Quantifying FCoV Viral Loads in FCWF-4 Cells

The number of copies of viral genomes in fcwf-4 cells infected with FCoV was determined using RT-qPCR [11 (link)]. In brief, the medium was removed 20 h after infection, and RNA from the cells was prepared using Isogen (Nippongene), according to the manufacturer’s protocol. Total RNA was reverse transcribed using the PrimeScript RT-PCR Kit (Perfect Real Time; Takara Bio). Viral cDNAs were quantified using qPCR with primers specific for the gene encoding FCoV-N (forward, 5′-TGGCCACACAGGGACAAC-3′; reverse, 5′-AGAACGACCACGTCTTTTGGAA-3′) and the TaqMan probe (FAM-TTCATCTCCCCAGTTGACG-BHQ-1). Reaction mixtures were prepared according to the manufacturer’s protocol using Premix EXTaq (Takara Bio), and sequences were amplified using the 7500 Sequence Detection System (Life Technologies). The viral RNA copy number was normalized to that of the feline gene encoding feline β2-microglobulin (β2M) (GenBank accession no. NM_001009876) using qPCR with forward (5′-CGCGTTTTGTGGTCTTGGTCTTGGT-3′) and reverse (5′-AAACCTGAACCTTTGGAGAATGC-3′) primers specific for β2M. The TaqMan probe, TAMRA-CGGACTGCTCTATCTGTCCCACCTGGA-BHQ-2, was used to detect β2M mRNA.