syed
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. wangchengbao@nwsuaf.edu.cn.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. wangchengbao@nwsuaf.edu.cn.
authors.forename: Chengbao
authors.initials: C
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agriculture Science, Harbin, 150001, China. aci139@sina.com.
authors.forename: Xuehui
authors.initials: X
authors.lastname: Cai
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. zhouem@nwsuaf.edu.cn.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, 712100, China. zhouem@nwsuaf.edu.cn.
authors.forename: En-Min
authors.initials: EM
authors.lastname: Zhou
mesh_terms: D000276:Adjuvants, Immunologic / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D015870:Gene Expression; D042822:Genomic Instability; D006651:Histocytochemistry; D007273:Injections, Intramuscular; D015847:Interleukin-4 / Q000096:biosynthesis* / Q000235:genetics; D019318:Porcine Reproductive and Respiratory Syndrome / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000302:isolation & purification*; D011995:Recombination, Genetic; D013552:Swine; D019562:Viral Load; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D000276:Adjuvants, Immunologic; D000914:Antibodies, Viral; D014765:Viral Vaccines; D015847:Interleukin-4