Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus

Abstract

A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK(12). To rescue infectious virus, capped RNA was transcribed in vitro from the pOK(12) clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5' end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.

FIG. 1.

Multistep strategy used to assemble full-length cDNA clone of VR-2332. In the top cartoon, the organization of the viral genome is shown, as are the positions of the unique restriction sites used for cloning purposes. The numbers 1a, 1b, and 2 through 7 indicate the PRRSV open reading frames. 5′ indicates the 5′ leader. and 3′ indicates the 3′ nontranslated region. At the 5′ end of the genome, XhoI and AscI restriction sites and a T7 RNA promoter were fused to the genome. The asterisk indicates the transcription start site of T7 RNA polymerase, resulting in the sequence 5′-m⁷G(5′)ppp(5′)G cap analog-TA TGA CGT ATA GGT…3′ as the predicted 5′ terminus of RNA transcribed in vitro with the T7 mMessage Machine kit. Downstream of the 3′ nontranslated region, a poly(A) tail of 38 A's and the restriction sites AclI and XbaI were inserted. The complete viral genome was divided into six fragments flanked by unique restriction sites, represented by the horizontal lines labeled a through f. The length of each fragment is indicated in parentheses below the horizontal lines (in nucleotides). As shown in the bottom cartoon, these fragments were individually cloned into the pOK₁₂ vector in the order indicated by the letters a to f. Prior to viral genome assembly, pOK₁₂ was prepared by inserting a stuffer fragment containing all the unique restriction sites shown in the top cartoon in the XhoI and XbaI sites.

FIG. 2.

Detection of cloned virus replication in Marc-145 cells. Supernatants from BHK-21C cells transfected with either (a) transfection reagent DMRIE-C without RNA as a negative control reaction or (b) RNA transcribed in vitro from the full-length cDNA clone were used to infect Marc-145 cultures. At day 3 after infection, the Marc-145 cultures were ethanol fixed and stained with monoclonal antibody SDOW17, directed against PRRSV nucleocapsid protein (a late marker of viral replication), and a horseradish peroxidase-conjugated secondary antibody (immunoperoxidase monolayer assay).

FIG. 3.

Differentiation between cloned virus and parental VR-2332 strain. A BstZ17I restriction site was introduced in the full-length cDNA clone of VR-2332 to allow discrimination between cloned virus (tagged with the BstZ17I site) and parental virus (lacks a BstZ17I site). RNA was extracted from lysates of cells infected with either the cloned virus or the parental VR-2332 isolate, and a 990-bp fragment was amplified by RT-PCR as described in Materials and Methods. The amplicons were digested with BstZ17I and analyzed on a 2.5% agarose gel. The presence of a BstZ17I restriction site resulted in fragments of 762 bp and 228 bp. As expected, the restriction site was found in the cloned virus but not in the parental VR-2332 virus isolate.

FIG. 4.

Growth curves of cloned virus and parental VR-2332 isolate. Parallel cultures of Marc-145 cells were infected at a multiplicity of infection of 0.002 TCID₅₀/cell with virus recovered from the full-length cDNA clone and the parental VR-2332 isolate. After 2 h of incubation at 37°C, the cells were washed twice and fresh medium was added (time 0), and the cells were incubated at 37°C. At 0, 3, 16, 20, 24, 48, and 72 h postinfection, samples of the supernatants were taken, and virus titers were determined as described in Materials and Methods.

FIG. 5.

Development of blue ears in pigs inoculated with cloned virus. A classical clinical sign of PRRSV infection, blue discoloration of the ears, was detected at day 9 postinfection in pigs experimentally infected with (a) the cloned virus or (b) the parental VR-2332 strain.