US10 Protein Is Crucial but not Indispensable for Duck Enteritis Virus Infection in Vitro

Figures

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  Figure 1. Schematic diagrams of US10 deletion. (a) The DEV genome consists of unique long (UL), unique short (US), internal repeat (IR) and terminal repeat (TR) regions. (b) Partial US region. (c) US10 ORF is replaced by kanR. (d) KanR deletion by the Flp-FRT recombination system.

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  Figure 2. Construction and identification of parental and recombinant viruses. (A) PCR analysis of recombinant BACs. The BAC DNAs of BAC-G, ΔUS10 + kanR, ΔUS10, US10 + kanR and US10FRT were extracted and amplified by PCR using the indicated primers. (B) Western blot analyses of recombinant viruses. Anti-US10 monoclonal antibody(MAb) and anti-β-actin Mab were used to detect US10 and β-actin in total proteins extracted from mock-, ΔUS10-, BAC-G or US10FRT-infected DEFs. (C) Passage of rescued viruses in DEFs. Enrichment of rescued viruses were obtained by the three times passage after transfection.

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  Figure 3. Viral titer and genome copies in multistep growth kinetics. Confluent DEF cells monolayers were infected with each virus shown at an MOI of 0.02. Viral titer and viral copies of infected supernatant, cells and mixture of cells cultures were determined at the indicated time points by measuring TCID50 on DEF cells. All titrations were carried out in three independent experiment. The titers and copies obtained were averaged, and the standard error of the mean was calculated each time point. (A) Viral titer in cytoplasm samples. (B) Viral titer in supernatant samples. (C) Total viral titer and genome copies. (D) Genome/TCID50 ratios.

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  Figure 4. Viral titer and genome copies in one-step growth kinetics. Confluent DEF cells monolayers were infected with each virus shown at an MOI of 2. Viral titer and viral copies of infected cells were determined at the indicated time points by measuring TCID50 on DEF cells. All titrations were carried out in three independent experiment. The titers and copies obtained were averaged, and the standard error of the mean was calculated each time point. (A) Viral copies. (B) Viral titer. (C) Viral copies and titer. (D) Genome/TCID50 ratios.

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  Figure 5. Transcription level of immune-related genes in virus-infected DEFs. Total RNA was collected and extracted at the indicated time points for reverse transcription, and cDNAs were used for qPCR detection. The relative expression levels of immune-related genes were calculated by the 2−ΔΔCt method. Statistical significance was analysed using Student’s t test and considered significant as follows: *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001.