Coding potential, transcription regulatory sequences and sequence comparisons of Ro-BatCoV GCCDC1 with Ro-BatCoV HKU9 strains, SARS-CoV, BatCoV HKU3 stains, MERS-CoV, BatCoV HKU4 strains and BatCoV HKU5 strains.

<p>Coding potential, transcription regulatory sequences and sequence comparisons of Ro-BatCoV GCCDC1 with Ro-BatCoV HKU9 strains, SARS-CoV, BatCoV HKU3 stains, MERS-CoV, BatCoV HKU4 strains and BatCoV HKU5 strains.</p

Syncytium formation and functional analyses of Ro-BatCoV GCCDC1 p10 gene.

<p>(A) The construction of transient expression plasmid of p10 gene based on a pCAGGS vector. (B) Transient expression of the p10 gene and syncytium formation. <b>Top:</b> the observation of syncytium formation with Wright-Giemsa staining on the monolayer BHK-21 cells transfected with recombinant plasmid of Pulau virus p10 gene, recombinant plasmid of Ro-BatCoV GCCDC1 p10 gene, and empty pCAGGS vector; <b>Bottom:</b> the observation of syncytium formation with indirect immunofluorescence staining on the cells treated as described above. (C) The construction of subgenomic plasmid of p10 gene. The putative subgenome of p10 was cloned into a pcDNA3.0-derived vector. (D) Transient expression of the p10 gene and syncytium formation with recombinant subgenomic p10 plasmid. <b>Top:</b> the observation of syncytium formation with Wright-Giemsa staining on the monolayer BHK-21 cells transfected with recombinant plasmid of Pulau virus p10 gene, recombinant plasmid of p10 subgenome of Ro-BatCoV GCCDC1 and empty pcDNA3.0 vector; <b>Bottom:</b> the observation of syncytium formation with indirect immunofluorescence staining on the cells treated as described above. (Wright-Giemsa staining: stained monolayers were imaged using an Olympus IX51FL+DP70 microscope under 100× magnification, scale bars = 200 μm; indirect immunofluorescence staining: stained monolayers were imaged using a Nikon DIAPHOT-TMD microscope under 200× magnification, scale bars = 50 μm).</p

Genome organization and phylogenetic history of Ro-BatCoV GCCDC1.

<p>Genome organization of Ro-BatCoV GCCDC1. Nonstructural genes and putative mature nonstructural proteins, structural genes, and 5’- and 3’-UTR are illustrated with yellow, dark blue and light blue colors, respectively. The remarkable p10 gene is shown in red. The potential origin of the p10 gene is indicated by a dotted arrow and a question mark. The leader sequence and leader transcription regulatory sequence (TRS) are directly shown with nucleobases. The bat, <i>Rousettus leschenaulti</i>, is used to show the host species that Ro-BatCoV GCCDC1 was discovered. The schematic virion of coronavirus is used to show the virus that identified in the present study. The schematic virion of orthoreovirus and the segment S1 of the genome that it contains are used to demonstrate the possible origin of the p10 gene.</p

Identification of the recombinant p10 gene and its TRS.

<p>(A) Confirmation of the “exotic” p10 gene. The sequences that cover the upstream junction site between the N and p10 genes, and downstream junction site between the p10 and NS7a genes, are illustrated with sequencing patterns. The length of the intergenic sequence between the N and p10 genes is indicated with a number. The TRS preceding the NS7a gene in the intergenic sequence is marked with red arrow. (B) Identification of the TRS of the p10 gene. The TRS of the p10 gene in the N gene is illustrated with a sequencing pattern. The distance from the TRS to the AUG codon of p10 gene is indicated with a number. The length of the intergenic sequence between the N gene and genes just downstream of N gene are indicated with numbers. The TRSs of genes just downstream of N gene are marked with red arrows.</p

Prediction of the putative pp1a/pp1ab cleavage sites of Ro-BatCoV GCCDC1 based on comparison with prototype coronaviruses^a.

<p>Prediction of the putative pp1a/pp1ab cleavage sites of Ro-BatCoV GCCDC1 based on comparison with prototype coronaviruses<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005883#t002fn001" target="_blank">^a</a>.</p

Comparison of the 3'-terminus of the N gene of Ro-BatCoV GCCDC1 with those of Ro-BatCoV HKU9 strains and Ro-BatCoV Kenya.

<p>Alignment of nucleotide and amino acid sequence of the 3'-terminus of the N gene among Ro-BatCoV GCCDC1, Ro-BatCoV HKU9 strains and Ro-BatCoV Kenya. The eight amino acid truncation and two-amino-acid deletion at the 3’-terminus of N protein of the Ro-BatCoV GCCDC1 are illustrated.</p

Subgenomic structures of Ro-BatCoV GCCDC1.

<p>(A) Schematic of the Ro-BatCoV GCCDC1 genome. The genome is represented by a black line; ORFs, and the 5’-UTR and 3’-UTRs are indicated by yellow and grey arrows, respectively. The TRSs are marked with small red triangles. The genomic locations of the leader and body TRS(s) are shown with blue and red arrows, respectively. (B) Schematic structures of putative transcribed subgenomic mRNAs. Subgenomes are represented by a black rectangles and the common leader sequence is denoted by a blue box. The target sites of forward and reverse primers are marked and indicated with letter F and R, respectively. Two numbers are shown in front of each subgenomic mRNA. The black number to the right of the slash indicates the potential number of fragment(s) that could be amplified using this set of primers, while the red one to the left represents the actual numbers of the fragment(s) obtained in this experiment which corresponds to the number of band(s) on each lane marked with a red arrow(s) on the agarose gel. (C) Agarose gel electrophoresis of the PCR products of subgenomic mRNA. The lowest band marked with a red arrow on each lane is the specific amplicon of each subgenomic mRNA. Other marked bands are amplicons of upper subgenomic mRNAs as shown in Fig 6B. (D) mRNA junctions of the detected subgenomic mRNAs. The TRSs and fusion sites are shown in a black frame. The bias of the TRS of p10 gene is highlighted with a yellow block. The leader sequence and CDS are indicated. The lengths of intergenic sequences are shown with numbers.</p

Phylogenetic analyses of p10 from representative reoviruses and Ro-BatCoV GCCDC1.

<p>The tree was inferred using the maximum likelihood method available in PhyML. Bootstrap values are shown at relevant nodes. The GenBank accession numbers used in this analysis are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005883#ppat.1005883.s009" target="_blank">S3 Table</a>.</p