Identification Of 5'UTR CIS-Acting Element For Replication Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome (PRRS) is a severe infectious disease. It is characterized by mild to severe respiratory failure in sows and gilts, and respiratory problems in piglets. The causative agnt is identified as PRRS virus (PRRSV), which was first isolated almost simultaneously in Europe and North American; the strains are designated Lelystad and VR-2332. PRRS is a economically significant infectious disease in the swine industry worldwide, but the current control for PRRS is insufficient. More in-depth studies on the process of virus life cycle are warrantied to better control the disease. Based on our established PRRSV infectious clone of North American strain, we intended to functionally dissect the 5 untranslational region (UTR), and define the key cis-acting elements for that regulate the discrete steps of the viral life cycle. The detailed experimentations are discussed as follows.1. Substitution of the PRRSV 5'UTR with that of a different genotypeBased on the porcine reproductive and respiratory syndrome virus (PRRSV) full length infectious cDNA clone pCBC2, a mutant full length cDNA clone was constructed, which was named pTLNd4. Using PCR-based mutagenesis, a Nde I restrict endonulease site was inserted between the end of the 5'UTR and the translation initiation codon of ORF1. Rely on the Nde I restrict endonulease site, North American type 5'UTR was substitute with that of European type to obtain the chimeric clone pTLV8. The original pCBC2 was used as wild-type control in all experiments. These plasmids were lineared by Xho I, and the gel-purified DNAs were used as templates for in vitro RNA transcription. The RNA transcripts were transfected into MARC-145 cells. Cytopathic effects (CPE) were observed in pTLNd4 transfection, while no visible CPE seen in that of pTLV8. Genomic RNA was analyzed by RT-PCR, and subgenomic mRNA was analyzed by Northern Blot. In order to investigate the effect induced by the engineered mutations for the process of virus genomic...