Molecular characterization of PL97-1, the first Korean isolate of the porcine reproductive and respiratory syndrome virus

Abstract

We determined the complete nucleotide and predicted amino acid sequence of the genomic RNA of PL97-1, the first Korean strain of porcine reproductive and respiratory syndrome virus (PRRSV), which was isolated from the serum of an infected pig in 1997. We found that the 15411-nucleotide genome of PL97-1 consisted of a 189-nucleotide 5' noncoding region (NCR), a 15071-nucleotide protein-coding region, and a 151-nucleotide 3'NCR, followed by a poly (A) tail. The 5'-end of PL97-1 began with 1ATG ACG TAT AGG12. Comparison of the PL97-1 genome with the 11 fully sequenced PRRSV genomes currently available revealed sequence divergence ranging from 0.3% (the VR-2332-derived vaccine MLV RespPRRS/Repro strain) to 38% (the Dutch Lelystad strain). To better understand the genetic relationships between these different strains, phylogenetic analyses were performed on the full-length PRRSV genomes. Significantly, the phylogenetic tree based on the ORF1b or ORF7 genes most closely resembled the tree based on the full-length genomes. Thus, these single genes will be the most useful in revealing the genetic relationships between the different strains relative to their geographical distribution. Extensive phylogenetic analyses using the ORF7 sequences of 111 PRRSV isolates available revealed that PL97-1 is most closely related to the North American genotype VR-2332, a VR-2332-derived vaccine strain, and Chinese BJ-4. It is distantly related to the European genotype Lelystad. This study provides the largest full-length genome phylogenetic analysis of PRRSV that has been published to date, and supports an earlier genetic grouping of the many temporally and geographically diverse PRRSV strains currently isolated.

Fig. 1

Nucleotide sequence alignment of the 5′NCR (A) and 3′NCR (B) regions of the 12 available fully sequenced PRRSV strains including PL97-1. Except for PL97-1, which was sequenced in this study, all other sequence information was obtained from the GenBank database indicated in Table 2. The consensus sequence of all 12 PRRSV strains is shown on top, and only differences from that sequence in the PRRSV strains are indicated. Deletions are indicated by hyphens. Dash-line boxes indicate a highly conserved region in the 5′NCRs (A) and 3′NCRs (B) present in all 12 PRRSV genomes. (A) The open box indicates a stretch of 23–24 nucleotides at the utmost 5′-end of the NVSL 97-7985 and MLV RespPRRS/Repro PRRSV strains, respectively, which were not reported. (B) The open box represents a string of deletions of 3, 18, and 17 nucleotides in the beginning of the 3′NCR of the Lelystad PRRSV strain.

Fig. 2

Distribution of nucleotide (A) and amino acid (B) differences throughout the entire PRRSV genome. Nucleotide and amino acid sequences were compared by the multiple sequence alignment method using ClustralX. The number of differences throughout the entire genome was plotted. (A) Schematic diagram of the full-length PRRSV genome is schematically depicted on top. The four subregions containing higher nucleotide sequence variation are indicated as graded bars on the top of the PRRSV genome structure. (B) Each ORF is drawn on two separate lines, and linearly illustrated on top for the purpose of discussion. Arrowheads indicate predicted proteolytic cleavage sites in the ORF1 polyproteins.

Fig. 3

Phylogenetic tree constructed with the nucleotide sequence of the full-length genome (A), or the ORF1b (B), ORF7 (C), or ORF5 (D) gene of all 12 available PRRSV strains. Phylogenetic trees were constructed using the neighbor-joining method in ClustralX (Thompson et al., 1997). The scale bars at the bottom of each tree represent the number of nucleotide substitutions per site. The numbers at each node indicate bootstrap replicate support. The trees were rooted using the nucleotide sequence of EAV, a member of the Arteriviridae family. The genomes of LDV and SHFV were included in the phylogenetic analyses to provide information on divergence levels in comparison to other arteriviruses. The strain name is followed by the country and the year of isolation in two digits.

Fig. 4

Phylogenetic relationships predicted from the ORF7 nucleotide sequences of 111 selected PRRSV strains isolated from different geographic regions worldwide at different time periods. (A) All 111 PRRSV strains used in this analysis were classified into two distinct phylogenetic groups corresponding to the European and North American genotypes. (B–C) A branch of the European genotype (B) or the North American genotype (C) in (A) was magnified for the purpose of illustration. Detailed information regarding the PRRSV strains used in this analysis is provided in Table 2. The 12 fully sequenced PRRSV strains are boxed. Numbers at each node indicate bootstrap replicate values greater than 500 (1000 replicates). For details of the trees, see legend to Fig. 3.