Quantification of Viral DNA Copies

Viral or total cellular DNA was extracted with a DNAeasy kit (Qiagen, Courtaboeuf, France) and the DNA concentration and purity were determined using a Nanodrop spectrophotometer. Real-time PCR was performed using different dilutions of the purified total DNA and the QuantiTect SYBR green PCR master (Qiagen, Courtaboeuf, France) with a LightCycler 96 thermal cycler (Roche Applied Science, Meylan, France), using the E11L primers previously described [51 (link)]. For relative quantification, the 2^−ΔΔCt method was used [52 (link)]. The quantification of copies per millilitre was performed against a standard curve of WR virions. The number of viral DNA copies was evaluated either by Nanodrop (1 ng DNA viral DNA = 4.74 × 10⁶ copies) or directly by the qPCR assay.