Susceptibility of Ferrets, Cats, Dogs, and Other Domesticated Animals To SARS-coronavirus 2
Susceptibility of Ferrets, Cats, Dogs, and Other Domesticated Animals To SARS-coronavirus 2
Susceptibility of Ferrets, Cats, Dogs, and Other Domesticated Animals To SARS-coronavirus 2
China. 2National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, People’s Republic of China. 3National High Containment Laboratory for
Animal Diseases Control and Prevention, Harbin 150069, People’s Republic of China.
*These authors contributed equally to this work.
†Corresponding author. Email: buzhigao@caas.cn (Z.B.); chenhualan@caas.cn (H.C.); wugz@ivdc.chinacdc.cn (G.W.)
In late December 2019, an unusual pneumonia emerged in most precisely to model the efficacy of such control measures
humans in Wuhan, China, and rapidly spread internationally, in humans? To address these questions, we evaluated the sus-
raising global public health concerns. The causative pathogen ceptibility of different model laboratory animals, as well as
was identified as a novel coronavirus (1–16) that was named companion and domestic animals to SARS-CoV-2.
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- All experiments with infectious SARS-CoV-2 were per-
CoV-2) on the basis of a phylogenetic analysis of related coro- formed in the biosafety level 4 and animal biosafety level 4
naviruses by the Coronavirus Study Group of the Interna- facilities in the Harbin Veterinary Research Institute (HVRI)
tional Committee on Virus Taxonomy (17); the disease it of the Chinese Academy of Agricultural Sciences (CAAS),
causes was subsequently designated COVID-19 by the World which was approved for such use by the Ministry of Agricul-
Health Organization (WHO). Despite tremendous efforts to ture and Rural Affairs of China. Details of the biosafety and
control the COVID-19 outbreak, the disease is still spreading. biosecurity measures taken are provided in the supplemen-
As of March 11, 2020, SARS-CoV-2 infections have been re- tary materials (19). The protocols for animal study and ani-
ported in more than 100 countries, and 118,326 human cases mal welfare were reviewed and approved by the Committee
have been confirmed, with 4,292 fatalities (18). COVID-19 has on the Ethics of Animal Experiments of the HVRI of CAAS
now been announced as a pandemic by WHO. (approval number 2020-01-01JiPi).
Although SARS-CoV-2 shares 96.2% identity at the nucle- Ferrets are commonly used as an animal model for respir-
otide level with the coronavirus RaTG13, which was detected atory viruses that infected humans (20–26). We therefore
in horseshoe bats (Rhinolophus spp) in Yunnan province in tested the susceptibility of SARS-CoV-2 in ferrets. Two viruses
2013 (3), it has not previously been detected in humans or [SARS-CoV-2/F13/environment/2020/Wuhan, isolated from
other animals. The emerging situation raises many urgent an environmental sample collected in the Huanan Seafood
questions. Could the widely disseminated viruses transmit to Market in Wuhan (F13-E), and SARS-CoV-
other animal species, which then become reservoirs of infec- 2/CTan/human/2020/Wuhan (CTan-H), isolated from a hu-
tion? The SARS-CoV-2 infection has a wide clinical spectrum man patient] were used in this study. Pairs of ferrets were
in humans, from mild infection to death, but how does the inoculated intranasally with 105 plaque forming unit (PFU) of
virus behave in other animals? As efforts are made for vaccine F13-E or CTan-H, respectively, and euthanized on day 4 post-
and antiviral drug development, which animal(s) can be used inoculation (p.i.). The nasal turbinate, soft palate, tonsils,
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trachea, lung, heart, liver, spleen, kidneys, pancreas, small in- palate of one of the two ferrets that were euthanized on days
testine, and brain from each ferret were collected for viral 2 and 4 p.i.; in the soft palate of one ferret and in the nasal
RNA quantification by qPCR and virus titration in Vero E6 turbinate, soft palate, tonsil, and trachea of the other ferret
cells. Viral RNA (Fig. 1, A and B) and infectious virus were that were euthanized on day 8 p.i.; and was not detected in
detected in the nasal turbinate, soft palate, and tonsils of all either of the two ferrets that were euthanized on day 14 p.i.
four ferrets inoculated with these two viruses, but was not (fig. S5). These results indicate that SARS-CoV-2 can replicate
detected in any other organs tested (Fig. 1, C and D). These in the upper respiratory tract of ferrets for up to eight days,
results indicate that SARS-CoV-2 can replicate in the upper without causing severe disease or death.
respiratory tract of ferrets, but its replication in other organs Cats and dogs are in close contact with humans, and
is undetectable. therefore it is important to understand their susceptibility to
To investigate the replication dynamics of these viruses in SARS-CoV-2 for COVID-19 control. We first investigated the
ferrets, groups of three animals were inoculated intranasally replication of SARS-CoV-2 in cats. Seven subadult cats (aged
with 105 PFU of F13-E or CTan-H, and then placed in three 6-9 months, outbred domestic cats) were intranasally inocu-
separate cages within an isolator. Nasal washes and rectal lated with 105 PFU of CTan-H. Two animals were scheduled
swabs were collected on days 2, 4, 6, 8, and 10 p.i. from the to be euthanized on days 3 p.i. and 6 p.i., respectively, to eval-
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cats and one exposed cat by use of an ELISA and neutraliza- not detected in lung lobes, even after the ferrets were in-
tion assay (Fig. 3, C and D). tratracheally inoculated with the virus. It remains unclear
We replicated the replication and transmission studies in whether the virus causes more severe disease in male ferrets
juvenile cats (aged 70–100 days) (Figs. 2, E to H, and 3, E to than in female ferrets, as has been observed among humans
G, and fig. S6). Histopathologic studies performed on samples (13, 28).
from the virus-inoculated juvenile cats that died or eu- Several studies have reported that SARS-CoV-2 uses angi-
thanized on day 3 p.i. revealed massive lesions in the nasal otensin-converting enzyme 2 (ACE2) as its receptor to enter
and tracheal mucosa epitheliums, and lungs (fig. S7). These cells (3, 29–31). ACE2 is mainly expressed in type II pneumo-
results indicate that SARS-CoV-2 can replicate efficiently in cytes and serous epithelial cells of tracheo-bronchial submu-
cats, with younger cats being more permissive and, perhaps cosal glands in ferrets (25). Ferrets and cats have only two
more importantly, the virus can transmit between cats via the amino acid differences in the SARS-CoV-2 spike-contacting
airborne route. regions of ACE2 (table S1); therefore, the underlying mecha-
We next investigated the replication and transmission of nism that prevents the replication of SARS-CoV-2 in the lower
SARS-CoV-2 in dogs. Five 3-month-old beagles were intrana- respiratory tract of ferrets remains to be investigated. The
sally inoculated with 105 PFU of CTan-H, and housed with fact that SARS-CoV-2 replicates efficiently in the upper res-
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SUPPLEMENTARY MATERIALS
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Materials and Methods
Supplementary Text
Figs. S1 to S10
Table S1
References and Notes (33–35)
MDAR Reproducibility Checklist
View/request a protocol for this paper from Bio-protocol.
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Fig. 1. Replication of SARS-CoV-2 viruses in ferrets. Viral RNA in
organs or tissues of ferrets inoculated with (A) F13-E virus or (B) CTan-
H virus. Viral titers in organs or tissues of ferrets inoculated with F13-E
(C) and CTan-H (D). The viral RNA negative organs in panels A and B
were also virus titration negative, which were indicated as “Others” in
panels C and D. Viral RNA (E and F) and viral titer (G and H) in nasal
washes of ferrets inoculated with F13-E (E, G) and CTan-H (F, H).
Antibodies against SARS-CoV-2 tested by an ELISA (I and J) and
neutralization assay (K and L) with the sera derived from ferrets
inoculated with F13-E (I, K) and CTan-H (J, L). Each color bar represents
the value from an individual animal. The gray bars in the panels I to L
indicate the antibody values of sera collected from each animal before
the virus was inoculated. Asterisks indicate animals that were
euthanized on day 13 after virus inoculation, the other four animals were
euthanized on day 20 p.i. The horizontal dashed lines in the panels I and
L show the cutoff value for seroconversion, and the horizontal dashed
lines in other panels indicate the lower limit of detection.
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Fig. 2. Replication of SARS-CoV-2 in cats. Subadult cats and juvenile
cats inoculated with CTan-H virus were euthanized on day 3 post-
inoculation (p.i.) and day 6 p.i., and their organs were collected for viral
RNA detection and virus titration. (A) Viral RNA and (B) viral titers of
subadult cats on day 3 p.i. (C) Viral RNA and (D) viral titers of subadult
cats on day 6 p.i. (E) Viral RNA and (F) viral titers of juvenile cats on day
3 p.i., the values of red bars in panels E and F are from the cat that died
on this day. (G) Viral RNA and (H) viral titers of juvenile cats on day 6 p.i.
Others: viral negative organs, including brain, heart, submaxillary lymph
nodes, kidneys, spleen, liver, and pancreas. Each color bar represents
the value from an individual animal. The horizontal dashed lines indicate
the lower limit of detection.
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Fig. 3. Transmission of SARS-CoV-2 in cats. Transmission of CTan-H virus was evaluated in subadult
cats (A to D) and juvenile cats (E to G). (A) Viral RNA in the feces of virus inoculated or exposed
subadult cats. (B) Viral RNA in tissues or organs of virus inoculated or exposed subadult cats that were
euthanized on day 11 p.i. (pair one, red bars) or on day 12 p.i. (pair 2 and pair 3). Antibodies against
SARS-CoV-2 of these euthanized subadult cats were detected by using an ELISA (C) and neutralization
assay (D). (E) Viral RNA in nasal washes of juvenile cats. Sera of the juvenile cats were collected on day
20 p.i., except for one virus inoculated animal died on day 13 p.i. and the antibody values of this cat
(indicated by asterisks) were detected from the sera collected on day 10 p.i., their antibodies against
SARS-CoV-2 were detected by using an ELISA (F) and neutralization assay (G). Each color bar
represents the value from an individual animal. The horizontal dashed lines in the panels C and F show
the cutoff value for seroconversion, and the horizontal dashed lines in other panels indicate the lower
limit of detection.
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Table 1. Susceptibility of dogs, pigs, chickens, and ducks to SARS-CoV-2a.
Viral RNA detection in animals inoculated with
SARS-CoV-2 isolate CTan-H;
no. of positive/total (copies, log10) Seroconversion:
Animal Treatment No. of
Oropharyngeal swab Rectal swab
positive/totalc
Day 2 Day 4 Day 6 Other Day 2 Day 4 Day 6 Other
p.i. p.i. p.i. timepoints p.i. p.i. p.i. timepoints
2/5 1/4
Inoculated 0/5 0/5 0/4 0/4 0/5 0/4 2/4
Dog b (6.5, 5.4) (4.2)
Contact 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2
Inoculated 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Pig
Contact 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Inoculated 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Chicken
Contact 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Inoculated 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Duck
Contact 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
virus, and two (beagles) or three (pigs, chickens, and ducks) uninfected animals were housed in the same room with
their infected counterparts to monitor the transmission of the CTan-H virus. Oropharyngeal and rectal swabs from all
animals were collected on the indicated days post inoculation (p.i.) for viral RNA detection. Other timepoints include
days 8, 10, 12, and 14 p.i..
b
One virus-inoculated beagle was euthanized on day 4 p.i., but viral RNA was not detected in any of its collected or-
gans, which included lung, trachea, nasal turbinate, soft palate, brain, heart, tonsil, kidneys, spleen, liver, pancreas,
and small intestine (fig. S6).
c
Sera were collected from all animals on day 14 p.i., and antibodies against SARS-CoV-2 were detected by using a Dou-
ble Antigen Sandwich ELISA kit (ProtTech, Luoyang, China).
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Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS−coronavirus 2
Jianzhong Shi, Zhiyuan Wen, Gongxun Zhong, Huanliang Yang, Chong Wang, Baoying Huang, Renqiang Liu, Xijun He, Lei Shuai,
Ziruo Sun, Yubo Zhao, Peipei Liu, Libin Liang, Pengfei Cui, Jinliang Wang, Xianfeng Zhang, Yuntao Guan, Wenjie Tan, Guizhen
Wu, Hualan Chen and Zhigao Bu
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