Construction of infectious cDNA clones of PRRSV:Separation of coding regions for nonstructural and structural proteins

摘要

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV),the causative agent of the ongoing "porcine high fever syndrome" in China,is capable of genetic and antigenic mutations at high frequency.How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest.We developed an infectious cDNA clone of an attenuated strain of Type II PRRSV,and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2,encoding for nonstructural- or structural-protein,respectively.The cDNA was generated from the cell-attenuated virus strain,APRRS,via RT-PCR,and followed by nucleotide sequencing and molecular cloning.The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail,which has a 99.7% nucleotide identity with that of PRRSV Nsp strain,also a vaccine strain.Based on the nucleotide sequencing results,the full-length cDNA clone was assembled in pBlueScript vector,under the control of T7 promoter at the immediate 5' terminus of genome.To discern the rescued viruses from that of parental virus,a Mlu I restriction site was engineered into ORF5 coding region.Upon transfection of the in vitro transcripts of both the original and Mlu I-tagged cDNAs into MA-104 cells,typical PRRSV cytopathic effects were observed.The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties.Subsequently,PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes,ORF1,and structural proteins,ORF2-ORF7.The synthetic RNA of such mutant clone,pCSA,was infectious and the rescued virus shared similar properties with that of the parental virus.This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering cross-protection to various genetically diversified PRRSV strains,and a platform for further development of PRRSV as a gene expression vector for recombinant vaccines against other significant swine diseases.