key: cord-0917546-253i2s8v
authors: McAloose, Denise; Laverack, Melissa; Wang, Leyi; Killian, Mary Lea; Caserta, Leonardo C.; Yuan, Fangfeng; Mitchell, Patrick K.; Queen, Krista; Mauldin, Matthew R.; Cronk, Brittany D.; Bartlett, Susan L.; Sykes, John M.; Zec, Stephanie; Stokol, Tracy; Ingerman, Karen; Delaney, Martha A.; Fredrickson, Richard; Ivančić, Marina; Jenkins-Moore, Melinda; Mozingo, Katie; Franzen, Kerrie; Bergeson, Nichole Hines; Goodman, Laura; Wang, Haibin; Fang, Ying; Olmstead, Colleen; McCann, Colleen; Thomas, Patrick; Goodrich, Erin; Elvinger, François; Smith, David C.; Tong, Suxiang; Slavinski, Sally; Calle, Paul P.; Terio, Karen; Torchetti, Mia Kim; Diel, Diego G.
title: From people to Panthera: Natural SARS-CoV-2 infection in tigers and lions at the Bronx Zoo
date: 2020-07-24
journal: bioRxiv
DOI: 10.1101/2020.07.22.213959
sha: 451450a93154cb3a1c9fae41a3190b1ae711bcec
doc_id: 917546
cord_uid: 253i2s8v

We describe the first cases of natural SARS-CoV-2 infection detected in animals in the United States. In March 2020, four tigers and three lions at the Bronx Zoo developed mild respiratory signs. SARS-CoV-2 RNA was detected by rRT-PCR in respiratory secretions and/or feces from all seven affected animals; viral RNA and/or antibodies were detected in their keepers. SARS-CoV-2 was isolated from respiratory secretions or feces from three affected animals; in situ hybridization co-localized viral RNA with cellular damage. Whole genome sequence and haplotype network analyses showed tigers and lions were infected with two different SARS-CoV-2 strains, suggesting independent viral introductions. The source of SARS-CoV-2 infection in the lions is unknown. Epidemiological data and genetic similarities between keeper and tiger viruses indicate human to animal transmission.

3 Abstract: We describe the first cases of natural SARS-CoV-2 infection detected in animals in the United States. In March 2020, four tigers and three lions at the Bronx Zoo developed mild respiratory signs. SARS-CoV-2 RNA was detected by rRT-PCR in respiratory secretions and/or feces from all seven affected animals; viral RNA and/or antibodies were detected in their 40 keepers. SARS-CoV-2 was isolated from respiratory secretions or feces from three affected animals; in situ hybridization co-localized viral RNA with cellular damage. Whole genome sequence and haplotype network analyses showed tigers and lions were infected with two different SARS-CoV-2 strains, suggesting independent viral introductions. The source of SARS-CoV-2 infection in the lions is unknown. Epidemiological data and genetic similarities between 45 keeper and tiger viruses indicate human to animal transmission.

COVID-19, a severe respiratory disease caused by a novel coronavirus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) (1), was first reported in Wuhan, Hubei 50 province, China at the end of December 2019 (2) . Within weeks the virus caused a global pandemic, and by July, 2020, over 10 million people were infected and more than 500,000 had died (https://www.who.int/emergencies/diseases/novel-coronavirus-2019; accessed 7/1/2020).

An early cluster of human cases had an epidemiological link to the Huanan Seafood Wholesale market in Wuhan where a variety of live wild animals were sold (3). Genome sequence analysis 55 revealed SARS-CoV-2 to be most closely related to a bat coronavirus (RaTG13-2013), and bats are considered the likely source of the ancestral virus from which the currently circulating 4 SARS-CoV-2 virus was derived (3, 4) . Subsequent genetic adaptation in an intermediate animal host(s) or after human transmission has been proposed (4, 5).

Given the suspected zoonotic origin of SARS-CoV-2, identifying susceptible animal 60 species, reservoirs, and transmission routes are topics of global scientific and public interest.

Natural SARS-CoV-2 infections in animals have been reported in dogs, cats and farmed mink in Hong Kong, Europe, China and the United States (6) (7) (8) . Infection in most of these cases has been linked to households or settings in which human owners or caretakers have tested positive for SARS-CoV-2. Experimental inoculation studies have shown that SARS-CoV-2 infects and 65 replicates with high efficiency in domestic cats, ferrets and fruit bats and poorly in dogs, pigs, and chickens; ducks do not seem to support productive SARS-CoV-2 infection (9, 10).

Importantly, virus shedding and horizontal transmission have been shown in cats and ferrets (9, 10)following experimental inoculation.

In this study, we report natural infection of tigers (Panthera tigris), lions (P. leo) and 70 keepers who provided care for the animals with SARS-CoV-2 at the Wildlife Conservation Society's (WCS) Bronx Zoo, New York NY. We provide a detailed characterization of viruses obtained from infected animals and keepers who had close contact with the SARS-CoV-2 positive animals. The animals were infected in March 2020, when, due to widespread community transmission (11) , NY had become a global SARS-CoV-2 epicenter. 75 On March 27, 2020, a 4-year-old, female Malayan tiger (P. t. jacksoni) (Tiger 1) developed an intermittent cough and audible wheezing despite remaining eupneic. By April 2, an additional Malayan (Tiger 2) and two Amur tigers (P. t. altaica) (Tigers 3, 4) housed in the same building as Tiger 1 but in different enclosures, and three African lions (P. l. krugeri) (Lions 1, 2, 5 3) housed in a separate building developed similar respiratory signs. All animals otherwise 80 exhibited normal behavior and activity. Clinical respiratory signs resolved within one week in all but Tiger 1, whose clinical signs lasted 16 days. An additional Amur tiger (Tiger 5) in the same building as Tigers 1-4 did not develop clinical respiratory disease.

Clinical evaluation and a broad diagnostic investigation were performed in Tiger 1.

Thoracic radiography revealed small, multifocal regions of peribronchial consolidation. 85 Cytologic examination of tracheal wash fluid identified necrotic epithelial and inflammatory cells consistent with tracheitis ( Fig. 1A and B) . In situ hybridization (ISH) co-localized SARS-CoV-2 RNA within necrotic epithelial and inflammatory cells in tracheal wash fluid (Fig. 1C, D and S1). Targeted PCR testing and metagenomic analysis on respiratory samples (nasal, orpharyngeal, tracheal) were negative for common feline pathogens (Table S1 Results were confirmed by amplicon sequencing (Table S5) .

Virus isolation was performed in respiratory and/or fecal samples from the animals. 100 Cytopathic effect (CPE) was observed in Vero cells inoculated with tracheal wash fluid from Tiger 1 ( Fig. 2A and B ) and fecal samples from Tiger 3 and Lion 2 ( Table S6) . Results were 6 confirmed by rRT-PCR (CDC N1 assay) and/or ISH and immunofluorescence assays ( Fig. 2C and D). A neutralizing antibody titer of 64 detected in Tiger 1 (Table S7 ) confirmed infection and development of an immunologic response against SARS-CoV-2. 105 An epidemiologic investigation of zoo staff identified ten zoo keepers and two managers who provided care for and had close but not direct contact with the tigers or lions between March 16, 2020 (the date the zoo was closed to the public due to the pandemic) and March 27 to April 1, 2020 (timeline of disease onset in the animals). Four staff (2 tiger Nine complete SARS-CoV-2 genome sequences (four from tigers, three from lions, two from keepers) and eight full-length S gene sequences (seven symptomatic and one asymptomatic 120 animals) were generated directly from respiratory and/or fecal samples. Compared to the Wuhan-Hu-1 (GenBank accession number NC_045512), all sequences obtained from the tigers and keepers contained six single-nucleotide polymorphisms (SNPs) with nine additional ambiguous sites (Fig. S2, Table S8 ). A total of 20 sites differed between the three lion sequences and Wuhan-Hu-1 (Fig. S2, Table S8 ).

125 7 Viral sequences in the tigers, lions and keepers clustered into common SARS-CoV-2 clades (Fig. 3A) . Those from tigers and tiger keepers clustered with clade G (defined by the D614G substitution in the spike protein); the lion sequences clustered with clade V (defined by the G251V substitution in ORF3a) (Fig. 3A) . Median-joining haplotype network analysis of the viral sequences in the animals and keepers corroborated results of the phylogenetic analyses 130 (Fig. 3B) . Genomic and epidemiological data support a close evolutionary relationship between the viral strains recovered from tigers and tiger keepers. Notably, the genetic differences and the distant phylogenetic relationship between sequences recovered from the tigers/tiger keepers and lions, and the relationship of these strains in the context of global sequences (Fig. 3B) indicate that tigers and lions were infected by two different viral strains. Furthermore, this data suggest 135 that at least two independent SARS-CoV-2 introductions occurred, one in tigers and another in lions.

The SARS-CoV-2 genome sequence from Tiger 1 was identical to the viral sequence recovered from Keeper 1 (a tiger keeper) and to other human SARS-CoV-2 strains detected in New York (NY_2929 [MT304486] and NY-QDX-00000001 [MT452574.1]) (Fig. 3) . These 140 observations, temporal overlap in animal and human infections, and a lack of new animal introductions to the collection support the conclusion of transmission from an infected keeper(s) to the tigers. Whether this was direct or indirect (e.g. fomite, food handling/preparation) and whether subsequent tiger to tiger transmission (aerosol, respiratory droplet, etc.) occurred was not determined. A clear association and transmission source was not identified for the lions. The 145 lion SARS-CoV-2 sequences were more divergent than those in the tigers and keepers (Fig. 3) .

However, nine of the 12 SNPs (relative to the Wuhan-Hu-1 reference strain) shared by all three lion viruses were also found in the closest human strain (GISAID accession: EPI_ISL_427161), 8 which was detected nearby in Connecticut, US. Two lion keepers were serologically positive for SARS-CoV-2, but viral RNA was not detected and SARS-CoV-2 strain(s) could not be 150 confirmed in their respiratory samples. However, given regular close contact between keepers and animals it is possible that the lions were also directly or indirectly infected by asymptomatic keepers or staff.

The host range of SARS-CoV-2 and other coronaviruses is determined primarily by the interaction of the virus S glycoprotein, specifically the spike 1 subunit (S1), and the cellular 155 receptor, angiotensin-converting enzyme II (ACE2) (12). Recently, in silico predictions have shown high binding potential between the S receptor binding domain (RBD) and domestic cat ACE2 receptor, and that three of five amino acid residues that are critical for interaction with the SARS-CoV-2 S glycoprotein are conserved between human and domestic cat ACE2 (12). These observations are supported by reports describing natural and experimental infection of domestic 160 cats with SARS-CoV- 2 (9, 13) and the data herein that shows a high degree of conservation between ACE2 in humans and domestic and wild felids (Fig. S3) . Compared with the Wuhan-Hu-1 strain, the tiger and lion SARS-CoV-2 S gene sequences have 1-3 nucleotide and 0-2 nucleotide differences, respectively (Fig. 4, Tables S9 and S10). These changes resulted in eight non-synonymous substitutions (P139L, L455S, F456Y, G496D, Q613R, D614G, A623T, and 165 T716I). Notably, of three non-synonymous substitutions (L455S, F456Y, G496D) in the tiger strains, only one (G496D) was found in available human SARS-CoV-2 strains. These changes were not observed in the viral sequences from the lions (Fig. 4) . Further work is needed to determine if these changes affect SARS-CoV-2 receptor binding and pathogenicity in felids and humans. 170 9 Infections in the tigers and lions occurred at a time before testing was widely available in the US and there was limited evidence of pre-or asymptomatic viral shedding (14) . They also preceded Centers for Disease Control and Prevention (CDC) guidance recommending face coverings (issued on April 3, 2020) to limit SARS-CoV-2 transmission. Keepers caring for the tigers and lions did not generally wear personal protective equipment (PPE) given the ( 

Figs. S1 to S3 305 Tables S1 to S9 Data S1 to S4 

The species Severe acute 205 respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

A novel coronavirus from patients with pneumonia in China

A pneumonia outbreak associated with a new coronavirus of probable bat origin

The proximal origin of SARS-CoV-2

Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event

Infection of dogs with SARS-CoV-2

First reported cases of SARS-CoV-2 infection in companion animals

SARS-CoV-2

Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARScoronavirus 2

Experimental transmission etudies of SARS-CoV-2 in fruit bats, ferrets, pigs and chickens. SSRN Electron

Introductions and early spread of SARS-CoV-2 in New York City area

Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection

SARS-CoV-2 neutralizing 255 serum antibodies in cats: a serological investigation. bioRxiv

Presymptomatic transmission of SARS-CoV-2 -Singapore

Sustainable development must account for pandemic risk

Eric Nelson at South Dakota State University and Beth Plocharczyk at Cayuga Medical Center for providing reagents used in the study. The following reagent was deposited by 275 the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52285. Funding: Diagnostic testing at the University of Illinois and Cornell University was supported in part by USDA:NAHLN infrastructure APHIS award AP19 VS NVSL00C020 and NIFA award 2018-37620-28832. Sequencing capacity at the University of Illinois and Cornell University was 280 funded in part by the Food and Drug Administration Veterinary Laboratory Investigation and Response Network (FOA PAR-18-604) under grants 1U18FD006673-01, 1U18FD006866-01, 1U18FD006714-01 and 1U18FD006716-01 Author contributions: Conceptualization, Project administration

Analysis, Investigation, Data Curation, Writing -Review & Editing: Melissa Laverack

Resources: Denise McAloose

Writing -Original Draft Preparation

Funding Acquisition

/generated from tigers, lions and keepers. Numbers in parentheses indicate differences between unique sequences (haplotypes)

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) (SnapGene v4.2.4). The nucleotide changes in each strain result in non-synonymous substitutions (pink lines) that are listed in line with the amino acid change. Schematic representation of the genome organization and functional domains of the S protein for SARS-CoV2 Wuhan

Homology modeling of the Tiger 1 (MT365033) spike protein (I-TASSER

Q613R, warm pink; D614G, purple/blue; A623T/I, magenta; T716I, slate)

The authors declare no competing interests. This manuscript represents