| In the study, the non-structural protein coding regions, including ORFI, 5'Non-coding region (NCR) and 3'NCR of porcine reproductive and respiratory syndrome virus (PRRSV) strain CH-la were sequenced and analyzed. The results showed that 5'NCR of PRRSV CH-Ia is l9Ont in length. It shared 94% nucleotide identity with that of strain VR2332, a representive of North American genotype. It was shorter than that of strain LV, a sepresentive of European genotype, and shared only 61% nucleotide identity. There were some conserved motifs in this region, including a 12-nucleotide stretch in the 5?end of 5'NCR. Forty-three nucleotides in the 3?end of 5'NCR exhibited high homology between CH-Ia and LV. There is also a most conserved motif at immediately upstream of ORFIa, i.e. 5UUAACC3 which may be critical for joining the 5eader to each of subgenomic mRNAs. Replicase-encoded ORF1, including ORFIa and ORFIb, was also determined. The results showed that ORF1 is ll882nt in length, ORFIa and ORF1b are 7512nt, 4373nt in length, respectively. ORF1a of CH-Ia shared 89% nucleotide identity with that of VR2332 and ORF1b shared 92% identity with that of VR2332. However, the sequence of CH-la ORFI was distinct from LV, ORFIa and ORFIb of CH-Ia exhibited only 54% and 63.3% nucleotide identities with that of LV. ORF1a of PRRSV CH-Ia codes a polyprotein. Six putative NSPs (NSPlct, NSPII3 and NSP2-5) are predicted. NSP2 is the most divergent among different PRRSV strains. The protein has been considered to be species-specific.lt seems to be more tolerable to mutations and insertions. NSP2 of CH-la shared 81% and 41% amino acid identities with those of VR2332 and LV, respectively. ORFIb codes another polyprotein. Four small putative proteins (CP1.4) are predicted, including RdRp. Compared with ORF1a, ORF1b is more conserved. But the carboxyl-terminal of CP4 is distinct, sharing only 48% amino acid identity with that of LV, and 96% amino acid identity with that of VR2332. This will be useful for distinguishing different genotypes. The ORFIa-Ib frame shift region of PRRSV is highly conserved. A hepta-nucleotide lippery sequence located just upstream of the stop codon of ORF Ia, and a pseudo-knot structure downstream of the slippery sequence were believed to be essential for the expression of ORF1b via a mechanism of ribosomal frame shifting. The 3'NCR of PRRSV CH-Ia is 151 nucleotides in length, and was 37 nucleotides longer than that of LV. It shared 78.2% nucleotide identity with that of LV, and 95.4% nucleotide identity with that of VR2332. There is a poly (A) tail in the 3'-end. A motif, consisting of eight nucleotidesimmediately upstream of the poly (A), is identical to those of LV and VR-2332. The motif ishighly conservative among arteriviruses, which suggested that the motif might be impoI-tantfor RdRp recognition and/or replication initiation.In summary' the non-structural protein regions of PaxSV CH-la were sequenced andanalyzed for the first time. The resuIts showed that the genetic distance between CH-la andLV was further than that between qH-la and VR2332, indicating that CH-la and VR2332can be classified to the same genotype.The study has provided material basis for constructing infectious cDNA clone ofPRRSV which is expected to provide basic knowIedge about PRRSV replication,transcription and translation, as well as to improve our understanding of the function ofPRRSV genome. |
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