Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3'-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5'-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5' leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.

FIG. 1

(A) Primer extension analysis of strain VR-2332. RNA from VR-2332-infected MA-104 cells was hybridized to γ-³²P-radiolabeled VR-2332 leader reverse primer /658P4 and reverse transcribed. The primer extension products were electrophoresed alongside the known sequencing products obtained from clone 712 and forward primer P71/. The primer extension product migrated with the thymidine residue located at nucleotide 2965 of clone 712, resulting in an extension product of 98 nucleotides. (B) Comparison of PRRSV leader sequences. VR-2332 leader (190 bases in length) and LV leader (221 bases in length) sequences exhibit 61.0% identity as analyzed by the GCG GAP program, with a gap weight of 5 and a length weight of 5 (lines between the sequences indicate identity). The leader-body junction sequence utilized for transcription of each mRNA is boxed.

FIG. 2

(A) Schematic of the 15,409-base viral genome of VR-2332 (open boxes) with sequenced cDNA clones (shaded boxes). (B) Sequence of the VR-2332 region between ORF 1a and 1b and its resulting predicted RNA pseudoknot tertiary structure involved in ribosome frameshifting, as modeled on the predicted pseudoknot of LV (1, 38). The proposed heptanucleotide slippery sequence (boxed), UAG stop codon of ORF 1a (bold), and differences between VR-2332 and LV (italics) are indicated.

FIG. 3

(A) Schematic of amino acid comparisons of VR-2332 (upper bars) and LV (lower bars) by domain, showing regions of similarity and dissimilarity (GCG GAP program alignments; see text). (B) ORF 1a alignment of the following: (1) arteriviral PCPα and -β domains and their respective catalytic residues (α or β) (with blosum62.cmp and pam250.cmp, a gap weight of 12, and a gap length weight of 4); (2) an unusual arteriviral cysteine protease domain with putative catalytic residues (•) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); and (3) the poliovirus 3C-like protease (PV1) domain with catalytic H and D and C/S residues (∗) (with blosum62.cmp, a gap weight of 1, and a gap length weight of 2). (C) ORF 1b with MHV-A59 ORF 1b residues used to align the following: (1) a putative polymerase region with amino acids conserved among positive strand RNA viruses (48) shown (∗) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); (2) a domain with conserved cysteine and histidine residues (•) (with blosum62.cmp, a gap weight of 6, and a gap length weight of 2); (3) helicase domains showing conserved amino acids (•) in Sindbis virus-like RNA plant virus group A2 (22) (with blosum62.cmp, a gap weight of 2, and a length weight of 2); and (4) a coronaviruslike domain (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5). In all panels, amino acids conserved in aligned sequences are shown in boldface, those conserved among arteriviruses are shown in uppercase (except for the PCPα and -β -alignment which shows conservation between VR-2332, LV, and LDVP in uppercase), and similar amino acids between the two PRRSV strains are boxed.

FIG. 3

(A) Schematic of amino acid comparisons of VR-2332 (upper bars) and LV (lower bars) by domain, showing regions of similarity and dissimilarity (GCG GAP program alignments; see text). (B) ORF 1a alignment of the following: (1) arteriviral PCPα and -β domains and their respective catalytic residues (α or β) (with blosum62.cmp and pam250.cmp, a gap weight of 12, and a gap length weight of 4); (2) an unusual arteriviral cysteine protease domain with putative catalytic residues (•) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); and (3) the poliovirus 3C-like protease (PV1) domain with catalytic H and D and C/S residues (∗) (with blosum62.cmp, a gap weight of 1, and a gap length weight of 2). (C) ORF 1b with MHV-A59 ORF 1b residues used to align the following: (1) a putative polymerase region with amino acids conserved among positive strand RNA viruses (48) shown (∗) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); (2) a domain with conserved cysteine and histidine residues (•) (with blosum62.cmp, a gap weight of 6, and a gap length weight of 2); (3) helicase domains showing conserved amino acids (•) in Sindbis virus-like RNA plant virus group A2 (22) (with blosum62.cmp, a gap weight of 2, and a length weight of 2); and (4) a coronaviruslike domain (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5). In all panels, amino acids conserved in aligned sequences are shown in boldface, those conserved among arteriviruses are shown in uppercase (except for the PCPα and -β -alignment which shows conservation between VR-2332, LV, and LDVP in uppercase), and similar amino acids between the two PRRSV strains are boxed.

FIG. 3

(A) Schematic of amino acid comparisons of VR-2332 (upper bars) and LV (lower bars) by domain, showing regions of similarity and dissimilarity (GCG GAP program alignments; see text). (B) ORF 1a alignment of the following: (1) arteriviral PCPα and -β domains and their respective catalytic residues (α or β) (with blosum62.cmp and pam250.cmp, a gap weight of 12, and a gap length weight of 4); (2) an unusual arteriviral cysteine protease domain with putative catalytic residues (•) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); and (3) the poliovirus 3C-like protease (PV1) domain with catalytic H and D and C/S residues (∗) (with blosum62.cmp, a gap weight of 1, and a gap length weight of 2). (C) ORF 1b with MHV-A59 ORF 1b residues used to align the following: (1) a putative polymerase region with amino acids conserved among positive strand RNA viruses (48) shown (∗) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); (2) a domain with conserved cysteine and histidine residues (•) (with blosum62.cmp, a gap weight of 6, and a gap length weight of 2); (3) helicase domains showing conserved amino acids (•) in Sindbis virus-like RNA plant virus group A2 (22) (with blosum62.cmp, a gap weight of 2, and a length weight of 2); and (4) a coronaviruslike domain (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5). In all panels, amino acids conserved in aligned sequences are shown in boldface, those conserved among arteriviruses are shown in uppercase (except for the PCPα and -β -alignment which shows conservation between VR-2332, LV, and LDVP in uppercase), and similar amino acids between the two PRRSV strains are boxed.

FIG. 3

(A) Schematic of amino acid comparisons of VR-2332 (upper bars) and LV (lower bars) by domain, showing regions of similarity and dissimilarity (GCG GAP program alignments; see text). (B) ORF 1a alignment of the following: (1) arteriviral PCPα and -β domains and their respective catalytic residues (α or β) (with blosum62.cmp and pam250.cmp, a gap weight of 12, and a gap length weight of 4); (2) an unusual arteriviral cysteine protease domain with putative catalytic residues (•) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); and (3) the poliovirus 3C-like protease (PV1) domain with catalytic H and D and C/S residues (∗) (with blosum62.cmp, a gap weight of 1, and a gap length weight of 2). (C) ORF 1b with MHV-A59 ORF 1b residues used to align the following: (1) a putative polymerase region with amino acids conserved among positive strand RNA viruses (48) shown (∗) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); (2) a domain with conserved cysteine and histidine residues (•) (with blosum62.cmp, a gap weight of 6, and a gap length weight of 2); (3) helicase domains showing conserved amino acids (•) in Sindbis virus-like RNA plant virus group A2 (22) (with blosum62.cmp, a gap weight of 2, and a length weight of 2); and (4) a coronaviruslike domain (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5). In all panels, amino acids conserved in aligned sequences are shown in boldface, those conserved among arteriviruses are shown in uppercase (except for the PCPα and -β -alignment which shows conservation between VR-2332, LV, and LDVP in uppercase), and similar amino acids between the two PRRSV strains are boxed.

FIG. 3

(A) Schematic of amino acid comparisons of VR-2332 (upper bars) and LV (lower bars) by domain, showing regions of similarity and dissimilarity (GCG GAP program alignments; see text). (B) ORF 1a alignment of the following: (1) arteriviral PCPα and -β domains and their respective catalytic residues (α or β) (with blosum62.cmp and pam250.cmp, a gap weight of 12, and a gap length weight of 4); (2) an unusual arteriviral cysteine protease domain with putative catalytic residues (•) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); and (3) the poliovirus 3C-like protease (PV1) domain with catalytic H and D and C/S residues (∗) (with blosum62.cmp, a gap weight of 1, and a gap length weight of 2). (C) ORF 1b with MHV-A59 ORF 1b residues used to align the following: (1) a putative polymerase region with amino acids conserved among positive strand RNA viruses (48) shown (∗) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); (2) a domain with conserved cysteine and histidine residues (•) (with blosum62.cmp, a gap weight of 6, and a gap length weight of 2); (3) helicase domains showing conserved amino acids (•) in Sindbis virus-like RNA plant virus group A2 (22) (with blosum62.cmp, a gap weight of 2, and a length weight of 2); and (4) a coronaviruslike domain (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5). In all panels, amino acids conserved in aligned sequences are shown in boldface, those conserved among arteriviruses are shown in uppercase (except for the PCPα and -β -alignment which shows conservation between VR-2332, LV, and LDVP in uppercase), and similar amino acids between the two PRRSV strains are boxed.

FIG. 3

(A) Schematic of amino acid comparisons of VR-2332 (upper bars) and LV (lower bars) by domain, showing regions of similarity and dissimilarity (GCG GAP program alignments; see text). (B) ORF 1a alignment of the following: (1) arteriviral PCPα and -β domains and their respective catalytic residues (α or β) (with blosum62.cmp and pam250.cmp, a gap weight of 12, and a gap length weight of 4); (2) an unusual arteriviral cysteine protease domain with putative catalytic residues (•) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); and (3) the poliovirus 3C-like protease (PV1) domain with catalytic H and D and C/S residues (∗) (with blosum62.cmp, a gap weight of 1, and a gap length weight of 2). (C) ORF 1b with MHV-A59 ORF 1b residues used to align the following: (1) a putative polymerase region with amino acids conserved among positive strand RNA viruses (48) shown (∗) (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5); (2) a domain with conserved cysteine and histidine residues (•) (with blosum62.cmp, a gap weight of 6, and a gap length weight of 2); (3) helicase domains showing conserved amino acids (•) in Sindbis virus-like RNA plant virus group A2 (22) (with blosum62.cmp, a gap weight of 2, and a length weight of 2); and (4) a coronaviruslike domain (with blosum62.cmp, a gap weight of 5, and a gap length weight of 5). In all panels, amino acids conserved in aligned sequences are shown in boldface, those conserved among arteriviruses are shown in uppercase (except for the PCPα and -β -alignment which shows conservation between VR-2332, LV, and LDVP in uppercase), and similar amino acids between the two PRRSV strains are boxed.

FIG. 4

Predicted junction site motifs (bold and underlined) in VR-2332 and LV ORF 7. VR-2332 mRNA 7.1 utilizes the first junction site motif (JS 1) and mRNA 7.2 utilizes the third junction site motif (JS 2). LV uses the third junction site motif exclusively (37) (JS). A potential junction site motif at nucleotide 14577 (VR-2332) appears not to be used (Fig. 5). The beginning ORF 7 nucleotide and predicted protein sequences are shown in bold type (with the GAP program [fastadna.cmp], a gap weight of 16, and a length weight of 4).

FIG. 5

(A) Northern blot analysis of PRRSV mRNA 7. Total RNA from cells infected with VR-2332 (lane 1) or LV (lane 2) were electrophoresed through an agarose gel and blotted onto a nylon membrane. The membrane was probed sequentially with a VR-2332 ORF 7 oligomer (lane 1) and then an oligomer to LV ORF 7 (lane 2). No nonspecific hybridization was detected in several analyses. (B) Northern blot analysis of total RNA from VR-2332-infected MA-104 (CL2621) cells and alveolar macrophages (AM) shows that junction site 1 is used to transcribe the majority of mRNA 7 (7.1) and that junction site 2 is used to transcribe a minority of mRNA 7 (7.2). Mock-, VR-2332-, and LV-infected-cell total RNA populations were electrophoresed through a 2% agarose gel and transferred to membranes. The membranes were probed with reverse complement oligomers to VR-2332 ORF 7 (a), VR-2332 ORF 7 junction site 1 (b), VR-2332 ORF 7 junction site 2 (c), and LV ORF 7 (d). An RNA ladder (Gibco BRL) was used to assess sgmRNA size.