SARS-CoV genome nucleotide sequence comparison.

a<p>GenBank accession number SARS-CoV Frankfurt-1: AY291315; SARS-CoV HKU-39849: AY278491; SARS-CoV HKU-39849 UOB: JQ316196 and recSARS-CoV HKU-39849: JN854286.</p>b<p>Introduced nucleotide change to create <i>Sfi</i>I restriction site for cloning purposes.</p

Construction of a vaccinia virus based SARS-CoV reverse genetic system.

<p>The genome structure of SARS-CoV is shown at the top of the figure. Nine cDNA clones produced from the genomic RNA of SARS-CoV isolate HKU-39849 are shown below. The region of the SARS-CoV genome encompassed by each clone is indicated by the nucleotide number (using the recSARS-CoV sequence; GenBank: JN854286) at the beginning and end of each clone. Restriction enzyme sites used to join the clones are shown, with restriction enzymes sites added to the clones shown in bold. The cDNA fragments isolated from the clones and gpt PCR products covering regions of the genome unstable as cDNA clones were ligated with each other and vaccinia virus DNA to produce two vaccinia virus recombinant clones spanning nts 1–20288 and 20272–29727 of the SARS-CoV genome respectively. The first 2012 nts of the former vaccinia virus recombinant was derived from the SARS-CoV isolate Frankfurt-1 (shaded in dark grey). Vaccinia virus mediated homologous recombination was then used to reconstitute the SARS-CoV subgenomic fragments, introducing regions of cDNA that were unstable in <i>E. coli</i> and repairing errors (*) introduced during the cloning process. This resulted in the vaccinia virus clones vSARS-CoV-5prime and vSARS-CoV-3prime. The SARS-CoV cDNA fragments were isolated from the two vaccinia virus recombinants by restriction enzyme digestion and then joined using unique <i>SfiI</i> and <i>BglI</i> sites that had been introduced into the cDNA. The ligated cDNA fragments were used as a template for <i>in vitro</i> transcription using a T7 polymerase promoter introduced at the 5′ end of the SARS-CoV 5′ cDNA clone to produce a RNA transcript representing the SARS-CoV genome.</p

Recovery and analysis of recSARS-CoV by RT-PCR and plaque assay.

<p><b>A.</b> Immunofluorescence microscopy analysis of Vero-E6 cells infected with P0 virus harvests obtained from cells electroporated with full-length recSARS-CoV RNA. Cells were fixed at 8 hours post infection (p.i.) and stained for nonstructural protein 3 (green) as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032857#pone.0032857-Snijder1" target="_blank">[40]</a>. Nuclei were stained using Hoechst 33258 (blue). <b>B.</b> RT-PCR analysis and restriction enzyme digestion confirm the recovery of recSARS-CoV. Vero-E6 cells were infected with P0 culture medium from cells transfected with recSARS-CoV RNA, (harvested 48 hours post transfection, lanes 2–5), SARS-CoV Frankfurt-1 (SARS-CoV, lanes 6 and 7) or mock infected (mock, lanes 8 and 9). At 48 hours p.i., RNA was isolated from culture supernatants (lanes 4 and 5) or infected Vero-E6 cells (lanes 2, 3, 6, 7, 8 and 9). The RNA samples were used for RT-PCR analysis. Lanes 2, 4, 6 and 8 show the products obtained from RT-PCR reactions designed to amplify a genomic region containing a <i>BglI</i> restriction site that had been engineered into recSARS-CoV genome at the cDNA level, whereas lanes 3, 5, 7, and 9 show corresponding control reactions without reverse transcriptase. The RT-PCR products shown in lanes 2, 4 and 6 were then further analyzed by <i>Bgl</i>I digestion to verify the presence of this marker mutation in the recSARS-CoV progeny. The 2.5 kb PCR products derived from recSARS-CoV (lanes 11 and 12) were digested into two expected fragments of similar size (1266 bp and 1285 bp), whereas the wildtype PCR product remains undigested. The sizes of the PCR products were determined by comparison to a 1 Kb Plus DNA ladder (Invitrogen) (lanes 1 and 10). Bacteriophage λ DNA was cleaved with <i>Bgl</i>I (lane 14) as a digestion control. <b>C.</b> Comparative plaque assays of different SARS-CoV variants on Vero-E6 cells. Upon complete CPE, progeny virus was harvested from Vero-E6 cells infected with recSARS-CoV, SARS-CoV-Frankfurt-1, the original SARS-CoV HKU-39849 (HKU), and the SARS-CoV HKU-39849 used to produce the recSARS-CoV cDNAs used in this study (HKU-B). Tenfold serial dilutions were plated on Vero-E6 cells under a semisolid overlay and cell layers were fixed and stained with crystal violet after two days.</p

Analysis of SARS-CoV gene expression in hDCs using a recSARS-CoV expressing <i>Renilla</i> luciferase.

<p><b>A.</b> The genome structure of recombinant SARS-CoV and HCoV-229E viruses expressing <i>Renilla</i> luciferase (HCoV-229E-luc and SARS-CoV-luc) is shown. White boxes represent ORFs encoding virus replicase and accessory proteins, grey boxes represent ORFs encoding virus structural proteins. The regions of HCoV-229E ORF4a/b and SARS-CoV ORF7a are enlarged to illustrate the ORF encoding <i>Renilla</i> luciferase and surrounding nucleotides. Nucleotide numbers depict CoV nucleotides at the border to non-CoV sequences. The dashed line in the upper panel depicts nucleotides derived from restriction sites <i>BamHI</i> and <i>EcoRI</i> that have been introduced to facilitate cloning of the recombination plasmid containing the <i>Renilla</i> luciferase gene. <b>B.</b> Pairwise comparison of the replication of recSARS-CoV and HCoV-229E with the corresponding luciferase encoding viruses. RecSARS-CoV/SARS-CoV-luc and HCoV-229E/HCoV-229E-luc were used to infect Vero-E6 and Huh-7 cells, respectively, at an MOI of 0.01. The culture supernatants were harvested at 24, 48 and 72 hrs p.i and the titers of virus in the supernatants determined by plaque assay. The peak viral titres for recSARS-CoV/SARS-CoV-luc (at 72 hours p.i) and HCoV-229E/HCoV-229E-luc (at 48 hours p.i) are shown. The average titers from 3 independent experiments are shown together with error bars. <b>C.</b> Analysis of SARS-CoV-luc- and HCoV-229E-luc-mediated <i>Renilla</i> luciferase expression in infected (MOI = 1) Vero-E6, Huh-7 and hDCs. <i>Renilla</i> luciferase expression was assessed at 12 hours (black bars) and 24 hours p.i. (white bars), The fold increase in <i>Renilla</i> luciferase expression levels in virus-infected cells represents the ratio of luciferase activity in virus-infected cells compared to that in mock-infected cells.</p

Growth curve and intracellular RNA and protein synthesis of recSARS-CoV and SARS-CoV Frankfurt-1.

<p><b>A.</b> Vero-E6 cells were infected with a MOI of 5 for both viruses and cell culture supernatant samples harvested at the indicated time points. Virus titers were determined by plaque assay. SARS-CoV Frankfurt-1 and recSARS-CoV titers are shown as (▪) and (□) respectively. <b>B.</b> Vero-E6 cells were infected with a MOI of 5 for both viruses, and intracellular RNA was isolated at the indicated time points. Following separation in an agarose gel, hybridization with a [³²P]-labeled DNA probe recognizing the 3′ end of all viral mRNAs was used to visualize viral RNA bands. <b>C.</b> Vero-E6 cells were infected with a MOI of 5 for both viruses and cells were formaldehyde fixed at the indicated time points. Following permeabilization, cells were double-labeled for nonstructural protein 3 (in red) and nucleocapsid protein (in green) as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032857#pone.0032857-Snijder1" target="_blank">[40]</a>.</p