sybr green plus reagent kit  (TaKaRa)

Journal: Poultry Science

Article Title: Pioneering discovery: US3-phosphorylated sites on VP22 orchestrate duck plague virus release and pathogenicity

doi: 10.1016/j.psj.2025.105908

Figure Lengend Snippet: The Role of VP22 phosphorylation in the in vitro viral life cycle. A. Generation of recombinant viruses carrying VP22 phosphorylation site mutations. Infectious clone plasmids containing VP22 phosphorylation site mutants (pDPV-VP22 QA ) or the corresponding revertant (pDPV-VP22 QA -Rev) were transfected into DEF cells. The recombinant viruses DPV-VP22 QA (phosphorylation-deficient virus) and DPV-VP22 QA -Rev (revertant virus) were obtained through plaque purification and lacked fluorescent markers. B. Multistep growth curve analysis of the recombinant viruses. DEF cells were infected with the virus at an MOI of 0.1, and samples were collected at 24, 48, 72, and 96 hpi for viral titer determination. The data represent the mean viral titers from three independent experiments, with standard deviations included. C-E. Quantitative PCR (qPCR) was performed to determine the impact of VP22 phosphorylation deficiency on viral adsorption (C), invasion efficiency (D), and viral DNA replication (E) by quantifying viral genome copy numbers. F. Viral assembly. Transmission electron microscopy (TEM) was used to visualize the secondary envelopment process of viral particles. G. Viral particle release capacity. Supernatants collected at different time points were analyzed using TCID₅₀ assays to assess the impact of VP22 phosphorylation deficiency on viral particle release efficiency (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed using SYBR qPCR Premix (Cat No: RR047A; Takara, Japan).

Techniques: Phospho-proteomics, In Vitro, Recombinant, Transfection, Virus, Purification, Infection, Real-time Polymerase Chain Reaction, Adsorption, Transmission Assay, Electron Microscopy

Journal: Poultry Science

Article Title: Pioneering discovery: US3-phosphorylated sites on VP22 orchestrate duck plague virus release and pathogenicity

doi: 10.1016/j.psj.2025.105908

Figure Lengend Snippet: Accumulation of most viral gene mRNAs requires VP22 phosphorylation. A. Quantification of viral gene expression following infection. DEF cells were infected with 1 MOI of the virus, and total RNA was extracted at 24 hpi. RT-qPCR was performed to assess the expression levels of target genes, with the data normalized using GraphPad Prism 9. The relative gene expression levels were calculated using the 2 -ΔΔCt method. B. Dynamic expression analysis of viral genes. RT-qPCR was used to analyze the expression patterns of viral genes at different transcriptional stages: immediate-early (UL54, ICP4, US1), early (US3, UL23, UL29), and late (UL41, UL47, UL48). 18S rRNA served as an internal reference for normalization. Relative expression levels in the DPV CHv50-infected group were calculated using the 2 -ΔΔCt method, and the data were presented as the fold change relative to the DPV CHv50 group. C. Protein-level analysis of viral gene expression. DEF cells were infected with virus at an MOI of 1, and protein samples were collected at 24 hpi for detection by Western blotting.

Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed using SYBR qPCR Premix (Cat No: RR047A; Takara, Japan).

Techniques: Phospho-proteomics, Gene Expression, Infection, Virus, Quantitative RT-PCR, Expressing, Western Blot