title: Addis Ababa virus preparation from raw sewage
organism: metagenomes
description: Two samples (10L each) of untreated wastewater  were obtained from the wastewater treatment plant in Addis Ababa, Egypt in June 2009 and processed on site. Virions were concentrated from the wastewater sample by organic flocculation based on the procedure previously described (Calgua et al., J. Virol Methods, 2008, vol 153, p.79-83). Briefly, 100ml of pre-flocculated skim milk solution (pH 3.5) was added to 10 L acidified raw sewage (pH 3.5) and mixed for 8 hours. Flocculants were allowed to settle and then centrifuged. The flocculated viral concentrate was resuspended in 15 ml phosphate buffer and then eluted in 30 mL of 0.25 M glycine pH 9.5 for 45 min at 4* C by slow agitation with vortexing. Suspended solids were separated by low speed centrifugation at 7,500 g for 30 min at 4* C and the high pH of the supernatant was stabilized by adding 20 mL of 2X PBS. Virions present in the supernatant were concentrated by ultracentrifugation at 100,000 g for 1 hr at 4* C and resuspended in phosphate buffer (1:2 v/v of 0.2 M Na2HPO4 and 0.2 M NaH2PO4).