2.5 MTN for BCoV
MTN was performed by modifying the protocols previously described (Alenius et al., 1991; Tsunemitsu et al., 1991). Advanced RPMI 1640 (1×; Gibco Ref 12633-012) was used in this protocol.
Samples were heat-inactivated by incubation at 56°C for 30 min, and then 50 μL per well of serum samples in duplicates were subjected to 2-fold serial dilutions from 1:20 through 1:320, and from 1:4 through 1:64 in 50 μL culture media.
Each dilution was mixed with an equal volume of a viral suspension containing 100 TCID50 of BCoV strain Nebraska, which was kindly provided by Dr. S. Reiche (Friedrich-Loeffler-Institut, Insel Reims, Germany) (Ulrich et al., 2020), and incubated for 1 hour at 37 C. Finally, 100 μL of HRT-18 (a cell line derived from human rectal adenocarcinoma) cell suspension, adjusted with maintenance medium to 105 cells/mL, was added to each well and cultured in 5% CO2 at 37°C. After 6–7 days of incubation, the cells were fixed and stained with a 0.1% crystal violet solution in 5% PFA for 30 min to detect cytopathic effects. The neutralisation endpoint titre was determined as the endpoint serum dilution that inhibited BCoV-induced CPE in at least two out of three parallel wells. The complete inhibition of virus propagation in an individual well was accepted as a positive result (Tuncer & Yeşilbağ, 2015).