3.2 Whole genome sequencing and bioinformatics
NGS processing yielded SARS-CoV-2 sequences for both C2 and C3 in oropharyngeal and fecal swabs, as well as in the oropharyngeal swab from the owner (Table 2). Multiple sequence alignment revealed an absolute overlap of amino-acid sequences. Only one difference among 5 samples was observed; a synonymous T-to-C SNP in position 24544 in the oropharyngeal sample of C3, at a ratio of 0.57 (Table 3). A statistically significant imbalance of quasispecies abundance at the aforementioned nucleotide position was observed among samples. Specifically, the oropharyngeal swab of C3 showed an increased C variant ratio compared to the fecal swab of the same cat (Odds ratio: 11.96, p <0.0001), since the presence of this variant in the fecal sample of this animal was also revealed, but the respective ratio was low (0.10). This variant was also identified in the owner’s pharyngeal swab at an even lower ratio (0.07). Comparison with the oropharyngeal swab of C3 revealed an odds ratio of 17.72 (p <0.0001). On the contrary, this variant was not identified neither in the oropharyngeal swab, nor the fecal swab from C2 and comparison with the oropharyngeal swab of C3 revealed an odds ratio of 1061.57 (p <0.0001). Two other sites (nucleotide positions 22685 and 23335) showed within-sample T/C variability in at least one additional sample (Table 3).
Comparison of the sequence obtained from the oropharyngeal swab of C3 against the “Wuhan-Hu-1” reference isolate indicated 17 SNPs (Table 4). Eleven of the polymorphisms resulted in an amino-acid substitution at the respective protein. Out of the 17 SNPs, 4 occurred in sequential nucleotides (28881-28884), with 3 of them being located at codon 203 of N and resulting in the N-R203K amino-acid substitution. The fourth SNP is associated with the N-G204L amino-acid substitution. In total, 6 out of 9 amino-acid substitutions were located at the structural/accessory protein-encoding moiety of the genome (Figure 2). Phylogenetic analysis using the GISAID and NextStrain platforms showed that the strains sequenced herein are placed in the GISAID GR clade, in the NextStrain 20B clade, and in the B1.1 lineage, according to the PANGOLIN SARS-CoV-2 lineage assigner interface (Figure 3).