Characterization and Pathogenicity of the Porcine Deltacoronavirus Isolated in Southwest China

Figures

• 

  Figure 1. Isolation and identification of PDCoV CHN-SC2015 in LLC-PK cells by cytopathic effect (CPE) and immunofluorescence assay (IFA). The confluent LLC-PK cells were infected with PDCoV CHN-SC2015 and the CPE was recorded at 24 h post-infection. (A): Micro examination of the PDCoV-infected LLC-PK cells; (B): Micro examination of the mock-infected LLC-PK cells. (C–H): LLC-PK Cells were fixed and stained with rabbit anti-PDCoV N protein and FITC-goat anti-rabbit IgG at 24 h post-infection (green); nuclei were counterstained with DAPI (blue). Magnification = 10×.

• 

  Figure 2. Electron microscopy of PDCoV strain CHN-SC2015. The virus particles were resuspended in PBS and negatively stained with 2% sodium phosphotungstic and examined with a transmission electron microscope. The arrow indicates the crown-shaped projections on the virus surface. Bar: 200 nm.

• 

  Figure 3. PDCoV CHN-SC2015 replication kinetics in LLC-PK cells was determined by qRT-PCR. The total viral RNA from PDCoV CHN-SC2015 infected LLC-PK cells at different times (12 h, 24 h, 36 h, 48 h and 60 h) was extracted and subjected to qRT-PCR. The quantity of PDCoV viral RNA was calculated based on the standard curve and is expressed as the copies per milliliter of the cell culture. * indicates that the significant difference compared to 6 h post-infection.

• 

  Figure 4. Heat map of nucleotide identities and differences between CHN-SC2015 and 59 reference strains in GenBank. The level of nucleotide identity (top right) and the number of nucleotide differences (bottom left) between CHN-SC2015 and other 59 reference strains were analyzed using Clustal W software in MEGA X. The nucleotide identity and difference are represented by a gradation of color; the deeper the color, the greater the identity or difference.

• 

  Figure 5. Analysis of deletions and insertions in the complete genome of PDCoV strain CHN-SC2015. Alignments, ORF1ab gene (A) and the S gene and 3′ UTR (B) were performed with ClustalW software in MEGA X. PDCoV strain CHN-SC2015 is outlined with a box. A dash indicates that the deleted nucleotide is present in the other 59 sequences compared to the consensus sequences.

• 

  Figure 6. Phylogenetic trees based on the complete genome (A) and S gene (B) of PDCoV strain CHN-SC2015. Phylogenetic trees were constructed using the neighbor-joining method in MEGA7. CHN-SC2015 is indicated with a red asterisk. The branches in red, green, yellow, purple, light blue, and dark blue represent the clades from China, Southeast Asia, USA, South Korea, and Hong Kong, respectively. The trees were constructed using the ML method and a bootstrap analysis (n = 1000) in Mega X software. Bootstrap values > 50% are shown at the branch points.

• 

  Figure 7. Recombination analysis of the complete genome of PDCoV strain CHN-SC2015. Potential recombination events were identified using Recombination Detection Program 4 (RDP4) and then examined using similarity plots and bootstrap analysis in Simplot 3.5.1. in the case of p-values less than 10−8. Major and minor parents are represented by red and black, respectively. The green arrows indicate the recombination breakpoints.

• 

  Figure 8. Diarrhea scores of the challenged piglets. The five-old-day piglets were inoculated orally with PDCoV CHN-SC2015 passage 12 and DMEM. Clinical signs were observed daily, and diarrhea was scored for challenged piglets as followed: 0 = normal feces,1 = soft, 2 = liquid with some solid content,3 = watery with no solid content.

• 

  Figure 9. Pathology of PDCoV strain CHN-SC2015 in five-day-old piglets. The images are representative of experimental and control piglets. Clinical observation of a piglet challenged with PDCoV CHN-SC2015 passage 12 (A) or DMEM (D) at 5 dpi. Macroscopic lesions of gastrointestinal tissue from the infected piglet (B) and uninfected (E) at 5 dpi. Macroscopic lesions of the stomach from the infected piglet (C,F) at 5 dpi.

• 

  Figure 10. Histological analysis of gastrointestinal sections and stomach from piglets infected with PDCoV CHN-SC2015. The tissues from the piglets incubated with CHN-SC2015 or DMEM were collected at 5 DPI and were stained with hematoxylin and eosin. The different colors of arrows and boxes indicated the typical histological lesion in the different tissues. Scale bars are also shown in each picture.

• 

  Figure 11. PDCoV N-protein was detected in sections of jejunum by immunofluorescence staining. Jejunum samples from five-old-day piglets infected with PDCoV CHN-SC2015 were fixed and then stained with the rabbit anti-PDCoV N protein monoclonal antibody and FITC-conjugated goat anti-rabbit IgG (green); the nucleus was counterstained with DAPI (blue). Magnification = 2×.

• 

  Figure 12. Viral shedding and distribution in the challenged piglets. (A) The five-day-old piglets were infected with PDCoV CHN-SC2015 and viral shed in feces was quantitated daily from two or three of the challenged piglets. (B) The five-day-old piglets were infected with PDCoV CHN-SC2015 and distribution of CHN-SC2015 in the intestines and internal organs was assayed after necropsy.