Construction of coxsackievirus B5 viruses with luciferase reporters and their applications in vitro and in vivo

Figures

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  Fig. 1. Generation and characterization of CVB5 infectious clone. A Diagram of CVB5 genome structure and cloning strategy of the full-length infectious clone of CVB5. B CPE caused by pCVB5 or rCVB5. RD cells infected with indicated CVB5 (MOI ​= ​0.1) were observed for CPE at indicated time points. Scale bar, 100 ​μm. C RD cells infected with pCVB5 or rCVB5 were immunoblotted at indicated time points with anti-VP3 antibody. β-actin was used as a loading control. D Uninfected or RD cells infected with pCVB5 or rCVB5 (MOI ​= ​1) were immunostained with anti-dsRNA(J2) antibody at 12 ​h.p.i. Nuclei were counterstained by DAPI. Scale bar, 10 ​μm. E Plaque assay of RD cells infected with serial dilutions of pCVB5 or rCVB5. RD cells (1.2 ​× ​105 ​cells) were infected with ten-fold diluted pCVB5 or rCVB5 with staring MOI of 0.2. The plaque assay was performed at day 3 post infection. F Viral RNAs from RD cells (1.2 ​× ​105 ​cells) infected with pCVB5 or rCVB5 (MOI ​= ​0.1) were quantitated by q-RT-PCR at various time points. Data presented as mean ​± ​SD from three repeat experiments.

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  Fig. 2. Generation and characterization of NanoLuc-CVB5. A Diagram showing the gene structure of the NanoLuc-CVB5. NanoLuc luciferase reporter gene as well as 3C cleavage site were inserted immediately upstream of VP4. B RD cells (1.2 ​× ​105 ​cells) transfected with 0.5 ​μg viral RNAs from rCVB5 or NanoLuc-CVB5 were analyzed for luminescence at indicated time points. C RD cells (1.2 ​× ​105 ​cells) were infected with different passages of reporter virus (MOI ​= ​0.1). Luciferase activity was determined at 24 ​h.p.i. D qRT-PCR analysis of NanoLuc gene copy numbers from RD cells infected with different passages of reporter virus. E Uninfected or RD cells (1.2 ​× ​105 ​cells) infected with indicated virus (MOI ​= ​1) were immunostained with anti-dsRNA(J2) antibody at 12 ​h.p.i. Nuclei were counterstained by DAPI. Scale bar, 10 ​μm. F RD cells infected with NanoLuc-CVB5 were immunoblotted at indicated time points with anti-VP3 antibody. β-actin was used as a loading control. G RD cells (1.2 ​× ​105 ​cells) infected with indicated rCVB5 or NanoLuc-CVB5 (MOI ​= ​0.1) were observed for CPE at indicated time points. Scale bar, 100 ​μm. H Plaque assay of RD cells infected with serial dilutions…

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  Fig. 3. Use of NanoLuc-CVB5 for antiviral screening. RD cells (1.5 ​× ​104) infected with NanoLuc-CVB5 reporter virus (MOI ​= ​10) were treated with indicated concentrations of GuHCl (A) or PI4KIIIβIN-9 (B) for 24 ​h before NanoLuc luciferase was determined. Cellular ATP content was determined with Cell Titer Glo to indicate the toxicity of tested compound. Data presented as mean ​± ​SD from three repeat experiments. C Z-factor measurement of NanoLuc-CVB5 infection inhibition assay. The dotted lines represent three standard deviations above and below the mean of DMSO. The mean, standard deviation (SD) of DMSO or PI4KIIIβ-IN-9 treated cells from one representative of three repeat experiments were indicated in the table.

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  Fig. 4. Construction and characterization of subgenomic replicon of CVB5. A Diagram showing the structure of the CVB5 subgenomic replicons. Rluc-Neo fusion gene as well as 3C cleavage site were used to replace VP4, VP2 and part of VP3 gene. VP3′, partial VP3. Asterisk indicates the GND mutation in the 3D catalytic site. B The luminescence of RD cells (1.2 ​× ​105) transfected with 0.5 ​μg SGR-CVB5 or SGR-CVB5 (GND) were assayed at indicated time points. C Left panel: RD cells (1.2 ​× ​105) were stably transduced with control or DAF sgRNA lentivirus and infected with CVB5 (MOI ​= ​0.1). DAF and VP3 expression were determined 24 ​h.p.i with immunoblotting. Right panel: RD cells were stably transduced with control or PI4KB shRNA lentivirus and infected with CVB5. PI4KB and VP3 expression was determined 24 ​h.p.i with immunoblotting. D RD cells (1.2 ​× ​105) were stably transduced with DAF sgRNA (left panel) or PI4KB shRNA (right panel) lentivirus and infected with NanoLuc-CVB5 (MOI ​= ​0.1). The luciferase activity was determined at 24 ​h.p.i. E RD cells (1.2 ​× ​105) stably transduced with DAF sgRNA or PI4KB shRNA were either uninfected or infected with NanoLuc-CVB5 (MOI ​= ​0.1) and cell…

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  Fig. 5. In vivo imaging of NanoLuc-CVB5 infected mice. A Survival rate of CVB5 infected mice. 3-Day old mice (n ​= ​7) were inoculated with 2 ​× ​1010 TCID50 pCVB5 or NanoLuc-CVB5 and monitored for 7 days. B Viral titers from organs of pCVB5 or NanoLuc-CVB5 infected mice were quantified and expressed as TCID50. C Viral RNAs from organs of CVB5 or NanoLuc-CVB5 infected mice were quantified by qRT-PCR and expressed as viral copy number per mg tissue. D Indicated tissues from NanoLuc-CVB5 infected mice were homogenized and subjected to immunoblotting. VP3 expression for CVB5 infection was determined and GAPDH was detected as loading control. E Bioluminescence measurement of mice infected with parental CVB5 (upper right panels) or NanoLuc-CVB5 (lower panels) using furimazine substrate. PBS injected mice was shown as control (upper left panels). Mice infected with virus for 24 and 48 ​h were injected with furimazine. Four or eight minutes after injection, images were taken with VISQUE™ InVivo Smart-LF. Data presented as mean ​± ​SD from three repeat experiments.

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