Manipulation of the porcine epidemic diarrhea virus genome using targeted RNA recombination

Abstract

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3'-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.

Figure 1. Coronavirus genome organization and targeted RNA recombination scheme.

(A) Genomic organization of PEDV. (B) Targeted RNA recombination scheme to make the interspecies chimeric virus mPEDV (Stage 1) or recombinant PEDV derivatives e.g. lacking the ORF3 gene as shown here (Stage 2). The ectodomain-encoding region of the MHV S gene is shown as a light-grey box in the mPEDV genome. Synthetic RNAs transcribed from the transfer vectors (Fig. 2A) were electroporated into PEDV (Stage 1) or mPEDV (Stage 2) infected cells, respectively. A single recombination event (indicated by a curved line) anywhere within the 3′ region of ORF1b present in the donor RNA and viral genome generates a recombinant genome. Selection of recombinant progeny viruses against parental viruses was done on the basis of the acquired ability to form plaques in murine cell monolayers (Stage 1) or on the basis of the ability to infect VERO cells and the concomitantly lost ability to infect murine cells (Stage 2).

Figure 2. PEDV transfer vectors.

(A) The pPEDV transfer vector contains the 5′-proximal 605 nt fused to the 3′ approximately 8 kilobases of the PEDV genome. All other vectors are derivatives thereof. The red triangle indicates the T7 promoter in the transfer vectors from which synthetic RNAs were made in vitro using T7 RNA polymerase. (B) Nucleotide sequences of junctions in the PEDV transfer vectors. Encircled numbers correspond to the numbered positions in the vector maps as indicated in Fig. 2A. (upper panel) The stop codon of ORF1b is underlined, the start codon of S is in blue, the transcription regulatory sequences (XUA(A/G)AC; [4]) are in orange and the BamHI site is indicated in purple. (lower panel) The stop codon of the S gene is underlined, the start codon of the ORF3 gene is in blue, the start codon of E gene is in red, the transcription regulatory sequences are in orange and the unique PmlI and EcoRV sites are indicated in purple and green, respectively.

Figure 3. Characterization of a chimeric PEDV carrying MHV spikes.

(A) Immunofluorescence analysis of mPEDV, MHV and PEDV infected cells. L cells infected with mPEDV were fixed and double immunolabeled with a polyclonal antibody against MHV (green) and a monoclonal antibody against the PEDV nucleocapsid (red). MHV and PEDV infected L cells were taken along for comparison. Nuclei are visualized with DAPI (blue). Overlay pictures (Merge) and graphical presentation of the MHV, mPEDV and PEDV virions are indicated at the right. Of note, the α-MHV fluorescence signal for MHV is significantly stronger than that for mPEDV due to the contribution of antibodies directed against other MHV proteins in the polyclonal MHV serum. (B) Inhibition of syncytia formation by the MHV-S HR2-peptide fusion inhibitor. HR2 peptide (4 µM; [32]) was added to MHV and mPEDV infected cells at 2 hours p.i. and kept present until 6.5 hours p.i. when cells were fixed and immunolabeled with the polyclonal MHV serum (green). Nuclei are visualized with DAPI (blue).

Figure 4. Characterization of a PEDV recombinant virus lacking ORF3.

(A) Genetic analysis of PEDV-ΔORF3. RT-PCR was performed covering the S-ORF3-E-M region (primers 4538/4977) using RNA templates isolated from wtPEDV, r-wtPEDV and PEDV-ΔORF3, and analyzed by gel electrophoresis. The expected sizes of the RT-PCR products (numbered 1 to 3) are indicated in the genome maps. For primer sequences, see Table 1. (B) Multi-step growth kinetics of r-wtPEDV and PEDV-ΔORF3. VERO cells were infected with each recombinant PEDV (MOI = 0.01), washed after three hours and viral infectivity in the culture media was determined at different times p.i. by a quantal assay on VERO cells from which TCID₅₀ values were calculated.

Figure 5. Recombinant PEDVs carrying Renilla luciferase and GFP genes.

(A) Genetic analysis of recombinant viruses. RT-PCR was performed covering the 1b-S junction (primers 5109/4535) or the S-M region (primers 4538/4977) using RNA templates isolated from wild-type PEDV, PEDV-Rluc, PEDV-ΔORF3/Rluc or PEDV-ΔORF3/GFP, and analyzed by gel electrophoresis. The expected sizes of the RT-PCR products (numbered 1 to 5) are indicated in the genome maps. For primer sequences, see Table 1. (B) Luciferase expression by the recombinant PEDV-Rluc and PEDV-ΔORF3/Rluc viruses. Left panel: VERO cells were infected with PEDV-Rluc at an MOI of 0.01, 0.1 or 1. Right panel: VERO cells were infected with PEDV-Rluc and PEDV-ΔORF3/Rluc at an MOI of 0.01. Intracellular Renilla luciferase activity (y-axis; Relative Light Units [RLU]) was determined at different times postinfection. (C) GFP expression by the recombinant PEDV-ΔORF3/GFP virus. VERO cells were infected with PEDV-ΔORF3/GFP at an MOI of 0.01 or 0.1 and fluorescence images were taken at different times p.i. Nuclei of cells were stained with DAPI (blue). (D) A rapid virus neutralization assay based on recombinant PEDVs expressing reporter proteins. PEDV-ΔORF3/Rluc and PEDV-ΔORF3/GFP (8,000 TCID50) were mixed with subsequent dilutions of serum positive for PEDV antibodies and a negative control serum (N.C.) for 30 minutes at room temperature. Mixtures were incubated with VERO cells and Renilla luciferase (left panel) or GFP (right panel) expression was measured at 8 and 9 hours p.i., respectively.