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).