The two N-terminal cleavage products, nsp1a and nsp1b, of the replicase polyproteins of porcine reproductive and respiratory syndrome virus (PRRSV) each contain a papain-like autoproteinase domain, which have been named PCPa and PCPb, respectively. To assess their role in the PRRSV life cycle, substitutions and deletions of the presumed catalytic cysteine and histidine residues of PCPa and PCPb were introduced into a PRRSV infectious cDNA clone. Mutations that inactivated PCPa activity completely blocked subgenomic mRNA synthesis, but did not affect genome replication. In contrast, mutants in which PCPb activity was blocked proved to be non-viable and no sign of viral RNA synthesis could be detected, indicating that the correct processing of the nsp1b/nsp2 cleavage site is essential for PRRSV genome replication. In conclusion, the data presented here show that a productive PRRSV life cycle depends on the correct processing of both the nsp1a/nsp1b and nsp1b/nsp2 junctions.

The 5′-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5′-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

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The purpose of this study was to analyze the antigenic structure of the nucleocapsid protein N of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV) and to identify antigenic differences between this prototype European isolate and other North American isolates. To do this, we generated a panel of monoclonal antibodies (mAbs) directed against the N protein of Lelystad virus and tested them in competition assays with other N-specific mAbs described previously (Drew et al., 1995; Nelson et al., 1993; van Nieuwstadt et al., 1996). Four different competition groups of mAbs were identified. Pepscan analysis with solid-phase dodecapeptides was used to identify specific antigenic regions in the N protein that were bound by the mAbs. In this pepscan analysis, we found that the mAb of the first competition group reacted with linear peptides whose core sequences consisted of amino acids 2-12 (site A), the mAbs of the second group reacted with peptides whose core sequences consisted of amino acids 25-30 (site B), and the mAb of the third group reacted with peptides whose core sequences consisted of amino acids 40-46 (site C). However, the fourth group of mAbs binding to an antigenic region, provisionally designated as domain D, reacted very weakly or did not react at all with solid-phase dodecapeptides. To further characterize the structure of the epitopes in domain D, we produced chimeric constructs composed of the N protein sequences of Lelystad virus and another arterivirus lactate dehydrogenase-elevating virus, which was used because its N protein has similarity in amino acid sequence and hydropathicity profile but does not react with our mAbs. When the mAbs specific to domain D were tested for binding to the chimeric N proteins expressed by Semliki Forest virus, we found that the regions between amino acids 51-67 and amino acids 80-90 are involved in the formation or are part of the epitopes in domain D. Therefore, we conclude that the N protein contains four distinct antigenic regions. The epitopes mapped to sites A-C are linear, whereas the epitopes mapped to domain D are more conformation dependent or discontinuous. Sites A and C contain epitopes that are conserved in European but not in North American isolates; site B contains epitopes that are conserved in European and North American isolates; and site D contains epitopes that are either conserved or not conserved in European and North American isolates. The antigenic regions identified here might be important for the development of diagnostic test for PRRSV in particular tests that discriminate between different antigenic types of PRRSV.