Construction Of An Infectious Clone Of Porcine Reproductive And Respiratory Syndrome Virus BJ-4

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS. It causes an important disease in pigs characterized by reproductive failure in sows and gilts and respiratory dieases in piglets, resulting in great economic losses to the swine industry. The present article describes the establishment of an infectious full-length cDNA clone of PRRSV BJ-4 by utilizing reverse genetic manipulation technology. Reverse mutation of 23-nucleoitide mutations within the full-length cDNA clone were achieved. Infectious of the in vitro transcripts from the full-length cDNA clone after mutation were studied. The availability of such cDNA clones offers an opportunity for analysis and modification of viral genomes of PRRSV at the molecular level and has greatly aided research on virus replication, pathogenesis, and function of gene product. The infectious clone of BJ-4 provided fundamental materials for the development of novel viral vector, as well as for the development of a safe and effective live vaccine for use in pigs.Six sets of primers were designed according to the full-length genomic sequence of PRRSV BJ-4. By using the RT-PCR, 6 verlapping cDNA fragments covering the complete genome were amplified. These cDNA overlapping fragments Al, A2, Bl, B2, C, D with a size of 1,294 bp, 3,452 bp, 3,376 bp, 1,823 bp, 3,484 bp and 2,887 bp were cloned into pGEM-T Easy vector or pBluescript SK+ vector. Then the full-length cDNA clone pWSK DCBA of PRRSV BJ-4 were obtained when the 6 fragments were ligated in order and cloned into low-copy-number vector pWSK29. Unique restriction sites, which were absent from the viral sequence, were introduced at both end and within the full-length cDNA clone. At the 5' end, the transcription initiation sequence of the SP6 RNA polymerase and a nonviral G were introducd. A poly(T)g3 sequence was introduced to the 3' end of the cDNA clone.Six overlapping cDNA fragments covering the complete genome of PRRSV BJ-4 were sequenced, and compared with its parental virus and all other PRRSV sequence within the GenBank. All together 23-nucleoitide mutations were founded. Reverse mutation of these nucleoitide were achieved by using site-directed mutagenesis kit. Successful reverse of these 23-nucleoitide mutations was confirmed by sequencing. Sequencing analysis also showed that no other mutation was introduced into the six de novo clones. The post-mutated full-length cDNA clone PlP2mP3P4-63 of PRRSV BJ-4 were obtained whlie the 6 fragments were ligated in order and cloned into low-copy-number vector pWSK29.Full-length cDNA clone PlP2mP3P4-63 was cultured in mass volume, and plasmid DNA was purified according to the protocol of very low-copy-number plasmid. The plasmid DNA was linearized by cleavage with Vspl, then the linearized plasmids was used for in vitro transcription. Template DNA was digested by DNase I , then, BHK-21 cells were transfectd with in vrtro-transcribed RNA using transfection reagent DMRIE-C. Supernatants from cells at 24 h posttransfection were serially passaged on Marc-145 cells for three passages. Obvious cytopathiceffect was observed in the second passage. The infectious rescue viruses were obtained by RT-PCR and an indirect immuno fluorescence assay with the monoclonal antibody against N protein of PRRSV.