Subgenomic RNA7 is transcribed with different leader-body junction sites in PRRSV (strain VR2332) infection of CL2621 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV), like all members of the order Nidoviridae, is expressed in the infected cell as a nested set of subgenomic (sg) RNAs with a common 5'-leader sequence. We have determined that the 5'-leader sequence for the US prototype strain (VR2332, Collins, et al., 1992) is distinct from the European prototype strain [Lelystad (LV); Wensvoort, et al., 1991, Meulenberg et al., 1993a], yet these two strains use almost the same sequence for downstream sites of 5'-leader-body junction formation. Analysis of VR2332 genomic sequence identified several potential 5'-leader-body junction sequences upstream of open reading frame (ORF) 7, coding for the nucleocapsid protein, that could be used for generation of VR2332 sgRNA7 transcripts. Sequence determinations of RT-PCR-generated cDNA clones of sgRNA7 identified two species of RNA7 transcripts in infected cells, one utilizing a leader-body junction sequence (AUAACC) 123 nucleotides upstream of the AUG start site and one utilizing a sequence (UAAACC) 9 nucleotides upstream of the AUG start site for ORF7 translation.

Genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern United States.

The nucleotide sequence of a 3266 bp region encompassing open reading frames (ORFs) 2 through 7 of the porcine reproductive and respiratory syndrome virus (PRRSV) was determined for 10 isolates recovered from the midwestern United States. Pairwise comparisons showed that genetic distances between isolates ranged from 2.5% to 7.9% (mean 5.8% +/- 0.2%) whereas the Lelystad strain from Europe was, on average, 34.8% divergent from US clones. Thus, US and European PRRSV isolates represent genetically distinct clusters of the same virus. ORF 5, which encodes the envelope glycoprotein, was the most polymorphic [total nucleotide diversity (pi) = 0.097 +/- 0.007] and ORF 6, encoding the viral M protein, was the most conserved (pi = 0.038 +/- 0.003). The substantial differences in nucleotide diversity among ORFs suggests that the virus is evolving by processes other than simple accumulation of random neutral mutations. In support of this hypothesis, statistical analyses of the nucleotide sequence provided strong evidence for intragenic recombination or gene conversion in ORFs 2, 3, 4, 5 and 7, but not in ORF 6. An excess of synonymous (silent) substitutions was observed in all six ORFs, indicating an evolutionary pressure to conserve amino acid sequences. Taken together, the data indicate that despite intragenic recombination among extant PRRSV isolates, purifying selection has acted to maintain the primary structure of individual ORFs.

Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus.

The 3'-portion of the genome of a U.S. isolate of the porcine reproductive and respiratory syndrome (PRRS) virus, ATCCVR-2332, was cloned and sequenced. The resultant 3358 nucleotides contain 6 open reading frames (ORFs) with homologies to ORFs 2 through 7 of the European strain of the PRRS virus and other members of the free-standing genus of arteriviruses. Both VR-2332 and the European isolate (called the Lelystad virus) have been identified as infectious agents responsible for the swine disease called PRRS. Comparative sequence analysis indicates that there are degrees of amino acid identity to the Lelystad virus open reading frames ranging from 55% in ORF 5 to 79% in ORF 6. Hydropathy profiles indicate that the ORFs of VR-2332 and Lelystad virus correspond structurally despite significant sequence differences. These results are consistent with the biological similarities but distinct serological properties of North American and European isolates of the virus.