Fig. 3.

Increased viral titers (A) and pro-inflammatory (IL-6) cytokine levels (B) in the cell culture supernatants of IPEC-J2 cells during the 5^th serial passage of PDCoV OH-FD22. An estimated MOI of 0.1 of an OH-FD22-P8-I-P4 (passage 4 in IPEC-J2 cells) was inoculated in duplicate onto IPEC-J2 cell monolayers (2 × T75 flasks) for the 5^th passage, alongside a PDCoV-uninoculated, trypsin-treated negative control (1 × T75 flask). After virus adsorption, the wash step was omitted, and 15 ml of trypsin supplemented cell culture medium was added. The cell culture supernatants (1 ml per time-point) were harvested at PIHs 7, 24, 31, 48, 72, and 96. The cell culture supernatants harvested were centrifuged (1000 × g for 10 min at 4 °C) and stored at −70 °C until virus quantification by a TCID₅₀ assay (A) and cytokine quantification by ELISA (B). The samples were tested in duplicate, and cytokine levels or infectious virus titers were expressed as the mean values.