Generation and Characterization of a Replication-Competent Human Adenovirus Type 55 Encoding EGFP

Figures

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  Figure 1. Schematic representation of the construction of pAd55-FL and pAd55-dE3-EGFP. AmpR: ampicillin resistance gene; CMV: CMV promotor; E3: E3 gene of HAdV-55; E3L and E3R: left and right homologous arm to the E3 gene; EGFP: enhanced green fluorescence protein; gAd55: genome of HAdV-55 virus; Hind III, Pme I, EcoR I, Avr II, Asis I and Nde I represent restriction sites; HLA and HRA: left and right homologous arm to the virus genome of HAdV-55; ori: pBR322 origin of replication; pEGFP-N1: vector for fusing EGFP to the C-terminus of a partner protein; pAdBone-LRA: recombinant pAdBone with HAL and HAR homologous arm regions; pAd55-FL: plasmid harboring the full-length cDNA of HAdV-55 virus genome; rAd55-dE3-EGFP: recued HAdV-55 virus encoding EGFP without E3 gene.

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  Figure 2. Restriction enzyme analysis and PCR confirmation of recombinant plasmids pAd55-FL and pAd55-dE3-EGFP. (a) In silico restriction map generated by SnapGene. Lane 1: DL 15,000 DNA marker (TAKARA, China); Lane 2: pAd55-FL/Nde I; Lane 3: pAd55-dE3-EGFP/Nde I; (b) Restriction map of pAd5-FL and pAD55-dE3-EGFP. Lane 4: DL 15,000 DNA marker (TAKARA, China); Lane 5: pAd55-FL/Nde I; Lane 6: pAd55-dE3-EGFP/Nde I; (c) PCR amplification with E3D-Det-F2/AVRII-R. Lane 7: DL5000 DNA marker (TAKARA, China); Lane 8: PCR amplification with E3D-Det-F2/AVRII-R of pAd55-dE3-EGFP; Lane 9: PCR amplification with E3D-Det-F2/AVRII-R of pAd55-FL.

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  Figure 3. rAd55 and rAd55-dE3-EGFP rescue. (a) CPE and EGFP expression of rAd55 and rAd55-dE3-EGFP at 7 days post-transfection, scale bar: 50 μm. (b) EGFP expression of rAd55-dE3-EGFP at indicated time. Visualized by fluorescence microscopy under fluorescent light and visible light, scale bar: 100 µm.

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  Figure 4. Stability testing of rAd55-dE3-EGFP. (a) EGFP expression and CPE in cells infected with P1, P6, P12, and P20 viruses under fluorescent or visible light. (b) The EGFP gene identified by nucleic acid electrophoresis. Scale bar: 200 um.

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  Figure 5. Growth kinetics characteristics of rAd55 and rAd55-dE3-EGFP. (a) The viral growth of HAdV-55, rAd55, and rAd55-dE3-EGFP viruses in A549 cells. (b) Viral DNA replication of HAdV-55, rAd55, and rAd55-dE3-EGFP viruses in A549 cells. (c) Relative fluorescence of rAd55-dE3-EGFP virus.

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  Figure 6. Expression pattern of EGFP in virus-infected A549 cells. A549 cells were infected with HAdV-55, rAd55, and rAd55-dE3-EGFP viruses. Red: hexon, blue: DAPI nuclear stain, green: EGFP. Scale bar: 50 µm.

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  Figure 7. The validation of MN assays based on rAd55-dE3-EGFP. (a) Mouse antisera nAb titers were determined using CPE-based MN assays (red dots) and eGFP-based MN assays (blue dots), respectively. (b) Serial dilutions of HAdV-55-seropositive sera with nAb titers <200, 200–500, 500–1500, and >1500 were incubated with rAd55-dE3-EGFP and then infected A549 cells. Green: eGFP. Scale bar: 100 µm.

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  Figure 8. Antiviral activities of cidofovir and brincidofovir on rAd55-dE3-EGFP viruses. Green: eGFP. Scale bar: 100 µm.

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  Figure 9. Negative-stained EM images of adenoviruses. (a) HAdV-55; (b) rAd55; (c) rAd55-dE3-EGFP. Scale bar: 200 nm.