Assessing the Potential Role of Cats (Felis catus) as Generators of Relevant SARS-CoV-2 Lineages during the Pandemic

Abstract

Several questions regarding the evolution of SARS-CoV-2 remain poorly elucidated. One of these questions is the possible evolutionary impact of SARS-CoV-2 after the infection in domestic animals. In this study, we aimed to evaluate the potential role of cats as generators of relevant SARS-CoV-2 lineages during the pandemic. A total of 105 full-length genome viral sequences obtained from naturally infected cats during the pandemic were evaluated by distinct evolutionary algorithms. Analyses were enhanced, including a set of highly related SARS-CoV-2 sequences recovered from human populations. Our results showed the apparent high susceptibility of cats to the infection SARS-CoV-2 compared with other animal species. Evolutionary analyses indicated that the phylogenomic characteristics displayed by cat populations were influenced by the dominance of specific SARS-CoV-2 genetic groups affecting human populations. However, disparate dN/dS rates at some genes between populations recovered from cats and humans suggested that infection in these two species may suggest a different evolutionary constraint for SARS-CoV-2. Interestingly, the branch selection analysis showed evidence of the potential role of natural selection in the emergence of five distinct cat lineages during the pandemic. Although these lineages were apparently irrelevant to public health during the pandemic, our results suggested that additional studies are needed to understand the role of other animal species in the evolution of SARS-CoV-2 during the pandemic.

Keywords: SARS-CoV-2; cats; evolution; phylogenomics; variants.

Figure 1

General overview of the metadata information associated with the SARS-CoV-2 sequences from cats used in this study. A total of 179 reports related to cat sequences submitted to the GISAID database were considered. Reports included sequences submitted in 2020 (65 sequences), 2021 (103 sequences), and 2022 (11 sequences). (A) Viral sequences recovered from cat infections are described in 29 countries, covering multiple geographic regions. (B) The gender of the cats was reported in half of the reports (n = 90), of which 67.77% came from male cats. (C) The age of the cats ranged from 3 months to up to 18 years. Opened bars represent ages where cat deaths were reported (one report for each age). In this case, age was recorded in just 84 reports.

Figure 2

SARS-CoV-2 viral lineages infect cats (Felis catus) and other animal species. As a result of the analysis of a total of 2515 reports associated with infections of SARS-CoV-2 in multiple animal species, 155 pangolin lineages were identified as responsible for these infections. The proportion of cases within species related to these lineages was analyzed by hierarchical cluster analysis to assess the relationship between lineages that affected Felis catus and other animal species during the pandemic. These species include the dog (Canis lupus familiaris), white-tailed deer (Odocoileus virginianus), lion (Panthera leo), tiger (Panthera tigris), Western gorilla (Gorilla gorilla), American mink (Neogale vison), house mouse (Mus musculus), Syrian hamster (Mesocricetus auratus), and pangolin (Manis javanica). Two main clusters were identified in the analysis. Cluster 2 (green) comprises the 12 more prevalent lineages among different species. The Y-left axe shows the SARS-CoV-2 pangolin lineages included in the analysis. Identification of these lineages included the genetic group/pangolin lineage. The number in the parenthesis next to each lineage indicates the proportion of this specific lineage among the total number of sequences reported in the GISAID database. Black asterisks reflect lineages where mortality was reported in cats. Numbers below animal species in the X-axis show the number of lineages reported during the infection in different species. At the same time, green asterisks indicate species where the prevalence of positive cases was statistically significantly higher in the lineages associated with cluster 2. The spectrum of colors ranges from cream to brown and reflects a low to high proportion of the reports related to specific pangolin lineages, respectively.

Figure 3

Circulation dynamics of the SARS-CoV-2 lineages during the pandemic. (A) The graphic shows the circulation dynamics of different genetic groups of SARS-CoV-2 (based on the GISAID classification) described during the pandemic and their relationship with those circulating in cat populations. The coefficient of determination (R²) analysis is presented between the circulation of specific genetic groups affecting humans and cat populations during the same pandemic year. Asterisks indicate significant correlations. (B) The graphic shows the overall number of clinical reports in cat populations associated with specific genetic groups of SARS-CoV-2 (GISAID classification).

Figure 4

Phylogenomics of SARS-CoV-2 lineages affecting cats. A phylogenetic analysis was conducted using 105 full-length viral sequences retrieved from the GISAID database (specific details about the sequences can be found in Supplementary File S1). The analysis was performed using the maximum likelihood method and the general reversible model. In the tree, labels B.1.497, B.1, B.1.2, B.1.177, AY.69, AY.20, AY.103, AY.44, AY.3, and B.1.1.7 identify clusters associated with the most prevalent SARS-CoV-2 pangolin lineages related to cat infections. The right pangolin lineages are grouped in the context of GISAID genetic group classification. The dN/dS ratios are indicated for the most prevalent cat lineages. Calculations were conducted using the FEL algorithm. Asterisks of different colors reflect specific codon sites at different genes evolving under positive selection in SARS-CoV-2 lineages affecting cats. Information about the codon sites associated with specific asterisk colors is presented in Figure 5 Black bars label lineages carrying premature stop codons at gen ORF8 codon positions 27 and 64.

Figure 5

Comparison between the evolutionary dynamics of SARS-CoV-2 lineages that affected cat and human populations during the pandemic. (A) A total of 105 sequences were evaluated by MEME analysis to infer potentially relevant codon sites evolving under positive selection in SARS-CoV-2 lineages that affected cat populations during the pandemic. Sites under positive selection were identified with asterisks of different colors (see the tree in Figure 4 to identify lineages displaying specific sites under positive selection). In the column identified as “codon population composition at this site,“ the numbers in parenthesis represent the number of viral sequences in the population carrying this specific codon. The letters in the parenthesis indicate amino acids encoded by specific codons. (B) MEME analysis was conducted on 117 viral sequences recovered from humans during the pandemic. Information about the countries where mutations at specific codon sites under positive selection were found and the overall percentage of human sequences carrying these mutations was obtained from the GISAID database. For MEME analysis, α = synonymous substitution rate at the site, β+ = nonsynonymous substitution rate at the site for the positive/neutral evolution component, and LRT = likelihood ratio test statistic for diversifying selection. (C) Comparison of dN/dS ratios at different gene segments of SARS-CoV-2 between cat and human populations. Calculations were conducted using MEME.

Figure 6

Phylogenomic dynamics between the cat and human SARS-CoV-2 lineages. The phylogenetic analysis was conducted using 222 SARS-CoV-2 lineages (cats n = 105, humans n = 117). The SARS-CoV-2 lineages associated with cats and humans were identified in red and green, respectively. Because of size restrictions, the version of this phylogenetic tree showing the ID of cats and human-recovered viral lineages is shown in Supplementary File S3. This figure was created using BioRender.com under the academic license number WW25XVH794.

Figure 7

Identification of potential SARS-CoV-2 lineages evolving under positive selection from cat populations. (A) Results of the aBSREL analyses identify potential cat lineages evolving because of natural selection. (B) Phylogenetic relationship between identified cat SARS-CoV-2 lineages (red branches) 22 AY.69 GK 21, 29 AY.69 GK 21, and its closest related ancestral SARS-CoV-2 human lineage (green branch, sequence OV832299.1 2021 GK human). (C) Phylogenetic relationship between identified cat SARS-CoV-2 lineages (red branch) 34 BA.1.1 GRA 22, and its closest related ancestral SARS-CoV-2 human lineage (green branch, sequence EPI ISL 11686014 2022 GRA human). (D) Phylogenetic relationship between the identified cat SARS-CoV-2 lineage (red branch) 55 B.1 GH 20 (similar to 56 B.1 GH 20) and its closest related ancestral SARS-CoV-2 human lineage (green branch, sequence MW268725.1 2020 GH human). (E) Phylogenetic relationship between the identified cat SARS-CoV-2 lineage (red branches) 88 B.1.1.298 GR 20 and its closest related ancestral SARS-CoV-2 human lineage (green branch, sequence OW425144.1 2022 GR Human). The percentage in bold between cat and human sequences reflects their nucleotide identity. Below, multiple trees display the polymorphic codon sites at different genes impacting the identity between human (information in green) and cat (information in red) sequences. All these short trees were obtained from the phylogenetic analysis in Supplementary File S3. (F) Busted analysis was conducted to determine evidence ratios (ER Busted) for specific codon sites associated with the branches linked to the emergence of cat lineages showing ω > 1. The relevance of these sites was assessed by FEL and MEME (cutoff 0.1). FEL analysis was conducted at both internal (selection at the population level) and external (selection at the individual level) nodes. The overall number of sequences in the GISAID database showing the alleles in cat lineages and human ancestral sequences at specific codon positions are shown. Frequencies represented by different alleles in humans and cats at specific codon positions were obtained from the GISAID database. The phylodynamics of multiple codon positions identified by BUSTED analysis are presented in Supplementary File S4.