Establishment of an infectious clone of the porcine transmissible gastroenteritis virus and a study on the location and function of accessory protein 3

Figures

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  Figure 1. Construction of the infectious cDNA clones of TH-98. (A) The cDNA segments of TGEV TH-98 were cloned into the BAC plasmid to assemble the full-length cDNA clones. (B) The TH-98 genome was divided into six segments (A, B, C, D, E, and F) and ligated into a pBlunt-simple vector. Electrophoresis identification of the pBlunt-simple vectors was conducted. (C) Construction order of the TGEV reverse genetic system.

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  Figure 2. Recovery and identification of the rescued virus. (A) Indirect immunofluorescence staining of the rescued virus with mouse monoclonal antibody to TGEV N protein. (B) The expression of the TGEV N protein in ST cells infected with the rescued virus, WT TGEV, or mock-infected was analyzed by Western blotting using mouse monoclonal antibody to TGEV N protein. (C) Detection of the genetic marker by sequencing of the rescued virus. (D) Comparison of plaque sizes and morphology of WT TGEV and the infectious clone-derived viruses on ST cells at 72 h post-infection. (E) Growth curve of rTH-98 and TH-98 in ST cells (MOI=0.01).

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  Figure 3. Construction of the infectious cDNA clones of TH98-Δ3-COE-HA and the rescued recombinant virus. (A) Model for the construction of the TH98-Δ3-COE-HA infectious clones. (B) Electrophoresis detection of pBAC-TH98-Δ3-COE-HA by bacterial PCR. (C) Schematic map of pBAC-TH98-Δ3-COE-HA.

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  Figure 4. Recovery and identification of the recombinant virus. (A) Indirect immunofluorescence staining of rescued virus with mouse monoclonal antibody to TGEV N protein (green) and rabbit polyclonal antibody to HA protein (red). (B) Comparison of plaque sizes and morphology of WT TGEV and the rescued virus on ST cells at 72 h post-infection. (C) Identification of several passages of the recombinant virus genetic marker. (D) Multi-step growth curve of WT and rescued viruses on ST cells at an MOI of 0.01. (E) Complementation assay of rTH-98-Δ3-COE-HA infection in ST cells transiently expressing ORF3a and ORF3b. Values are the mean ± SD of three independent tests. *** represents p <0.001 between different groups.

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  Figure 5. Localization of ORF3 in TGEV. (A) Observation of the recombinant virus by immunoelectron microscopy. (B) The expression of the TGEV N protein in the recombinant virus cell cultures, purified recombinant virus, or mock-infected was analyzed by Western blotting using a mouse monoclonal antibody to TGEV N protein. M, prestained protein marker; 1, mock; 2, purified recombinant virus; 3, unpurified recombinant virus. (C) The expression of the COE-HA protein in recombinant virus cell cultures, purified recombinant virus, or mock-infected was analyzed by Western blotting using a rabbit polyclonal antibody to the HA protein. M, prestained protein marker; 1, mock; 2, purified recombinant virus; 3, unpurified recombinant virus.

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  Figure 6. Recombinant virus immunogenicity analysis. (A) mRNA expression levels of IL-1β, IL-6, IL-8, and TNF-α in infected rescue virus IPEC-J2 cells. Values are the mean ± SD of three independent tests. * represents p<0.05; *** represents p <0.001 between different groups. The changes in anti-TGEV-N protein-specific IgG antibody levels in immunized mice. (B) Recombinant virus cell cultures and purified recombinant virus. (C) Recombinant virus and parental rescued virus cell cultures. The changes in anti-PEDV-COE protein-specific IgG antibody levels in immunized mice. Values are the mean ± SD of three independent tests. *** indicates a significant difference between the rTH-98-Δ3-COE-HA cell culture group and PBS group (P<0.001). ### indicates a significant difference between the rTH-98-Δ3-COE-HA purified particle group and PBS group (P<0.001). (D) Recombinant virus cell cultures and purified recombinant virus. (E) Recombinant virus and parental rescued virus cell cultures. Values are the mean ± SD of three independent tests. *** indicates a significant difference between the rTH-98-Δ3-COE-HA cell culture group and PBS group (P<0.001). ### indicates a significant difference between the…