From People to Panthera: Natural SARS-CoV-2 Infection in Tigers and Lions at the Bronx Zoo

Abstract

Despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. Among these, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and ebolaviruses have killed thousands; the human immunodeficiency virus (HIV) has killed millions. Zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. The current SARS-CoV-2 pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. In March 2020, New York City was a global epicenter for SARS-CoV-2 infections. During this time, four tigers and three lions at the Bronx Zoo, NY, developed mild, abnormal respiratory signs. We detected SARS-CoV-2 RNA in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral RNA with cellular damage in one. We produced nine whole SARS-CoV-2 genomes from the animals and keepers and identified different SARS-CoV-2 genotypes in the tigers and lions. Epidemiologic and genomic data indicated human-to-tiger transmission. These were the first confirmed cases of natural SARS-CoV-2 animal infections in the United States and the first in nondomestic species in the world. We highlight disease transmission at a nontraditional interface and provide information that contributes to understanding SARS-CoV-2 transmission across species.IMPORTANCE The human-animal-environment interface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important aspect of the coronavirus disease 2019 (COVID-19) pandemic that requires robust One Health-based investigations. Despite this, few reports describe natural infections in animals or directly link them to human infections using genomic data. In the present study, we describe the first cases of natural SARS-CoV-2 infection in tigers and lions in the United States and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. Our data show that tigers and lions were infected with different genotypes of SARS-CoV-2, indicating two independent transmission events to the animals. Importantly, infected animals shed infectious virus in respiratory secretions and feces. A better understanding of the susceptibility of animal species to SARS-CoV-2 may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health.

Keywords: One Health; Panthera leo; Panthera tigris; SARS-CoV-2; in situ hybridization; lion; rRT-PCR; tiger; virus isolation; whole-genome sequencing; zoo; zoonotic infection.

FIG 1

Tracheal wash cytology (A and B) and in situ hybridization (ISH) (C and D). Tiger 1. (A) Flocculent material from the trachea consists of stringy mucus with enmeshed degenerate cells characterized by condensed nuclei and loss of distinct cellular features (arrows). (B) Few intact cells (short arrow) and degenerate epithelial cells (long arrow) are admixed with abundant round to amorphous cellular debris and granular degenerate mucus (arrowheads). Inset: degenerate epithelial cell (upper right) with nuclear fragmentation (karyolysis) and an adjacent intact neutrophil (lower left). Modified Wright’s stain. (C and D) Incubation with SARS-CoV-2-specific probe is positive (red puncta) throughout the mucinous material, in the cytoplasm of intact and degenerate epithelial and inflammatory cells, and in cellular debris. Red chromogenic assay; hematoxylin counterstain. (Note: a software or equipment malfunction produced a faint horizontal line that may be visible in panels A and B.)

FIG 2

SARS-CoV-2 isolation from respiratory specimens from a tiger (Tiger 1). (A and B) Mock-infected control Vero cells (A) and Vero cells inoculated with tracheal wash fluid showing typical CPE at 48 h postinoculation (B). (C) ISH using a SARS-CoV-2 S-specific probe shows cytoplasmic red puncta. Red chromogenic assay with hematoxylin counterstain. (D) Immunofluorescence assay using a SARS-CoV N-specific monoclonal antibody shows SARS-CoV-2 replication in inoculated cells (green). Evans blue counterstain (red).

FIG 3

Phylogenetic and median-joining haplotype network analysis of SARS-CoV-2 strains in tigers, lions, and keepers. (A) Whole-genome phylogeny of zoo sequences with Wuhan-Hu-1 reference genome (NC_045512.2) and consensus sequences of other publicly available sequences from New York clustered at 99.99% identity (ML tree was unrooted and then midpoint rooted). (B) Haplotype analysis shows relatedness and levels of genetic variation between zoo and a global data set of SARS-CoV-2 genomes. Differences are indicated by one-step edges (lines) between black dots (hypothetical or unsampled haplotypes). Numbers in parentheses indicate differences between unique sequences (small panel; black dot = hypothetical sequence not sampled).

FIG 4

Nucleotide sequence and amino acid changes in the spike (S) protein of SARS-CoV-2 tigers and lions. (A) Comparison with Wuhan-Hu-1 (NC_045512). Nucleotide changes in each strain result in nonsynonymous substitutions (pink lines) that are listed in line with the amino acid change. Schematic representation of the organization and functional domains of the S protein for SARS-CoV2 Wuhan-Hu-1 (IBS online software). (B) Structural modeling of the SARS-CoV-2 spike protein. Homology modeling of the Tiger 1 spike protein (I-TASSER). S protein amino acid changes in the tiger and lion strains versus the Wuhan-Hu-1 strain are depicted in the structure.