A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus

Abstract

The nucleotide sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) was determined. Transfection of MARC-145 cells with capped in vitro transcripts derived from a full-length cDNA clone of the viral genome resulted in infectious PRRSV with growth characteristics similar to that of the parental virus. Primer extension analysis revealed that during replication, the viral polymerase corrected the two nonviral guanosine residues present at the 5' terminus of the transfected transcripts. Animal studies showed that the cloned virus induced hyperthermia, persistent viremia, and antibody response, similar to that observed with the parental virus. Contact transmission occurred rapidly within 3 days of introduction of naïve pigs into the group of clone virus-inoculated pigs. These results suggest that the cloned virus retains the in vivo virulence and contagion properties of the parental virus, thus, providing the background for reverse genetics manipulation in systematic examination of attenuation and virulence phenotypes.

Fig. 1

Construction of PRRSV NVSL #97-7895 full-length cDNA. (A) Five overlapping regions of the viral genome were amplified to generate the cDNA fragments A, B, 5C, 3C, and D. Fragment D was amplified to introduce 41 adenosines at the 3′ terminus. The T7 RNA polymerase promoter (Φ10) was inserted upstream of viral genomic 5′-terminal sequences in fragment A. Restriction enzyme sites used for cloning are listed below the fragments. (B) cDNA fragments with the consensus amino acid sequence were selected for assembly of the full-length clone in pBR322. An AclI site was incorporated immediately downstream of the poly(A) tail. The artificially introduced genetic marker, a BsrGI restriction enzyme site at position nt 1170, is shown. Bent arrow shows the position and direction of transcription by T7 RNA polymerase. (C) Original bacterial stock carrying pFL12 was propagated six times in E. coli DH5α. Plasmid DNA from passage 1 (P1) and 6 (P6) bacterial cultures was extracted, digested with MfeI, and resolved by electrophoresis on a 0.9% agarose gel along with a size marker (M). Expected sizes of the products of digestion are 1525, 1704, 1882, 1906, 2617, 4527, and 5034 bp. Gel image is inverted.

Fig. 2

Rescue and passage of PRRSV. (A) MARC-145 cells were transfected with in vitro transcripts derived from pFL12Pol⁻ or pFL12, or with total RNA from cells infected with parental virus. At 2 days posttransfection, cells were examined for expression of N protein by immunofluorescence. (B) Supernatants from parallel cultures of transfected cells were collected 2 days posttransfection, clarified, and passaged onto naïve MARC-145 cells. Forty-eight hpi, the cell monolayer was examined for expression of N protein as before. Cells in panel B were photographed at higher magnification than in panel A.

Fig. 3

Characterization of rescued virus. (A) An amplicon of 1740 bp, spanning the genetic marker, was amplified by RT-PCR using total RNA from cells infected with either vFL12 (lanes 3 and 4) or parental virus (lanes 5 and 6). pFL12 DNA also was used in PCR amplification as a positive control (lanes 1 and 2). The amplicons were digested with BsrGI. DNA was electrophoresed on 1% agarose gel and an inverted image was acquired. (B) Growth kinetics of cloned and parental virus. MARC-145 cells (2.5 × 10⁶) were infected with either vFL12 or parental virus. At 6, 12, 24, 48, and 72 hpi, supernatant was collected and titrated by plaque assay. PAMs were infected and cultures were collected at 6, 12, and 24 hpi for titration of infectivity. Error bars represent standard deviation from replica experiments.

Fig. 4

Nonviral 5′ guanosine residues in the in vitro transcripts from pFL12 are corrected by PRRSV polymerase during RNA replication. Primer extension analysis of RNA, isolated from cells mock-infected (lane 5) or infected with vFL12 (lane 7) or parental virus (lane 8) or capped in vitro transcripts alone (lane 6), was performed using a negative-sense oligonucleotide. Lanes 1–4 show the sequence ladder obtained with the same oligonucleotide using pFL12 DNA template. Nucleotides marked by * correspond to migration positions of extended cDNAs due to addition of cap. Arrow and arrowhead show the positions of 5′ends of in vitro transcripts and replicating viral RNAs, respectively.

Fig. 5

The cloned virus retains in vivo markers of virulence and is genetically stable. Three groups of pigs (four pigs per group) were sham-inoculated or inoculated with either vParental or vFL12 at TCID₅₀/ml of 10^5.2 and 10^4.8, respectively. (A) Temperatures were recorded daily from 2 days before inoculation to 16 dpi. Average temperatures from four different animals in each group are shown. (B) Serum samples were collected at 4, 7, 14, and 22 dpi and virus titers were determined and expressed as TCID₅₀/ml. Values represent average titers from four animals. (C) Serum antibody titers were determined with the Idexx ELISA kit. Values represent average titers from four animals. Dashed line at 0.4 S/P ratio designates threshold value above which titers are considered positive for anti-PRRSV antibodies. Error bars in panels A, B, and C represent standard deviation. (D) Serum from 14 dpi, recovered from inoculated animals, was used to distinguish between vFL12 and vParental by examining for the presence of the BsrGI genetic tag (left panel). Viral RNA extracted from sera was amplified by RT-PCR and the products were digested with BsrGI. Similar analysis (lane 7) was performed with virus in serum from a sentinel pig at 8 days postcohabitation with pigs infected with vFL12 (right panel). Gel image is inverted. Error bars represent standard deviation.