3.2 Amplification and sequence analysis of FCoV S gene
The S protein, encoded by the FCoV S gene, is considered the primary viral regulator in host cell entry; it possesses a receptor-binding domain, while mutations in amino acids of the FCoV S protein are associated with the conversion of FECV to FIPV (Bosch et al., 2003; Jaimes & Whittaker, 2018; Millet & Whittaker, 2015). To further understand the molecular characteristics and genetic variation of the FCoV S gene in southwest China, 18 type I FCoV and five type II FCoV strains were selected to amplify the whole gene of FCoV S according to various years, living environments, and regions. The 18 type I FCoV S genes were 4395–4419 bp in length, each encoding a protein of 1465–1473 aa residues (GenBank accession number MW316830-MW316847). Five type II FCoV S genes were 4353–4362 bp in length, each encoding a protein of 1451–1454 aa residues (GenBank accession number MW316848-MW316852).
A heatmap was constructed using HemI software based on the sequence homology of type I FCoV with classical strain Black and type II FCoV with classical strain 79–1146, respectively (Figure 1). The results showed that the NTD variation in S1 subunit was the highest. The NTD homology between 23 strains and the reference strain was 73.6%–80.3%; the S2 subunit was relatively conserved, the S2 subunit homology between 23 strains and the reference strain was 93.9%–94.2%. The 18 type I FCoV strains shared 85.9%–100% nucleotide sequence identities and 89.4%–100% aa identities. They shared 87.7%–93.8% nucleotide sequence identity and 87.9%–93.1% aa sequence identities with all 64 of the complete type I FCoV S genes available in the GenBank database. Sequence analysis of 105 FCoV strains showed that seven type I FCoV strains (SMU-CD86, SMU-CD77, SMU-CD9, SMU-CD8, SMU-CD60, SMU-CDF12, SMU-CDF19) identified in this study had two aa insertions in the NTD (159HL160), and strains with this insertion pattern also appeared in strains that have been reported in China and the Netherlands (JN183882, KF530123, KY566209, KY566210, KY566211). The five type II FCoV strains shared 97.4%–98.9% nucleotide sequence identities and 95%–98.8% aa identities between one another and shared 86.9%–97.8% nucleotide sequence identity 84.9%–97.7% aa sequence identities with the 18 type II FCoV reference strains. The type II FCoV strain SMU-CQ18 showed three discontinuous unique aa deletions (209N, 214M/L, 217R) in the NTD. These data showed that the nucleotide of the carbohydrate-bound NTD in the 23 S genes had significant mutations, and there were also insertions and deletions of aa in the NTD.