key: cord-0765620-09qybafy
authors: Martins, Mathias; Fernandes, Maureen H. V.; Joshi, Lok R.; Diel, Diego G.
title: Age-Related Susceptibility of Ferrets to SARS-CoV-2 Infection
date: 2022-02-09
journal: J Virol
DOI: 10.1128/jvi.01455-21
sha: a2b2d858443a1ce85e610cb8968d0de18f2b4f1b
doc_id: 765620
cord_uid: 09qybafy

Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the outcome of coronavirus disease 2019 (COVID-19) have been linked to underlying health conditions and the age of affected individuals. Here, we assessed the effect of age on SARS-CoV-2 infection using a ferret model. For this, young (6-month-old) and aged (18- to 39-month-old) ferrets were inoculated intranasally with various doses of SARS-CoV-2. By using infectious virus shedding in respiratory secretions and seroconversion, we estimated that the infectious dose of SARS-CoV-2 in aged animals is ∼32 PFU per animal, while in young animals it was estimated to be ∼100 PFU. We showed that viral replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets compared to young animals. Similar to observations in humans, this was associated with higher transcription levels of two key viral entry factors, ACE2 and TMPRSS2, in the upper respiratory tract of aged ferrets. IMPORTANCE In humans, ACE2 and TMPRSS2 are expressed in various cells and tissues, and differential expression has been described in young and old people, with a higher level of expressing cells being detected in the nasal brushing of older people than young individuals. We described the same pattern occurring in ferrets, and we demonstrated that age affects susceptibility of ferrets to SARS-CoV-2. Aged animals were more likely to get infected when exposed to lower infectious dose of the virus than young animals, and the viral replication in the upper respiratory tract and shedding are enhanced in aged ferrets. Together, these results suggest that the higher infectivity and enhanced ability of SARS-CoV-2 to replicate in aged individuals is associated, at least in part, with transcription levels of ACE2 and TMPRSS2 at the sites of virus entry. The young and aged ferret model developed here may represent a great platform to assess age-related differences in SARS-CoV-2 infection dynamics and replication.

Intranasal inoculation of SARS-CoV-2 in young and aged ferrets results in subclinical infection. To assess the susceptibility of ferrets to SARS-CoV-2, a total of 40 (20 young [6-month-old] and 20 aged [18-to 39-month-old]) animals were allocated in 10 experimental groups (n = 4 per group). Animals were mock inoculated (control group) or inoculated intranasally with different doses of SARS-CoV-2 (10 1 , 10 2 , 10 3 , and 10 6 ) (Fig. 1A) . Virus-inoculated ferrets were housed in the animal biosafety level 3 (ABSL-3) facility at the East Campus Research Facility (ECRF) at Cornell University. Control animals (four young and four aged animals) were kept under ABSL-1 conditions. Both inoculated and control ferrets were housed individually in Horsfall HEPA filter cages throughout the 14-day experimental period. Following inoculation, clinical parameters, including temperature, body weight, activity, and signs of respiratory disease, were monitored daily. The body temperature in both age groups remained within physiological ranges throughout the experimental period, and no differences were observed between experimental groups ( Fig. 1B and C) . The body weight had slight variation in all groups, but no difference was noticed between SARS-CoV-2-inculated groups and the mock control ferrets (normalized; day 0 represents 100%) in both young and aged animals ( Fig. 1D and E) . No clinical signs were observed in young and aged ferrets, even when the highest viral dose (10 6 PFU) was inoculated. No marked differences in daily activity of mock control-or SARS-CoV-2-inoculated animals were noticed. It is important to note that young and aged ferrets naturally exhibit differences in behavior (18) . While young animals are more active and curious, aged ferrets are less active and are more restful and calmer than young animals, and this FIG 1 Experimental design, body weight, and temperature following SARS-CoV-2 inoculation. Forty ferrets (Mustela putorius furo), 20 young (6 months old) and 20 aged (18 to 39 months old [30.5 6 5.3; average 6 SD]) were allocated to 10 experimental groups (4 animals per group). Animals were inoculated intranasally with 1 ml (0.5 ml per nostril) MEM (mock control groups) or with a 1-ml virus suspension containing 10 1 , 10 2 , 10 3 , and 10 6 PFU of SARS-CoV-2 isolate NY67-20 on day 0. All animals were maintained individually in Horsfall HEPA-filtered cages. Clinical parameters, including temperature, body weight, activity, and signs of respiratory disease were monitored daily for 14 days postinoculation (pi). Oropharyngeal (OPS), nasal (NS), and rectal swab (RS) and blood samples were collected at various times postinoculation. Animals were humanely euthanized on day 14 pi. Following necropsy, tissues were collected and processed for rRT-PCR and virus isolation. (A) Black hatched squares represent actual collection/measure time points for each sample type/ parameter described. (B and C) Body temperature following intranasal SARS-CoV-2 inoculation recorded throughout the experimental period in young (B) or aged (C) ferrets. (D and E) Body weight measurements throughout the experimental period in young (D) or aged (E) ferrets expressed as percent weight on day 0 (individual ferret weight was normalized to day 0, which represents 100%).

was also observed in our study. Additionally, no clinical signs of respiratory disease were observed in any of the SARS-CoV-2-inoculated or control mock-inoculated ferrets throughout the experimental period.

Aged ferrets shed higher viral loads in respiratory secretions and feces than young ferrets. The dynamics of SARS-CoV-2 replication and shedding were monitored in respiratory secretions and feces by rRT-PCR following inoculation. Oropharyngeal (OPS), nasal (NS), and rectal (RS) swab samples were collected on days 0, 1, 3, 5, 7, 10, and 14 postinoculation (pi) (Fig. 1A) . Viral RNA was detected throughout the experiment in young and aged ferrets in SARS-CoV-2-inoculated animals at various levels until day 14 pi. Higher levels of viral RNA were detected in OPS than NS and RS (Fig. 2) . Detection of Detection of viral RNA in OPS, NS, and RS samples (J, K, and L, respectively) in young and aged ferrets (31 6 3 months; average 6 SD) inoculated with 10 6 PFU of SARS-CoV-2. Viral RNA loads are expressed as 45 rRT-PCR cycles minus the actual C T value. Day 0 represents swab samples collected prior to inoculation with SARS-CoV-2. ****, P , 0.0001; ***, P , 0.001; **, P , 0.002 *, P , 0.02. viral RNA in animals inoculated with 10 1 PFU of SARS-CoV-2 was restricted to a single aged ferret on day 3 pi in which viral RNA was detected in OPS and RS samples ( Fig. 2A and C) . In the groups inoculated with 10 2 PFU, 2/4 young ferrets tested positive by rRT-PCR on days 1 to 5 pi, while 4/4 aged animals were positive on days 1 to 7 pi. Importantly, viral RNA loads in OPS and RS samples were significantly higher in aged animals on days 3 (P , 0.02), 7, and 10 pi (P , 0.001 and P , 0.02) ( Fig. 2D and F ). In the groups inoculated with 10 3 PFU, all young (4/4) and aged (4/4) ferrets tested positive for SARS-CoV-2 RNA in OPS samples between days 1 and 5 pi, and viral RNA loads were similar between young and aged animals (Fig. 2G ). Viral RNA was detected in RS samples in 2 to 3 of 4 animals in both young and aged groups until day 10 pi (Fig. 2I ). In the groups inoculated with 10 6 PFU, SARS-CoV-2 RNA was consistently detected in OPS, NS, and RS samples between days 1 and 5 pi in both young and aged animals ( Fig. 2J to L). Interestingly, marked differences in SARS-CoV-2 RNA load were observed in NS on day 7 pi (P , 0.02) and in OPS and RS samples on day 10 pi (P , 0.0001 and P , 0.002, respectively) among young and aged ferrets.

To assess shedding of infectious virus in young and aged ferrets inoculated with SARS-CoV-2, rRT-PCR-positive respiratory and fecal samples were subjected to virus isolation in cell culture. Infectious SARS-CoV-2 was isolated from OPS samples from 2/4 young ferrets and from 4/4 aged ferrets inoculated with 10 2 PFU in at least one time point following infection ( Fig. 3A and B). Higher frequency of infectious virus shedding was detected between days 3 and 5 pi ( Fig. 3A and B ). In the group inoculated with 10 3 PFU, infectious virus shedding was detected in OPS of all young and aged ferrets ( Fig. 3C and D). No infectious virus was detected in NS or RS samples in the groups inoculated with 10 2 or 10 3 PFU (Fig. 3A to D). In the group inoculated with 10 6 PFU, SARS-CoV-2 was isolated in 4/4 young and aged ferrets, with all animals in the aged group shedding infectious virus between days 1 and 7 pi (Fig. 3F ). Infectious SARS-CoV-2 was also isolated from NS samples from 2/4 young and 4/4 aged animals ( Fig. 3E and F) .

In addition to virus isolation, all the OPS samples that tested positive by rRT-PCR were subjected to virus quantitation. Peak viral titers were detected between days 3 and 5 pi (Fig. 4) . In general, viral titers and the frequency of animals shedding detectable infectious SARS-CoV-2 were higher in the aged group than in young animals ( Fig.  4) . In animals inoculated with 10 2 PFU, statistically significant differences in virus shedding between young and aged ferrets were observed on day 5 pi (P , 0.002) (Fig. 4A ). While 4/4 aged ferrets shed virus (titers ranging from 1.0 to 2.8 log 10 TCID 50 Áml 21 [50% tissue culture infectious dose per milliliter]), only 1/4 young ferrets shed 1.0 log 10 TCID 50 Áml 21 (Fig. 4A ). In animals inoculated with 10 3 PFU, 4/4 young and aged animals shed infectious virus ( Fig. 3C and D) on day 3 pi. Viral titers were significantly higher in aged ferrets than young animals on day 5 pi (P , 0.001) (Fig. 4B ). Although differences in viral titers were observed between young and aged ferrets inoculated with 10 2 and 10 3 PFU at limited time points, these differences were more evident between the age groups in animals inoculated with the highest SARS-CoV-2 dose (10 6 PFU). In these groups, infectious virus was recovered from OPS and NS ( Fig. 3E and F). While young ferrets shed between 1.0 and 2.0 log 10 TCID 50 Áml 21 in OPS secretions, aged animals shed up to 3.8 log 10 TCID 50 Áml 21 of virus (days 3 to 5 pi, P , 0.0001; day 7 pi, P , 0.001; Fig. 4C ). Viral titers were above the limit of quantification (1.0 log 10 TCID 50 Áml 21 ) in 4/4 aged ferrets from day 1 to 7 pi; however, in young animals, viral titers were only detected in 2/4 on days 1 and 3 pi and 1/4 on day 5 pi (Fig. 4C) . These results indicate that SARS-CoV-2 replication was more efficient in the upper respiratory tract of aged animals, suggesting that aged ferrets are more susceptible to SARS-CoV-2 infection.

Viral load in tissues. Viral RNA load and tissue distribution of SARS-CoV-2 were assessed on day 14 pi. Nasal turbinate, soft palate/tonsil, and left and right lungs (cranial and caudal lobes) were collected and processed by rRT-PCR. SARS-CoV-2 RNA was only detected in nasal turbinate and soft palate/tonsil from young and aged ferrets inoculated with 10 6 PFU (Fig. 5 ). Viral RNA was detected with high threshold cycle (C T ) values in the nasal turbinate of 3/4 and soft palate/tonsil of 2/4 young ferrets, while in aged animals, the nasal turbinate of 4/4 and the soft palate/tonsil of 3/4 animals were rRT-PCR positive. While the tissues were positive for viral RNA, no infectious virus was recovered after three blind passages in cell culture.

Neutralizing antibody responses to SARS-CoV-2. The serological response to SARS-CoV-2 was assessed by virus neutralization (VN) assay. All control (mock-inoculated) animals and those inoculated with 10 1 PFU remained seronegative until the end of the experiment on day 14 pi (Fig. 6A ). In the groups inoculated with 10 2 PFU of SARS-CoV-2, 2/4 young and 4/4 aged ferrets seroconverted and presented neutralizing antibody titers on day 14 pi (Fig. 6B ) (P , 0.02). All animals in the 10 3 PFU group, regardless of age, seroconverted by day 14 pi, and no differences in NA titers between age groups were detected (Fig. 6C ). Among the animals inoculated with the highest dose (10 6 PFU), both young and aged ferrets seroconverted as early as day 7 pi, with higher antibody titers being detected on day 14 pi (Fig. 6D) . Together, these results confirm successful infection of 2/4 young ferrets and 4/4 aged ferrets inoculated with 10 2 PFU of SARS-CoV-2.

Aged ferrets are more likely to get infected when exposed to lower infectious dose of SARS-CoV-2 than young animals. The infectivity of SARS-CoV-2 in young and aged ferrets was assessed using three parameters: (i) rRT-PCR in oropharyngeal secretion, (ii) virus isolation in oropharyngeal secretion, and (iii) seroconversion to SARS-CoV-2 on day 14 pi. This resulted in three binary (i.e., positive or negative) response variables for each ferret in the study. Each animal was determined to be infected when at least two of the three parameters evaluated were positive. It is important to note that rRT-PCR results alone were not used as a definitive proof of infection; shedding of infectious virus or seroconversion was also required to define an animal as infected. Table 1) . None of the animals in the young or aged groups shed infectious virus or seroconverted when inoculated with 10 1 PFU of SARS-CoV-2, indicating no infection. In animals inoculated with 10 2 PFU, 2/4 young animals tested positive by rRT-PCR and virus isolation and the same two animals seroconverted, as determined by detection of NA on day 14 pi, while all aged ferrets inoculated with 10 2 PFU were rRT-PCR and virus isolation positive and seroconverted to SARS-CoV-2. All young and aged animals inoculated with 10 3 and 10 6 PFU tested positive by rRT-PCR and infectious virus and seroconverted to SARS-CoV-2 by day 14 pi. Based on the infection status of the animals, the estimated median infectious dose (ID 50 ) for SARS-CoV-2 in aged ferrets was 31.6 PFU (;32), while in young animals the ID 50 was estimated at 100.1 PFU (;100; Fig. 7 ).

Higher levels of ACE2 and TMPRSS2 transcription in the respiratory tract of aged ferrets compared to young animals. To investigate factors that could contribute to susceptibility of young and aged ferrets to SARS-CoV-2, we assessed transcription of ACE2 and TMPRSS2, two critical viral entry factors, in the upper (nasal turbinates) and lower (lungs) respiratory tracts of all animals in the study (young [n = 20] and aged ferrets [n = 20]) by qRT-PCR. Notably, ACE2 and TMPRSS2 mRNA levels were higher in the nasal turbinates of aged ferrets than in young animals (P , 0.05) ( Fig. 8A and B). No differences in transcription of ACE2 and TMPRSS2 between age groups were observed in the lungs ( Fig. 8C and D) . Additionally, transcription of ACE2 was higher in the URT than the LRT in both young and aged animals (P , 0.05 and P , 0.0001, respectively) ( Fig. 8E and G) . Transcription levels of TMPRSS2, on the other hand, were higher in the LRT than URT in both young and aged ferrets (P , 0.0001) (Fig. 8F and H) .

Here, we compared the susceptibility of young and aged ferrets to SARS-CoV-2 and assessed the infectivity of the virus by inoculating young and aged animals with increasing viral doses. As evidenced by SARS-CoV-2 replication in the upper respiratory tract, virus shedding in respiratory secretions (viral RNA and infectious virus), and seroconversion following intranasal inoculation, our study shows that aged ferrets are more susceptible than young ferrets to SARS-CoV-2 infection.

Intranasal inoculation of 10 1 PFU of SARS-CoV-2 did not result in productive infection in young or in aged ferrets. While all aged animals were successfully infected after inoculation with 10 2 PFU/animal, only 2 of 4 young ferrets were infected with this viral dose. Importantly, the infection rates observed in young and aged animals were consistent and supported by all three criteria used to define productive infection: (i) positive rRT-PCR, (ii) infectious virus shedding in respiratory secretions, and (iii) seroconversion (Table 1 ). These results suggest that the infectious dose of SARS-CoV-2 required to infect aged ferrets is lower than the dose required to infect young animals. Indeed, the ID 50 estimated using the three-parameter logistic dose-response model in aged animals was ;32 PFU, while in young animals it was ;100 (19) . Interestingly, when younger ferrets (7 months old) and a lower dose (5 Â 10 2 PFU per animal) were used, only 1 of 6 inoculated ferrets was infected after intranasal inoculation with a SARS-CoV-2 isolate from Australia (Victoria/1/2020) (20) . Despite inherent experimental differences, observations from these earlier studies are consistent with the results presented Age Affects Susceptibility of Ferrets to SARS-CoV-2 Journal of Virology here demonstrating a higher susceptibility of older ferrets to SARS-CoV-2. It is important to note, however, that both UCN19 and Victoria/1/2020 isolates belong to early SARS-CoV-2 lineages (lineage A) that do not contain the spike mutation D614G, which is present in the isolate NYI67-20 (lineage B.1) used in our study. This is relevant, as several studies have shown that the S D614G mutation is associated with increased infectivity and transmission of SARS-CoV-2 in animal models and in humans (21) (22) (23) (24) (25) .

Differences in viral RNA load between age groups inoculated with the same viral doses were observed mainly on days 7 and 10 pi, when aged ferrets remained positive, while viral RNA was no longer detected in most young animals. The highest viral loads were detected on OPS samples compared to NS and RS. Higher viral loads in OPS or nasopharyngeal swab (NPS) samples have also been described in humans compared to sputum or anterior nares samples (ANS) (26) (27) (28) (29) . Most importantly, shedding of infectious virus in aged ferrets inoculated with SARS-CoV-2 was prolonged compared to that in young animals, and the viral titers detected in this age group were higher than those detected in young animals.

Infectious virus was isolated from aged ferrets with a higher frequency and for prolonged time compared to young animals. These differences were more pronounced in animals inoculated with the highest viral dose (10 6 PFU), from which infectious virus was isolated from all four aged ferrets from day 1 to 7 pi. Additionally, the viral titers were significantly higher in aged animals than young ferrets on day 5 pi in the 10 2 PFU (P , 0.002) and 10 3 PFU (P , 0.001) groups and on days 3, 5, and 7 pi in the 10 6 PFU group (P , 0.001). Together, these results demonstrate that SARS-CoV-2 replicates more efficiently in aged ferrets than young animals. Regardless of the viral dose, after day 7 pi, no infectious virus was isolated from any animal inoculated with the different viral doses, despite detection of viral RNA by rRT-PCR up to day 10 to 14 pi. These results corroborate virus shedding patterns observed in humans in which the infectious period was shown to last 7 to 10 days following onset of clinical signs (30) . Importantly, in humans a decrease in SARS-CoV-2 infectivity parallels increased levels of neutralizing antibodies in serum (4, 30) . This was also observed here, which suggests that antibody responses play a role in viral clearance. Innate immune responses at the site of virus replication, however, may also play a role and contribute to control the infection in the respiratory tract.

Our findings showing higher susceptibility of aged ferrets to SARS-CoV-2 infection in the present study parallel clinical observations in humans, which point to increased susceptibility and higher levels of virus replication in the respiratory tract of older people compared to young children (8, 10) . Additionally, results presented here corroborate findings of a recent study by Kim and collaborators (31) , who showed age-related differences in viral load in the respiratory tract and lung histopathology in ferrets inoculated with SARS-CoV-2. This study also showed that expression levels of genes related to the interferon (IFN) pathway, activated T cells, and macrophage responses were increased in older ferrets following SARS-CoV-2 infection (31). These changes are FIG 9 Age-related differential susceptibility to SARS-CoV-2 infection. Aged ferrets were more likely to get infected when exposed to lower infectious doses of the virus than young animals. SARS-CoV-2 replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets compared to young animals. Notably, aged ferrets express higher levels of ACE2 and TMPRSS2 in the upper respiratory tract. Together these results suggest that the higher infectivity and enhanced ability of SARS-CoV-2 to replicate in aged individuals is associated with expression levels of two of the molecules that are critical for SARS-CoV-2 infection and host cell entry.

likely due to enhanced immune responses following higher viral replication in aged animals. Based on our ID 50 estimates indicating that the infectious dose of SARS-CoV-2 is ;3Â higher in young ferrets (;100 PFU) than aged animals (;32 PFU), we hypothesized that differential expression of key SARS-CoV-2 entry factors, such as the ACE2 receptor and the TMPRSS2 protease, could underlie age-related differences in their susceptibility to SARS-CoV-2. Notably, we showed that transcription of both ACE2 and TMPRSS2 were lower in nasal turbinates (primary site of SARS-CoV-2 replication in the URT) of young ferrets compared to mRNA levels in aged animals. Additionally, transcription of ACE2 was higher in the URT than the lung (LRT). These observations corroborate findings in humans (16) and suggest that differences in ACE2 and TMPRSS2 mRNA levels in the respiratory tract contribute to age-related susceptibility to SARS-CoV-2. In future studies it will be important to investigate expression of ACE2 and TMPRSS2 proteins in URT and determine the dynamic changes in their expression throughout the course of SARS-CoV-2 infection in young and aged ferrets. The spectrum of factors that can contribute to SARS-CoV-2 susceptibility, however, is broad, and further studies are needed to assess the function and involvement of other entry and immune response-related factors on SARS-CoV-2 infection and replication in different age groups. The young and aged ferret models developed here provide an excellent platform to investigate age-related differences in susceptibility to SARS-CoV-2 infection and replication and the host and viral factors that play a role in these dynamic interactions. This model may be particularly useful to dissect the mechanisms/functions emerging in several of the SARS-CoV-2 variants (e.g., P.1 and B.1.617.2), which seem to have an enhanced ability to infect younger individuals.

In summary, here we demonstrated that age affects susceptibility of ferrets to SARS-CoV-2, with aged animals being more likely to get infected when exposed to lower infectious doses of the virus than young animals. Additionally, SARS-CoV-2 replication in the URT and shedding in respiratory secretions is enhanced in aged ferrets compared to young animals. We also showed that, similar to what has been described in humans (16) , aged ferrets present higher levels of ACE2 and TMPRSS2 mRNAs, two key factors determining virus entry into cells, in the URT (Fig. 9 ). Together, these results suggest that the higher infectivity and enhanced ability of SARS-CoV-2 to replicate in aged individuals is associated, at least in part, with transcription/expression levels of ACE2 and TMPRSS2 at the sites of virus entry.

Virus and cells. Vero E6 (ATCC CRL-1586) and Vero E6/TMPRSS2 (JCRB1819; JCRB Cell Bank) cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 UÁml 21 ), streptomycin (100 mgÁml 21 ), and gentamicin (50 mgÁml 21 ). The cell cultures were maintained at 37°C with 5% CO 2 . The SARS-CoV-2 isolate NYI67-20 (B.1 lineage) was propagated in Vero E6 cells. Low-passage-number virus stocks (passage 3) were prepared, cleared by centrifugation (2,000 Â g for 15 min), and stored at 280°C. The whole-genome sequence of the virus stock was determined to confirm that no mutations occurred during passages in cell culture. The titer of virus stock was determined according to the Spearman and Karber method and expressed as PFU per milliliter.

Animal housing and experimental design. All animals were handled in accordance with the Animal Welfare Act, and the study procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Cornell University (IACUC approval number 2020-0064). A total of 40 ferrets (Mustela putorius furo) were obtained from a commercial breeder (Triple F Farms, Gillett, PA, USA). Twenty young (6-month-old ferrets) and 20 aged (18 to 39 months [30.5 6 5.3; average 6 standard deviation, or SD]) ferrets were randomly allocated to experimental groups (n = 4). The age and sex of the ferrets allocated in each group are presented in Table 2 . While the control animals were housed at the ASBL-1 facility, all the virus-inoculated ferrets were housed in the ASBL-3 facility at the East Campus Research Facility (ECRF) at Cornell University. After 72 h of acclimation, ferrets were sedated and inoculated intranasally with 1 ml (0.5 ml per nostril) of a virus suspension containing 10 1 , 10 2 , 10 3 , or 10 6 PFU of SARS-CoV-2 isolate NYI67-20 (groups 2, 3, 4 and 5, respectively [n = 4/group]). Ferrets of each age maintained as mock-inoculated control (group 1, n = 4) were mock inoculated with Vero E6 cell culture medium supernatant. All ferrets were maintained individually in Horsfall HEPA-filtered cages, connected to the ASBL-3 facility's exhaust system. Body temperatures and weight were measured on a daily basis. Oropharyngeal (OPS), nasal (NS), and rectal (RS) swabs were collected under sedation (dexmedetomidine) on days 0, 1, 3, 5, 7, 10, and 14 pi. Upon collection, swabs were placed in sterile tubes containing 1 ml of viral transport medium (VTM; Corning, Glendale, AZ, USA) and stored at 280°C until being processed for further analyses. Blood was collected under sedation (dexmedetomidine) through cranial vena cava venipuncture using a 3-ml sterile syringe and 23-gauge, 1-inch needle and transferred into serum separator tubes on days 0, 3, 7, and 14 pi. The blood tubes were centrifuged at 1,500 Â g for 10 min, and serum was aliquoted and stored at 220°C until further analysis. Ferrets were humanely euthanized on day 14 pi under deep inhalatory anesthesia using isoflurane followed by cardiac puncture. Following necropsy, tissues, including nasal turbinate, soft palate/tonsil, and left and right lung, were collected and processed for rRT-PCR and virus isolation.

Nucleic acid isolation and real-time reverse transcriptase PCR. Nucleic acid was extracted from OPS, NS, RS, and tissue samples collected at necropsy. For OPS, NS, and RS samples, 200 ml of cleared swab supernatant was used for nucleic acid extraction. For tissues, 0.2 g of each tissue was minced with a sterile scalpel, resuspended in 2 ml DMEM (10%, wt/vol), and homogenized using a stomacher (one speed cycle of 60 s; Stomacher 80 Biomaster). Next, 200 ml of the tissue homogenate supernatant was used for RNA extraction using the MagMax Core extraction kit (Thermo Fisher, Waltham, MA, USA) and the automated KingFisher Flex nucleic acid extractor (Thermo Fisher, Waltham, MA, USA) following the manufacturer's recommendations. The real-time reverse transcriptase PCR (rRT-PCR) was performed using the EZ-SARS-CoV-2 real-time RT-PCR assay (Tetracore Inc., Rockville, MD, USA), which detects both genomic and subgenomic viral RNA targeting the virus nucleocapsid protein (N) gene. The limit of detection (LOD) for this assay was previously established as 250 viral genome copies per ml or 1.75 viral genome copies per reaction (32) . At this viral concentration, the assay results in mean C T values of 35.17 in 20 replicates. Based on this LOD, a C T cutoff of 37.09 was established (35.17 1 3 SD), and all samples with initial C T values above the cutoff are retested. Only samples with reproducible detection upon repeated testing were considered positive. Viral RNA loads are expressed as 45 rRT-PCR cycles minus the actual C T value. An internal inhibition control was included in all reactions. Positive and negative amplification controls were run side by side with test samples.

Virus isolation and titrations. All OPS, NS, and RS and tissue samples that tested positive for SARS-CoV-2 by rRT-PCR were subjected to virus isolation under BSL-3 conditions. Twenty-four-well plates were seeded with ;75,000 Vero E6/TMPRSS2 cells per well 24 h prior to sample inoculation. Cells were rinsed with phosphate-buffered saline (PBS) (Corning, Glendale, AZ, USA) and inoculated with 150 ml of each sample, and the inoculum was adsorbed for 1 h at 37°C with 5% CO 2 . Mock-inoculated cells were used as negative controls. After adsorption, replacement cell culture medium supplemented with FBS as described above was added, and cells were incubated at 37°C with 5% CO 2 and monitored daily for cytopathic effect (CPE) for 3 days. SARS-CoV-2 replication in CPE-positive cultures was confirmed with an immunofluorescence assay (IFA) as previously described (33, 34) . Cell cultures with no CPE were frozen, thawed, and subjected to two additional blind passages/inoculations in Vero E6/TMPRSS2 cell cultures. At the end of the third passage, the cell cultures were subjected to IFA (33, 34) . OPS were subjected to endpoint titrations. For this, the original sample was subjected to limiting dilutions and inoculated into Vero E6/TMPRSS2 cell cultures in 96-well plates. At 48 h postinoculation, cells were fixed with 3.7% formaldehyde for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 10 min at room temperature (in phosphate buffered saline [PBS]), and subjected to an IFA using a rabbit polyclonal antibody (pAb) specific for the SARS-CoV-2 nucleoprotein (N) (produced in D. G. Diel's laboratory), followed by incubation with goat anti-rabbit IgG (goat anti-rabbit IgG; DyLight 594 conjugate; Immunoreagent Inc.). Virus titers were determined at each time point using endpoint dilutions and the Spearman and Karber's method and expressed as TCID 50 Áml 21 .

Serology. Neutralizing antibody responses to SARS-CoV-2 were assessed by VN assay performed under BSL-3 laboratory conditions. Twofold serial dilutions (1:8 to 1:1,024) of serum samples were incubated with 100 to 200 TCID 50 of SARS-CoV-2 isolate NYI67-20 for 1 h at 37°C. Following incubation of serum and virus, 50 ml of a cell suspension of Vero E6 cells was added to each well of a 96-well plate and incubated for 48 h at 37°C with 5% CO 2 . The cells were fixed, permeabilized, and subjected to IFA as described above. Unbound antibodies were washed from cell cultures by rinsing the cells PBS, and virus infectivity was assessed under a fluorescence microscope. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited SARS-CoV-2 infection/replication. FBS and positive and negative serum samples from white-tailed deer fawns (33) were used as controls.

Expression level of ACE2 and TMPRSS2 genes in the respiratory tract. RNA samples extracted from nasal turbinate and lungs (described above) were treated with DNA-free kit DNase treatment and removal (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The amount of RNA in each sample was measured using a Qubit RNA BR assay kit (Invitrogen, Carlsbad, CA, USA), and then all the samples were diluted in double-distilled water (ddH 2 O) to obtain a concentration of 5 ng/ml. 

The primer and probe sequences for ferret ACE2 were 59-GATGTGAGGGTGAGCGATTT-39, 59-GGGACTTCCTGATAGCTTCTTC-39, and 59-6-FAM/TGACATCAT/ZEN/TCCCAGAGCTGACGT-39IABkFQ based on Mustela putorius furo ACE2, GenBank accession number NM_001310190.1; for ferret, TMPRSS2 sequences were 59-CGGTGTTTACGGACTGGATTTA-39, 59-GGTGCCCAGAGAATGAAGAA-39, and 59-6-FAM/ACAGCTAAT/ZEN/CCATGTGCCCTGTGT-39-IABkFQ based on Mustela putorius furo serine transmembrane protease 2 (TMPRSS2), GenBank accession number NM _001386127.1; and for ferret, GAPDH sequences were 59-GATGCTGGTGCTGAGTATGT-39, 59-CAGAAGGAGCAG AGATGATGAC-39, and 59-6-FAM/TTCACCACC/ZEN/ATGGAGAAGGCTGG-39-IABkFQ/based on Mustela putorius furo GAPDH, GenBank accession number NM_001310173.1. Real-time RT-PCR amplifications were performed in 10-ml reaction mixtures, with 3 ml of RNA, 5 ml of TaqMan RT-PCR mix (TaqMan RNA-to-Ct 1-step kit) (Applied Biosystems

Given the consistency of virus shedding detected in oropharyngeal swabs, the frequency of young and aged ferrets shedding virus in oropharyngeal secretions that seroconverted to SARS-CoV-2 were then used to calculate ID 50 . The three-parameter logistic model (3PL) was used to estimate ID 50 . To determine the dose-response curve using 3PL model, viral dose was log transformed, the bottom response value was constrained to 0, and the top response value was constrained to 1. The three-parameter logistic model was implemented in GraphPad Prism software

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

Chapter 28-Coronaviridae

Overview of lethal human coronaviruses

SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis

Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China

Severe outcomes among patients with coronavirus disease 2019 (COVID-19)-United States

Clinical course and factors associated with outcomes among 1904 patients hospitalized with COVID-19 in Germany: an observational study

SARS-CoV-2 viral load distribution reveals that viral loads increase with age: a retrospective cross-sectional cohort study

Age-specific mortality and immunity patterns of SARS-CoV-2

Impact of severe acute respiratory syndrome coronavirus 2 viral load on risk of intubation and mortality among hospitalized patients with coronavirus disease 2019

Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Elevated ACE-2 expression in the olfactory neuroepithelium: implications for anosmia and upper respiratory SARS-CoV-2 entry and replication

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

A single-cell RNA expression map of human coronavirus entry factors

Nasal gene expression of angiotensin-converting enzyme 2 in children and adults

Ferret Mustela furo

Hamster and ferret experimental infection with intranasal low dose of a single strain of SARS-CoV-2

Dose-dependent response to infection with SARS-CoV-2 in the ferret model and evidence of protective immunity

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo

SARS-CoV-2 spike D614G change enhances replication and transmission

Spike mutation D614G alters SARS-CoV-2 fitness

Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus

Evaluating the effects of SARS-CoV-2 spike mutation D614G on transmissibility and pathogenicity

Self-collected anterior nasal and saliva specimens versus health care worker-collected nasopharyngeal swabs for the molecular detection of SARS-CoV-2

Relative sensitivity of anterior nares and nasopharyngeal swabs for initial detection of SARS-CoV-2 in ambulatory patients: rapid review and meta-Analysis

Comparison of nasopharyngeal and oropharyngeal swabs for SARS-CoV-2 detection in 353 patients received tests with both specimens simultaneously

Performance of saliva, oropharyngeal swabs, and nasal swabs

Estimating infectiousness throughout SARS-CoV-2 infection course

Age-dependent pathogenic characteristics of SARS-CoV-2 infection in ferrets

Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples

Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2

Severe SARS-CoV-2 infection in a cat with hypertrophic cardiomyopathy

One-step versus two-step real

We thank the Center for Animal Resources and Education (CARE) staff and Cornell Biosafety team for the support.