CORD-19:4977a01aec602236d923e970569adab5bdc5865e JSONTXT 7 Projects

Susceptibility of ferrets, cats, dogs, and different domestic animals to SARS-coronavirus-2 Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the infectious disease COVID-19, which was first reported in Wuhan, China in December, 2019. Despite the tremendous efforts to control the disease, COVID-19 has now spread to over 100 countries and caused a global pandemic. SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are completely unknown. Here, we investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. We found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but efficiently in ferrets and cats. We found that the virus transmits in cats via respiratory droplets. Our study provides important insights into the animal reservoirs of SARS-CoV-2 and animal management for COVID-19 control. isolated from a human patient] were used in this study. Pairs of ferrets were inoculated intranasally with 10 5 pfu of F13-E or CTan-H, respectively, and euthanized on day 4 postinoculation (p.i.). The nasal turbinate, soft palate, tonsils, trachea, lung, heart, spleen, kidneys, pancreas, small intestine, brain, and liver from each ferret were collected for viral RNA quantification by qPCR and virus titration in Vero E6 cells. Viral RNA (Fig. 1A, B) and infectious virus were detected in the nasal turbinate, soft palate, and tonsils of all four ferrets inoculated with these two viruses, but was not detected in any other organs tested (Fig. 1C, D) . These results indicate that SARS-CoV-2 can replicate in the upper respiratory tract of ferrets, but its replication in other organs is undetectable. To investigate the replication dynamics of these viruses in ferrets, groups of three animals were inoculated intranasally with 10 5 pfu of F13-E or CTan-H, and then placed in three separate cages within an isolator. Nasal washes and rectal swabs were collected on days 2, 4, 6, 8, and 10 p.i. from the ferrets for viral RNA detection and virus titration. Body temperatures and signs of disease were monitored for two weeks. As shown in Fig. 1 , viral RNA was detected in the nasal washes on days 2, 4, 6, and 8 p.i. in all six ferrets inoculated with the two viruses (Fig. 1E, F) . Viral RNA was also detected in some of the rectal swabs of the virus-inoculated ferrets although the copy numbers were notably lower than those in the nasal washes of these ferrets ( fig. S1A, B) . Infectious virus was detected from the nasal washes of all ferrets (Fig. 1G, H) , but not from the rectal swabs of any ferrets (fig. S1C, D). One ferret from each virus-inoculated group developed fever and loss of appetite on days 10 and 12 p.i., respectively. To investigate whether these symptoms were caused by virus replication in the lower respiratory tract, we euthanized the two ferrets on day 13 p.i., and collected their organs for viral RNA detection. However, viral RNA was not detected in any other tissues or organs of either ferret, except for a low copy number (10 5.4 copies/g) in the turbinate of the ferret inoculated with CTan-H ( fig S2) . Pathological studies revealed severe lymphoplasmacytic perivasculitis and vasculitis, increased numbers of type II pneumocytes, macrophages, and neutrophils in the alveolar septa and alveolar lumen, and mild peribronchitis in the lungs of the two ferrets euthanized on day 13 p.i. (fig.S3 ). Antibodies against SARS-CoV-2 were detected in all of the ferrets by an ELISA and a neutralization assay, although the antibody titers of the two ferrets that were euthanized on day 13 p.i. were notably lower than those of the ferrets euthanized on day 20 p.i. (Fig. 1I-L) . To further investigate whether SARS-CoV-2 replicates in the lungs of ferrets, we intratracheally . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint inoculated eight ferrets with 10 5 pfu of CTan-H, and euthanized two animals each on days 2, 4, 8, and 14 p.i. to look for viral RNA in the tissues and organs. Viral RNA was only detected in the nasal turbinate and soft palate of one of the two ferrets that were euthanized on days 2 and 4 p.i.; in the soft palate of one ferret and in the nasal turbinate, soft palate, tonsil, and trachea of the other ferret that were euthanized on day 8 p.i.; and was not detected in either of the two ferrets that were euthanized on day 14 p.i. (fig. S4 ). These results indicate that SARS-CoV-2 can replicate in the upper respiratory tract of ferrets for up to eight days, without causing severe disease or death. Cats and dogs are in close contact with humans, and therefore it is important to understand their susceptibility to SARS-CoV-2 for COVID-19 control. We first investigated the replication of SARS-CoV-2 in cats. Five 8-month-old outbred domestic cats (referred to here as "subadult cats") were intranasally inoculated with 10 5 pfu of CTan-H. Two of the subadult cats were scheduled to be euthanized on day 6 p.i. to evaluate viral replication in their organs. Three subadult cats were placed in separate cages within an isolator. To monitor respiratory droplet transmission, an uninfected cat was placed in a cage adjacent to each of the infected cats. It was difficult to perform regular nasal wash collection on the subadult cats because they were aggressive. To avoid possible injury, we only collected feces from these cats and checked for viral RNA in their organs after euthanasia. From the two subadult cats that were euthanized on day 6 p.i. with CTan-H, viral RNA was detected in the nasal turbinates, soft palates, and tonsils of both animals, in the trachea of one animal, and in the small intestine of the other; however, viral RNA was not detected in any of the lung samples from either of these animals ( Fig. 2A) . Infectious virus was detected in the viral RNA-positive nasal turbinates, soft palates, tonsils, and trachea of these cats, but was not recovered from the viral RNApositive small intestine (Fig. 2B ). In the transmission study, viral RNA was detected in the feces of two virus-inoculated subadult cats on day 3 p.i., and in all three virus-inoculated subadult cats on day 5 p.i. (Fig. 3A ). Viral RNA was detected in the feces of one exposed cat on day 3 p.i. (Fig. 3A) . The subadult cats with viral RNA-positive feces were euthanized on day 11 p.i., and viral RNA was detected in the soft palate and tonsils of the virusinoculated animal and in the nasal turbinate, soft palate, tonsils, and trachea of the exposed animal ( Fig. 3B ), indicating that respiratory droplet transmission had occurred in this pair of cats. We euthanized the other pairs of animals on day 12 p.i., and viral RNA was detected in the tonsils of one virus-inoculated subadult cat, in the nasal turbinate, soft palate, tonsils, and trachea of the other virus-inoculated . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint subadult cat, but was not detected in any organs or tissues of the two exposed subadult cats (Fig. 3B ). Antibodies against SARS-CoV-2 were detected in all three virus-inoculated subadult cats and one exposed cat by use of an ELISA and neutralization assay (Fig. 3C, D) . We replicated the replication and transmission studies in juvenile cats (aged 70-100 days) ( (Table 1, fig S7) . These results indicate that dogs have low susceptibility to SARS- We also investigated the susceptibility of pigs, chickens, and ducks to SARS-CoV-2 by using the same strategy as that used to assess dogs; however, viral RNA was not detected in any swabs collected from these virus-inoculated animals or from naïve contact animals (Table 1) , and all of the animals were seronegative for SARS-CoV-2 when tested by using the ELISA with sera collected on day 14 p.i. (Table 1 ). These results indicate that pigs, chickens, and ducks are not susceptible to SARS-CoV-2. In summary, we found that ferrets and cats are highly susceptible to SARS-CoV-2, dogs have low susceptibility, and livestock including pigs, chickens, and ducks are not susceptible to the virus. Ferrets have frequently been used as an animal model for the study of human respiratory viruses (20) (21) (22) (23) (24) (25) (26) . Unlike influenza viruses and other human SARS-coronavirus, which replicate in both the upper . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint and lower respiratory tract of ferrets (20, 22-24, 26, 27) , we found SARS-CoV-2 only replicates in the nasal turbinate, soft palate, and tonsils of ferrets. It may also replicate in the digestive tract, as viral RNA was detected in the rectal swabs of the virus-infected ferrets, but virus was not detected in lung lobes, even after the ferrets were intratracheally inoculated with the virus. Several studies have reported that SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as its receptor to enter cells (3, (28) (29) (30) . ACE2 is mainly expressed in type II pneumocytes and serous epithelial cells of tracheo-bronchial submucosal glands in ferrets (25) ; therefore, the underlying mechanism that prevents the replication of SARS-CoV-2 in the lower respiratory tract of ferrets remains to be investigated. The fact that SARS-CoV-2 replicates efficiently in the upper respiratory tract of ferrets makes them a candidate animal model for evaluating antiviral drugs or vaccine candidates against COVID-19. The cats we used in this study were outbred, and were highly susceptible to SARS-CoV-2, which replicated efficiently and transmitted to naïve cats. Surveillance for SARS-CoV-2 in cats should be considered as an adjunct to elimination of of COVID-19 in humans. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint inoculated with or exposed to CTan-H at the indicated timepoints. (B) Viral RNA in tissues or organs of subadult cats that were inoculated with or exposed to CTan-H, the pair one cats (red bars) were euthanized on day 11 p.i. and the other two pairs were euthanized on day 12 p.i. Antibodies against SARS-CoV-2 of these euthanized cats were detected by using an ELISA (C) and neutralization assay (D). (E) Viral RNA in nasal washes of juvenile cats that were inoculated with or exposed to CTan-H. Sera of the juvenile cats were collected on day 20 p.i., and their antibodies against SARS-CoV-2 were detected by using an ELISA (F) and neutralization assay (G). One virus inoculated cat 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. Each color bar represents the value from an individual animal. The horizontal dashed lines indicate the lower limit of detection. . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint b. One virus-inoculated beagle was euthanized on day 4 p.i., but viral RNA was not detected in any of its collected organs, which included lung, trachea, nasal turbinate, soft palate, brain, heart, submaxillary lymph nodes, tonsil, kidneys, spleen, liver, pancreas, and small intestine (data not shown). c. Sera were collected from all animals on day 14 p.i., and antibodies against SARS-Cov-2 were detected by using a Double Antigen Sandwich ELISA kit (ProtTech, Luoyang, China). Three-to four-month-old female ferrets (Wuxi Cay Ferret Farm, Wuxi, China) were used in this study. Groups of five ferrets were anesthetized with Zoletil 50 (Virbac, Carros, France) and inoculated intranasally with 10 5 PFU of SARS-CoV-2 CTan-H or F13-E in a volume of 1 mL and each ferret was housed in a separate cage inside an isolator. Two ferrets from each group were euthanized on day 4 post-inoculation (p.i.) and their organs and tissues, including lungs, tracheas, nasal turbinates, soft palates, brains, hearts, submaxillary lymph nodes, tonsils, kidneys, spleens, livers, pancreas, and small intestines, were collected for viral RNA and virus detection. Nasal washes and rectal swabs were collected from the other three ferrets on days 2, 4, 6, 8, and 10 p.i. for viral RNA and virus detection. Body weights and body temperature were monitored every other day. The ferrets were scheduled to . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint be euthanized on day 20 p.i. to assess them for antibodies against SARS-CoV-2 by using the Double Antigen Sandwich ELISA kit (ProtTech, Luoyang, China) and by use of a neutralization assay. Eight ferrets were also inoculated intratracheally with 10 5 PFU of CTan-H. On days 2, 4, 8, and 14 p.i., two ferrets were each euthanized, and their organs were collected for virus detection. Ten outbred domestic juvenile cats (aged between 70 days and three months) and eight outbred subadult cats (aged 6-9 months) obtained from the National Engineering Research Center of To investigate the transmissibility of SARS-CoV-2 in cats, six cats were inoculated intranasally with 10 5 PFU of CTan-H and each animal was placed in a separate cage within an isolator. A similar-aged naive cat was then placed in each cage adjacent to the one that held the virus-inoculated cat. Feces were collected on days 3, 5, 7, and 9 p.i. from the big cats, and nasal washes were collected from the juvenile cats for viral RNA detection or virus titration. One pair and two pairs of the subadult cats were euthanized on days 11 and 12 p.i., respectively, and their organs, including lungs, tracheas, nasal turbinates, soft palates, brains, hearts, submaxillary lymph nodes, tonsils, kidneys, spleens, livers, pancreas, and small intestines, were collected for viral RNA detection and virus titration. Sera were collected and antibodies against SARS-CoV-2 were detected by using the Double Antigen Sandwich ELISA kit (ProtTech, Luoyang, China) and a neutralization assay. To access the susceptibility of dogs, pigs, chickens, and ducks to SARS-CoV-2, three-month-old beagles The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint ducks) of CTan-H, and two (dogs) or three (pigs, chickens, ducks) uninfected animals were housed in the same room with their infected counterparts to monitor the transmission of CTan-H. Oropharyngeal and rectal swabs from all animals were collected every other day for viral RNA detection. One beagle was viral RNA positive by its rectal swab on day 2 p.i. and was euthanized on day 4 p.i.; its organs, including nasal turbinates, soft palates, tonsils, tracheas, lungs, brains, hearts, kidneys, spleens, livers, pancreas, and small intestines, were collected for viral RNA detection by qPCR. Sera were collected from all remaining animals on day 14 p.i., and antibodies against SARS-CoV-2 were detected by using a Double Antigen Sandwich ELISA kit (ProtTech, Luoyang, China). SARS-CoV-2 CTan strain (100 PFU) was incubated with two-fold serial dilutions of sera for 1 h at 37°C. A plaque assay was then performed in Vero E6 cells with the neutralization mixtures. Neutralizing antibody titers were calculated as the maximum serum dilution yielding a 50% reduction in the number of plaques relative to control serum prepared from uninfected animals. Tissues of animals were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into 4µm sections. The sections were stained with hematoxylin-eosin (H&E) or used in immunohistochemical (IHC) assays. The sections used for immunohistochemistry were dewaxed in xylene and hydrated through a series of descending concentrations of alcohol to water. For viral antigen retrieval, sections were immersed in citric acid/sodium citrate solution at 121 °C for 15 minutes. After cooling, the sections were treated with 3% hydrogen peroxide for 30 minutes to remove endogenous peroxidase activity and blocked with 8% skim milk to reduce nonspecific binding. After three 5-minute washes in TBS, the sections were incubated with rabbit anti-SARS-CoV-2 nucleoprotein monoclonal antibody The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.015347 doi: bioRxiv preprint