Influence of different treatment conditions of resveratrol on DEV infection.

<p>DEFs infected with DEV (MOI  = 0.1) were treated with resveratrol in various mode of actions. (1) Virus inactivation: DEV was treated with drug for 1 h at 37°C before infection. The virus and drug mixture was then added to cells for 1 h at 37°C. The media was removed and replaced with drug-free media. DEV suspended without drug was used as control (effect of temperature). (2) Inhibition of virus attachment: DEFs were infected in media containing the drug and virus for 1 h at 37°C, and then the mixture was removed. The cells were overlaid with drug-free media. (3) Pre-treatment effect: DEFs were pre-treatment with drug for 1 h at 37°C. And then DEV was added to the cells after resveratrol solution removal. After 1 h 37°C, the inoculum was removed and replaced with drug-free media. (4) Intracellular inhibition: after removing the unabsorbed virus, the media containing the resveratrol was added to the DEFs. At 48 h p.i., the total DNA was extracted from DEV-infected DEFs and copies of DEV were evaluated by the real-time FQ-PCR assay. Values are means ± SD (n = 3). Significance: different capital letters represent extremely significant differences among groups (P<0.01, n = 3).</p

Inhibition effects of resveratrol on DEV <i>in vitro</i>^a.

a<p>The inhibition effects on DEV were evaluated by MTT assay.</p>b<p>Inhibition concentration 50% (IC₅₀): concentration required to inhibit DEV at 72 h post-infection by 50%.(n = 3).</p>c<p>Cytotoxic concentration 50% (CC₅₀) concentration required to reduce cell viability by 50%. (n = 3).</p>d<p>SI: Selectivity index is defined as the radio of CC₅₀ to IC₅₀.</p

Standard curve of real-time FQ-PCR amplification.

<p>Serial 10-fold dilutions of standard DEV DNA from 5.25×10¹⁰ to 10² copies were amplified in this process. Amplification efficiency (E) was 99.8%. The standard curve equations was <i>C</i>t = −3.328×lg [virus copies/5.25]+40.138 (R² = 0.997).</p

Inhibition of viral replication studied by TEM.

<p>DEFs were infected (C, D, E and F, MOI  = 0.1) or infected (A and B) with DEV in the absence (A, B, C and D) or presence (E and F) of 31.25 μg/mL of resveratrol. At 48 h p.i., the replication of DEV in DEFs were studied by TEM. Pictures were taken using a Tecnai G2 F20 electron microscope (FEI, USA). (A and B): normal appearance of cell nucleus and cytoplasm. (C): DEFs infected with DEV. ↗: viral capsids in nucleus; →: a massive accumulation of virus nucleic acids in nucleus; <>\scale 80%\raster="rg1"<>: mature viral particles in nucleus; white arrows ↗: chromatin margination; white arrows →: nucleolus membrane burst). (D): DEFs infected with DEV. (↗: Maturating and mature particles in cytoplasmatic vacuoles; →: Maturating particles in nucleus; white arrows ↗: chromatin margination; white arrows →: an extensive vacuoles degeneration). (E and F): Infected DEFs treatment with resveratrol. (→: few viral particles were observed in nucleus and cytoplasm). n: nucleus; c: cytoplasm; ec: extracellular space.</p

Growth curves of DEV in DEFs in the presence or absence of reveratrol.

<p>The DEFs were firstly infected with DEV at MOI of 1 and test resveratrol solution for 1 h at 37°C. DEV proliferated in the DEFs in the presence or absence of reveratrol within 72 h. The total DNA was extracted at the indicated time points (2, 4, 6, 8, 10, 12, 24, 48, and 72 h p.i.) and copies of DEV were detected via real-time FQ-PCR assay. Copies of DEV were calculated according to the standard curve equations: <i>C</i>t = −3.328×lg [virus copies/5.25]+40.138 (R² = 0.997). Values are means ± SD (n = 3).</p