Primers.

a<p>The location of primers is relative to the full genome sequence of the PEDV CV777 strain (GenBank accession No. AF353511).</p>b<p>Endonuclease restriction sites used for cloning are indicated in bold.References.</p

Coronavirus genome organization and targeted RNA recombination scheme.

<p>(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).</p

Characterization of a PEDV recombinant virus lacking ORF3.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069997#pone-0069997-t001" target="_blank">Table 1</a>. (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.</p

PEDV transfer vectors.

<p>(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 <i>in vitro</i> 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; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069997#pone.0069997-Tobler1" target="_blank">[4]</a>) are in orange and the <i>Bam</i>HI 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 <i>Pml</i>I and <i>Eco</i>RV sites are indicated in purple and green, respectively.</p

Design of the virus entry assays.

<p>Schematic overview of binding- (left), internalization- (middle), and fusion assay (right). 1 - Binding of virus to cell membrane; 2 – Lysis of cells and surface-bound virus; 3 – Complementation of ΔM15 by intravirion α-peptide, substrate conversion yielding luminescent readout; 4 – Invagination and 5 – Budding of endosomal vesicles containing virus particles; 6- Lysis of cell, intracellular compartment, and virion (after removal of cell surface-bound virions by protease treatment); 7 - Complementation of ΔM15 by intravirion α-peptide, substrate conversion yielding luminescent readout; 8 – Fusion of virion with endosomal membrane, exposure of intravirion α-peptide to the cytosol; 9 – Complementation of intracellular ΔM15 by virion α-peptide in intact cells, substrate conversion yielding fluorescent readout.</p

Effects of drugs on binding, internalization, and fusion of MHV and VSV.

<p>(<b>a–f</b>) Cells were pretreated with cycloheximide (CHX), ammonium chloride (NH₄Cl), bafilomycin A1 (BafA1), dynasore (Dyn), chlorpromazine (Chlopro), monensin (Mon), or latrunculin A (LatA), as well as with solvents dimethyl sulfoxide (DMSO) and methanol (MeOH) for 30 min. MHV and VSV viruses without α-peptide were included as background controls (inf wt). Error bars represent 1SEM, n = 3. (<b>a, d</b>) MHV-αN or VSV-Gα* were bound to ΔM15 expressing cells in presence of compounds on ice for 90 min. Cells were washed, lysed and assayed with Beta-Glo substrate as described in <b>4a</b>. Binding was determined relative to the complementation luminescence signal generated by virus bound to ΔM15 cells, treated without compound added (untr inf). (<b>b,e</b>) After binding as described in <b>a</b>, MHV-αN and VSV-Gα* were allowed to internalize at 37°C in presence of compounds for 40 and 30 min, respectively. Internalization was determined relative to the complementation luminescence signal of virus internalized into ΔM15 cells, treated without compound added (untr inf). (<b>c,f</b>) After binding as described in <b>a</b>, MHV-αN or VSV-Gα* were allowed to internalize and fuse at 37°C in presence of compounds for 100 and 40 min, respectively. MHV fusion inhibitor HR2 peptide (HR2) was included as control. Fusion was determined relative to the number of positive cells showing complementation fluorescein signal of virus fused in ΔM15 cells, treated without compound added (untr inf).</p

Model of viruses carrying α-peptide tagged proteins and visual selection of recombinant viruses and plaque growth by α-complementation.

<p>(<b>a–c</b>) α-peptide is shown as blue squares. (<b>a</b>) Model of MHV-αN and western blot analysis of N protein in purified virus stock. (<b>b</b>) Model of MHV-Sα and western blot analysis of S protein in purified virus stock. (<b>c</b>) Model of VSVΔG-Gα* pseudovirus and western blot analysis of VSV structural proteins in purified virus stock. (<b>d</b>) Serial dilution plaque assay of recombinant MHV-αN on LR7ΔM15 cell monolayers. After inoculation cells were covered for 2 days with a X-Gal containing agar-medium overlay. (<b>e</b>) Visualization of plaque growth of MHV-αN in LR7ΔM15 cell monolayers after 16, 30 or 48 h incubation (from left to right). Size bar corresponds to 1 mm.</p

Binding and internalization assay.

<p>(<b>a</b>) Luminescent signal after virus binding at various MOI. Increasing amounts of MHV-αN were bound to LR7ΔM15 cells on ice for 90 min before removing the inoculum and washing-off of unbound virus with ice-cold PBS. Cells and bound viruses were lysed and binding was determined by measuring the β-galactosidase activity using Beta-Glo substrate conversion to a luminescent product. (<b>b</b>) Internalization signal relative to MOI. Increasing amounts of MHV-αN were bound to LR7ΔM15 cells on ice for 90 min. Inoculum was removed and samples transferred to 37°C for 40 min. Cell-surface bound virus was removed by trypsinization. Cells and intracellular viruses were lysed and internalization determined by measuring β-galactosidase activity using Beta-Glo substrate conversion to a luminescent product. (<b>c</b>) Controls of binding and internalization assay. Samples were treated as described in <b>a</b> (binding) and <b>b</b> (internalization). After binding, attached virus was removed by trypsin treatment (trypsin). Binding and internalization were inhibited by incubation of cells with MHV receptor CC1a blocking anti-CC1a antibody (anti-CC1a) 30 min prior to and during inoculation. Error bars in <b>a</b> - <b>c</b> represent 1 SEM, n = 3.</p

Fusion assay.

<p>(<b>a</b>) Virus-cell fusion measured by flow cytometry. Sorting of MHV-αN infected cells by flow cytometry showed increasing fluorescence at increasing MOI. Cells were treated as described in <b>b</b>. (<b>b</b>) Increase of fusion signal relative to MOI. Increasing amounts of MHV-αN, MHV-Sα, and VSVΔG-Gα* were bound to ΔM15 expressing cells on ice. 40 min (VSV) or 100 min (MHV) post warming to 37°C fusion was assayed by measuring β-galactosidase activity using FDG substrate and flow cytometry. Inlay highlights β-galactosidase activity at low MOI. Error bars represent 1 SEM, n = 3. (<b>c, d</b>) Kinetics of internalized α-peptide tagged protein in comparison to β-galactosidase activity. MHV-αN (MOI = 100) was bound to cells on ice. Unbound virus was removed, and samples shifted to 37°C with (<b>c</b>) or without addition of cycloheximide (<b>d</b>). At the indicated time points, cells were washed and trypsinized on ice, removing surface bound virus. Virus-cell fusion was measured by β-galactosidase activity using flow cytometry or cells were lysed and immunoblotted against N for quantification the internalized α-peptide proteins. (<b>e</b>) Fluorescence microscopy image of β-galactosidase activity in infected cells. MHV-αN was bound to LR7ΔM15 cells on ice. Inoculum was washed off and cultures shifted to 37°C for the indicated time periods. β-galactosidase activity was visualized by fluorescein production using fluorescence microscopy. Size bar corresponds to 250 µm.</p

RSV-specific IgG against individual RSV glycoproteins do not correlate with disease severity.

<p>RSV glycoprotein-specific antibody levels were determined by ELISA against recombinant, soluble ectodomains. Median IgG levels (± IQR) were compared between healthy and RSV-infected infants as well as between RSV patients with and without oxygen therapy. Glycoproteins used for ELISA coating were (A-B) RSV G protein, (C-D) prefusion F protein, and (E-F) postfusion F protein. (G-H) The relative abundance of antibodies against prefusion F compared to G protein as well as pre- compared to postfusion F protein were categorized assessed by subtracting their levels. The median (± IQR) difference was compared between plasma from healthy infants and RSV patients with or without oxygen therapy. No significant differences were observed by Mann Whitney <i>U</i> test for comparison between two groups and Kruskall-Wallis for comparison between more than two groups.</p