Viral genome enrichment:
The cell culture supernatant was harvested after three freeze-thaw
cycles and centrifuged at 4000 RPM for 10 min. The clarified culture
supernatants harvested at third passage were tested by real time PCR
(Fernández-Pinero et al., 2013) and used for further process. The viral
genome enrichment was carried out as described earlier, with minor
modifications (Chapman et al., 2011). Briefly, cell supernatant
containing virus was centrifuged at 118000 xg for 1h at 4°C. The pellet
resuspended in equilibrating buffer (NaCl 10mM, Tris-HCl 10mM, EDTA 1mM)
was treated with 75 units of DNase I for 1h at 37°C. The virus was
pelleted over 20% sucrose in equilibrating buffer at 62000xg for 1.5 h.
The pellet was treated with RNAse (40 µg/mL), proteinase K (200 µg/mL),
and 1% sodium dodecyl sulfateand incubated for 18 h at 37°C. The
genomic DNA was extracted from the enriched pellet by phenol-chloroform
method (Sambrook and Russell, 2006).