5' and 3' cis-Acting RNA Elements Required for RNA Replication of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae, is the most important viral pathogens in swine industry worldwide. Here, we have investigated 5' and 3' cis-acting RNA elements required for PRRSV genome replication. Using the infectious PRRSV cDNA, we have manipulated the genomic RNA to generate mutant genomic RNAs, transfected these mutants into susceptible MARC-145 cells, and examined the competence of RNA replication. We found three genetic factors that were essential for viral replication. First, the cap structure present at the 5'-end of the genome was absolutely required for RNA replication. Secondly, polyadenylation of the genomic RNA at the 3'-end was also essential for RNA replication. Thirdly, approximately 100-nucleotide region just upstream of the N protein-coding region was crucial for genomic RNA replication. Taken together, our findings indicate that replication of PRRSV genomic RNA requires three important cis-acting RNA elements: 5' cap structure, 3' poly(A) motif, and an internal sequence of about 100 nucleotides. Further investigation is needed to elucidate the molecular mechanism(s) of how these elements act on PRRSV genome replication.

Complete Nucleotide Sequence of Genomic RNA of a Large-Plaque Forming Porcine Reproductive and Respiratory Syndrome Virus PL97-1/LP1

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important animal pathogens in swine industry worldwide. In this study, we isolated the large-plaque forming variant virus designated PL97-1/LP1 from the parental strain PL97-1, the first Korean strain of PRRSV, isolated from the serum of an infected pig in 1997. We found that the 15411-nucleotide genome of PL97-1/LP1 consisted of a 189-nucleotide 5' untranslated region (UTR), a 15071-nucleotide protein-coding region, and a 151-nucleotide 3'UTR, followed by a poly (A) tail of about 50~60 nucleotides in size. The 5'-end of PL97-1/LP1 began with ATGACGTAT. Comparison of the PL97-1/LP1 genome with the 11 fully sequenced PRRSV genomes currently available revealed sequence similarity from 99.6~99.7% (the North American VR-2332 and two VR-2332-derived vaccine strains MLV RespPRRS/Repro and RespPRRS MLV) to 62.0% (the Dutch Lelystad strain). Phylogenetc analysis revealed that PL97-1/LP1 is most closely related to the North American genotype VR-2332, two VR-2332-derived vaccine strains, and Chinese BJ-4. It is distantly related to the European genotype Lelystad. The entire nucleotide sequence of PL97-1/LP1 was identical to that of the parental virus PL97-1 except for three silent nucleotide substitutions, one in ORF1a (U4230C), one in ORF1b (C10977U), and one in ORF5 (U13976A). This nucleotide sequence has been submitted to the GenBank database under the accession number AY612613.