SARS-CoV-2 within-host population expansion, diversification and adaptation in zoo tigers, lions and hyenas

Abstract

SARS-CoV-2 rapidly adapts to new hosts following cross-species transmission; this is highly relevant as novel within-host variants have emerged following infection of susceptible wild and domestic animal species. Furthermore, SARS-CoV-2 transmission from animals (e.g., white-tailed deer, mink, domestic cats, and others) back to humans has also been observed, documenting the potential of novel animal-derived variants to infect humans. We investigated SARS-CoV-2 evolution and host-specific adaptation during an outbreak in Amur tigers (Panthera tigris altaica), African lions (Panthera leo), and spotted hyenas (Crocuta crocuta) at Denver Zoo in late 2021. SARS-CoV-2 genomes from longitudinal samples collected from 16 individuals were evaluated for within-host variation and genomic signatures of selection. The outbreak was likely initiated by a single spillover of a rare Delta sublineage subsequently transmitted from tigers to lions to hyenas. Within-host virus populations rapidly expanded and diversified. We detected signatures of purifying and positive selection, including strong positive selection in hyenas and in the nucleocapsid (N) gene in all animals. Four candidate species-specific adaptive mutations were identified: N A254V in lions and hyenas, and ORF1a E1724D, spike T274I, and N P326L in hyenas. These results reveal accelerated SARS-CoV-2 adaptation following host shifts in three non-domestic species in daily contact with humans.

Keywords: SARS-CoV-2; spillover; virus evolution; within-host variants.

Figure 1.. SARS-CoV-2 RNA was persistently detected in tigers, lions and hyenas by qRT-PCR followed by next-generation sequencing.

qRT-PCR was performed on nasal swabs from tigers (N=2), lions (N=11) and hyenas (N=4) between October 2021 and January 2022. Each point represents a nasal swab sample tested for three SARS-CoV-2 gene targets, colored by result determined by diagnostic software. Points with a blue border indicate samples from which high-quality NGS data was generated (N=63).

Figure 2.. A single introduction of SARS-CoV-2 lineage AY.20 was transmitted from tigers to lions to hyenas.

Time-based phylogenetic tree of SARS-CoV-2 sequences from tigers, lions and hyenas, with (A) collection date or (B) divergence in number of mutations relative to the ancestral Wuhan-1 reference sequence is indicated along the x-axis. Tree was generated with Nextstrain and an interactive version is available at https://nextstrain.org/community/laurabashor/DZSARS2. Data include SARS-CoV-2 consensus sequences generated from the zoo outbreak (N=63), all human-derived sequences classified as AY.20 in the state of Colorado between September 23rd and November 4th, 2021 (N = 198), and a random subsample of human-derived sequences in Colorado from the days leading up to the zoo outbreak (N=1500). (C) A haplotype network of the last high-quality sequence obtained from each individual (N=16) was generated using the ‘pegas’ package in R. (D) Four candidate adaptive mutations were detected in SARS-CoV-2 genomes from lions and hyenas.

Figure 3.. SARS-CoV-2 within-host variation in lions and hyenas varies by species.

(A) Fourteen within-host variants throughout the SARS-CoV-2 genome were identified in lions and hyenas (N=58 samples). Each row represents one nasal swab sample, and collection date and animal identifier are indicated on the y-axis. Each tile represents a mutation detected in that sample relative to the consensus sequence of SARS-CoV-2 genomes recovered from the two infected tigers and is colored by allele frequency. Black dots indicate nonsynonymous single nucleotide variants. SARS-CoV-2 genome indicates the genomic loci of the eleven coding region mutations. Code to prepare plot was adapted from the R package ‘outbreakinfo’. Number of (B) within-host variants (Wilcoxon two-sample test, W=273, p= 0.000107), (C) transitions and transversions, and (D) predicted effects of mutation by species.

Figure 4.. SARS-CoV-2 within-host populations underwent expansion and diversification over time.

Each point indicates the nucleotide diversity of a SARS-CoV-2 virus population observed in one sample, calculated as the mean of two sequencing replicates. (A) Within-host nucleotide diversity (π) by the number of days after an individual’s first positive SARS-CoV-2 test. Lines show the predicted fit of a linear mixed model by species. (B) Within-host synonymous nucleotide diversity (πS) over time by individual animal.

Figure 5.. Strong signatures of positive selection in (A) full SARS-CoV-2 genomes from hyenas and (B) the nucleocapsid gene from all species.

(C) Mixed signatures of positive and purifying selection were detected in ORF1ab, S and ORF3a, and (D) no or exclusively purifying signatures of selection were detected in the remaining gene segments. The ratio of nonsynonymous (πN) to synonymous (πS) nucleotide diversity was calculated as a measure of the type and strength of selection acting on SARS-CoV-2 populations. Each point represents the πN/πS ratio of (A) a SARS-CoV-2 virus population observed in the last nasal swab sample collected for each individual (N=16 animals), or (B, C, D) a SARS-CoV-2 gene segment observed in one sample calculated as the mean of two technical sequencing replicates. Black dashed lines indicate πN/πS ratio of 1, and colored lines indicate the mean for each species. Infinite values (where πS = 0) are indicated with half points along plot borders and were not used in mean calculations. The abbreviated gene segment is indicated at the top of each plot (nucleocapsid (N), open reading frame (ORF), spike (S), envelope (E), membrane (M)).

Figure 6.. The SARS-CoV-2 AY.20 lineage with the N A254V mutation (detected in lions and hyenas) was found in humans in Colorado during the early stage of the zoo outbreak.

Each point indicates the number of SARS-CoV-2 sequences in Colorado classified as AY.20 per epidemiological week (epiweek), from the first observation of AY.20 on June 23^rd, 2021 (2021 epiweek 25) to the last observation on January 16^th, 2022 (2022 epiweek 3). Orange bars represent the number of SARS-CoV-2 sequences classified as AY.20 with the N A254V mutation. Light blue shaded area indicates the early outbreak period from October 7^th to October 30^th, 2021. Data were obtained from the GISAID database.