title: [An inverted-repeat RNA construct for silencing dengue virus type 2 pre-membrane gene suppresses viral replication in BHK-21 cells].
abstract: OBJECTIVE
  To evaluate the anti-viral effects of a plasmid expressing an inverted-repeat RNA targeting dengue virus type-2 (DENV-2) pre-membrane (prM) gene.
  
  
  METHODS
  Suckling mice were inoculated with live DENV-2 in the brain. The total RNA was extracted from the brain tissue of the infected mice, and the prM gene fragments were amplified by RT-PCR and then subcloned into XhoI/EcoR I of the pcDNA3.1(+) plasmid in antisense orientation to construct the plasmid pcDNA-asprM. DENV-2 prM sequences were also subcloned into pMD18-T-vector in sense orientation to construct the plasmid pMD18-T- prM. pcDNA-irRNA was constructed by inserting in sense orientation the prM fragment isolated from pMD18-T-prM into the NheI/Kpn I of pcDNA-asprM. The plasmid pcDNA-irRNA was transfected into BHK-21 cells and the anti-viral effects were analyzed by semi-quantitative PCR and real-time PCR.
  
  
  RESULTS
  Transfection with the plasmid pcDNA-irRNA caused a reduction of NS3 mRNA expression level by 28% in BHK-21 cells following a 96-h challenge with DENV-2 as compared to the cells without plasmid transfection (positive control). The viral copies in pcDNA-irRNA-transfected cells was 1.44-fold lower than those in the positive control cells following a 72-h virus challenge, and the mRNA expression levels of NS1 were also significantly lower in the transfected cells at 96 h after viral challenge (P<0.05) as shown by real-time quantitative PCR.
  
  
  CONCLUSIONS
  The inverted-repeat RNA for DENV-2 prM gene silencing can suppress DENV-2 replication in BHK-21 cells, which provides a basis for developing dengue virus gene vaccine.
authors.affiliation: Department of Pathogenic Biology, Southern Medical University, Guangzhou, China. ptzhu215@163.com
authors.forename: Pingting
authors.initials: P
authors.lastname: Zhu
authors.forename: Jing
authors.initials: J
authors.lastname: Pan
authors.forename: Xueli
authors.initials: X
authors.lastname: Zheng
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D006224:Cricetinae; D003716:Dengue Virus / Q000502:physiology*; D020868:Gene Silencing; D051379:Mice; D008815:Mice, Inbred Strains; D012367:RNA, Viral / Q000235:genetics; D020079:Terminal Repeat Sequences; D014759:Viral Envelope Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D012367:RNA, Viral; D014759:Viral Envelope Proteins