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).