Figure 2

ORF I215L encodes an early viral protein that accumulates in viral factories. (A) I215L transcripts were detected from 2 hpi onwards showing a maximum peak at 16 hpi. The mRNA levels of CP204L (vp32) and B646L (vp72) were measured in parallel and used as controls of early and late viral gene expression, respectively. Results are shown as mean ± standard error by dividing the number of transcripts of each viral gene by the number of Cyclophilin A mRNA molecules (housekeeping gene), obtained from three independent experiments run in duplicate. (B) ASFV-I215L gene encodes for an early protein detectable from 4hpi onwards, with expressing levels unchanged by the AraC treatment. Vero cells infected with ASFV/Ba71V isolate (MOI of 5) and harvested at the indicated time points. The cytosine arabinoside treatment (AraC, 50 µg/ml) was performed after the initial viral adsorption period (1 hour) and cells were collected at 20 hpi. (C) Vero-infected cells (MOI = 2) were fixed (4, 8, 12 and 16 hpi), immunostained and analyzed by fluorescent microscopy. pI215L was detected from 8 hpi onwards, being recruited to viral factories, co-localizing with other viral proteins (e.g. pA104R and VP72, data not shown) and showing a faint distribution pattern in cell nucleus. In the merged images, pI215L, ASFV and DAPI staining is shown in green, red and blue, respectively. Representative images of at least three independent experiments are shown.