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Abstract
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.
References
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Figure 1.
Synthesis of cDNA amplicons encompassing the entire genomic RNA of the PRRSV PL97-1/LP1 strain. The full-length viral genomic RNA is schematically drawn at the top and the four overlapping cDNA amplicons are shown below. PF1, nt 180-5297 PF2, nt 3708-9108 PF3, nt 7688-13051 PF4, nt 9610–15238.
Figure 2.
Synthesis of cDNA amplicons encompassing the entire genomic RNA of the PRRSV PL97–1 strain. (A) Synthesis by long RT-PCR of four long overlapping cDNA amplicons (PF1, PF2, PF3, and PF4) that cover the full-length viral genomic RNA except for the 5′ and 3′ termini. Four PRRSV-specific overlapping cDNA amplicons are synthesized in the presence of an RNaseH(–) RT (lanes 1, 3, 5, and 7) but not in its absence (lanes 2, 4, 6, and 8). The products were analyzed on a 1% agarose gel and visualized by staining with ethidium bromide. Lanes 1-2, PF1 amplicons; lanes 3-4, PF2 amplicons; lane 5-6, PF3 amplicons, lanes 7-8, PF4 amplicons. M, 1 kb DNA marker indicated in base pairs. (B) Synthesis of PRRSV-specific amplicons by a modified 5'RACE protocol from the OligoT-ligated PRRSV cDNAs to determine the 5′-terminal sequence. RT reaction used to synthesize the first-strand cDNA corresponding to the 5′-end portion of the genome was conducted in the presence (lanes 1-2) or absence (lane 3) of RNaseH(–) RT. The OligoT was ligated to the 3′-end of first-strand cDNA using T4 RNA ligase in the presence (lanes 1 and 3) or absence (lane 2) of OligoT. The OligoT-ligated first-strand cDNA was then PCR-amplified using a pair of primers. PRRSV-specific cDNA amplicons were analyzed on a 1.5% agarose gel and visualized by staining with ethidium bromide. M, 100 bp DNA ladder indicated in base pairs. (C) Synthesis of PRRSV-specific amplicons from the OligoT-ligated PRRSV genomic RNAs by a modified 3'RACE protocol to determine the 3′-terminal sequence. The OligoT was ligated to the 3′-end of viral genomic RNA using T4 RNA ligase in the presence (lanes 1 and 3) or absence (lane 2) of OligoT. The OligoT-ligated viral RNA was then used for cDNA synthesis in the presence (lanes 1-2) or absence (lane 3) of RNaseH(–) RT. After PCR amplification, PRRSV-specific cDNA amplicons were analyzed on a 1.5% agarose gel and visualized by staining with ethidium bromide. M, 100 bp DNA ladder indicated in base pairs.
Figure 3.
The genome organization and the predicted polypeptides encoded by the PRRSV PL97–1/LP1 viral genome RNA. UTR, untranslated region; ORF, open reading frame; Nsp, non-structural protein; NT, nucleotide.
Figure 4.
Pairwise comparisons of full-length genome of PRRSV isolates. Percentage identity of nucleotide and amino acid sequences are shown above and below the diagonal line, respectively.
Figure 5.
A phylogenetic tree constructed with the nucleotide sequence of the full-length genome of all 12 available PRRSV isolates. The phylogenetic tree was constructed using the neighborjoining method in ClustralX method. The scale bar at the bottom of the tree represent the number of nucleotide substitutions per site. The tree was rooted using the nucleotide sequence of EAV, a member of the Arteriviridae family. The strain name is followed by the country and the year of isolation in two digits.
Figure 6.
Predicted pseudoknot structure of the ribosomal frame-shift region of PRRSV PL97–1/LP1. The heptanucleotide sequence (UUUAAAC) slippery sequence is in a box. The stop codon UAG of ORF1a is in the boldface type. Numbers indicate nucleotide position.
Table 1.
Oligonucleotides used in this study
| Oligonucleotide | Sequencea | Postitionb | Polarity | Purpose |
|---|---|---|---|---|
| PR1RT | 5′-TAGGATGGTGAGGGGGTG | 5332∼5349 | – | RT |
| PR1F | 5′-CCCTTTAACCATGTCT | 180∼195 | + | PCR |
| PR1R | 5′-CAAAGCAACCAGGTAA | 5282∼5297 | – | PCR |
| PR2RT | 5′-GAGCATGTCCTCAAACTT | 9168∼9185 | – | RT |
| PR2F | 5′-CCGGATATCGTCGCGG | 3708∼3723 | + | PCR |
| PR2R | 5′-CCATATGCTGTGCATA | 9093∼9108 | – | PCR |
| PR3RT | 5′-CACATTCCCTATCCCGAA | 13074∼13091 | – | RT |
| PR3F | 5′-GTTTAAACTGCTAGCC | 7688∼7703 | + | PCR |
| PR3R | 5′-GTGTAGCTGAAGGACA | 13036∼13051 | – | PCR |
| PR4RT | 5′-CTAATTGAATAGGTGACT | 15342∼15359 | – | RT |
| PR4F | 5′-ATTATGAGGGGAAGAA | 9610∼9625 | + | PCR |
| PR4R | 5′-ACGCGGATCAGGCGCA | 15223∼15238 | – | PCR |
| PR41 | 5′-GGAGAAGCCCCATTTTCC | 15038∼15055 | + | PCR |
| PR49 | 5′-CGACCCGTACCATTCTTT | 476∼493 | – | PCR |
| PR50 | 5′-AAAAGTCTTCAGGCTTGG | 692∼709 | – | RT |
| OligoT | 5′-CCAGTGTTGTGGCCTGCAGGGCGAATT | RNA ligation | ||
| OligoTR | 5′-GATGAATTCGCCCTGCAGGCCACAACA | RT, PCR |
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