5.2. Virus replication and entry inhibition assay

To evaluate the replication kinetics of clinical and ATCC isolates of HCoV-OC43, 10⁸ copies of virus were inoculated onto HBTE-ALI cultures or HCT-8 cells. After incubation for 4 h, the cells were washed twice and incubated at 34 °C. After 1 day of incubation, the cells were collected and used for analysis of virus entry. Cell supernatants or cell washings obtained after 3 days of incubation were used to evaluate virus replication as described below. The amounts of replicated virus were expressed as RNA copy numbers in cell supernatants or cell washings minus those remaining in cell washings after virus adsorption.

For the entry inhibition assay using HBTE-ALI cultures, 10⁷ to 10⁸ copies of viruses were inoculated onto cells that had been pre-treated with entry inhibitors [EST (23,25)trans-epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester) (330005: Calbiochem, San Diego, CA, USA)] or camostat mesylate (3193: Tocris Bioscience, Bristol, UK) for 1 h. After incubation for 4 h in the presence of inhibitors, cells were washed twice and incubated at 34 °C. After 1 day of incubation, the cells were collected. Cell viability was determined using CellTiter-Glo and a GloMAX luminometer (Promega, Madison, WI, USA). Total cellular RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan) with ribonucleic acid from baker's yeast (R6750, Sigma) as carrier RNA. To assess virus entry, subgenomic RNA containing nucleocapsid protein was amplified by real-time RT-PCR using the following primer and probe sets: For HCoV-OC43, forward, 5′- TCACTGATCTCTTGTTAGATCTTTTTGTA-3′; reverse, 5′- TTTGCTTGGGTTGAGCTCTT-3′; and probe, 5′- GGCCGATCAGTCCGACCAGTTT-3′ (FAM-labeled): for HCoV-HKU1, forward, 5′- TATCAGCTTACGATCTCTTGTCAGA-3′; reverse, 5′- TGGTCAGCCCAAGAAGTTTT-3′; and probe, 5′- GAAGCTCCTCTGGAAATCGTTCAGGA-3′ (FAM-labeled). RNA copy numbers were calculated from standard curves obtained with the aid of control RNA templates.

The data were statistically analyzed using the unpaired t-test and are presented as log RNA copies per well.