key: cord-0293176-osh3deg5
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: 2021-08-16
journal: bioRxiv
DOI: 10.1101/2021.08.16.456510
sha: cdff671578a4f056e08c6b31486259e31766ad1f
doc_id: 293176
cord_uid: osh3deg5

Susceptibility to SARS-CoV-2 and the outcome of 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 plaque forming units (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 when compared to young animals. Similar to observations in humans, this was associated with higher expressions levels of two key viral entry factors - ACE2 and TMPRSS2 - in the upper respiratory tract of aged ferrets.

In late December 2019, several cases of viral pneumonia of unknown etiology were described in a cluster of people in Wuhan, Hubei province, China. The causative virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was subsequently discovered and the disease named coronavirus disease 19 (COVID-19) 1 . Genome sequence analysis determined that SARS-CoV-2 is closely related to the bat SARS-like CoV RaTG13, a virus that was identified in horseshoe bats in the same geographic region in China. Given the close genetic and phylogenetic relationship of SARS-CoV-2 and SARS-like CoV found in bats, these animals are currently considered the likely source of the ancestral virus that originated SARS-CoV-2 [2] [3] [4] . Importantly, epidemiological investigations on the early clusters of COVID-19 revealed a strong link of the initial human cases with the Huanan Seafood Wholesale Market in Wuhan, where presumably SARS-CoV-2 may have been transmitted to humans through a yet unknown intermediate animal host 2, 5 . Since the description of first cases, SARS-CoV-2 spread across the world causing an unprecedented global pandemic that by August 2021 incurred in over 200 million human cases and more than 4.3 million deaths (https://covid19.who.int/).

Coronaviruses are positive sense, single stranded RNA viruses of the family Coronaviridae. Although coronaviruses usually cause mild respiratory infection in humans, they can also infect a range of other species resulting in broad clinical outcomes, varying from subclinical or mild-to-severe respiratory-to gastroenteric infections, which in some cases can lead to fatal disease 6 . Historically, several zoonotic coronaviruses have jumped into humans, including severe acute respiratory syndrome coronavirus (SARS-CoV), which was described in China in 2002, and Middle East respiratory syndrome coronavirus (MERS-CoV) identified in Saudi Arabia in 2012. Interestingly, SARS-CoV, MERS-CoV and SARS-CoV-2 present remarkable differences in infection outcomes. The fatality rates of SARS-CoV and MERS-CoV, for example, are much higher (~10% and ~35%, respectively) than that described for SARS-CoV-2 (<3%) 7, 8 . The clinical outcomes of SARS-CoV-2 infection are variable, ranging from asymptomatic infections -which represent most cases -to multiple organ failure and death 9 . Importantly, the most severe cases of COVID-19 resulting in death have been associated with other underlying health conditions 10, 11 . In addition, age is another risk factor that has been associated with distinct and often more severe outcomes of SARS-CoV-2 infection. While most human infections with SARS-CoV-2 lead to subclinical disease or only mild symptoms in young healthy adults, disease severity and mortality rates increase with age among individuals older than 30 years 12, 13 . Additionally, viral load, which is a direct measure of virus replication and that has been linked to severe disease outcomes, has also been shown to increase with the age of affected individuals 12, 14 . The mechanisms underlying the different SARS-CoV-2 infection outcomes and the contribution of comorbidities or age to these diverse outcomes, however, remains unknown.

The first step of SARS-CoV-2 infection involves binding of the viral spike (S) protein, more precisely the S receptor binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) -the viral cognate receptor 15, 16 . Following S RBD-ACE2 binding, the S protein is cleaved by host proteases (e.g. furin) into the S1/S2 subunits. The next step that takes place is cleavage of the S2 subunit at the S2' site by the transmembrane serine protease 2 (TMPRSS2), which leads to conformational changes that expose the fusion peptide enabling membrane fusion and completion of viral entry into host cells 16 . In humans, ACE2 is expressed in various cells and tissues, with high levels of the protein being expressed in the upper respiratory tract (URT) [17] [18] [19] . Notably, differential ACE2 and TMPRSS2 expression have been described in young and old people, with a higher percentage of ACE2 and TMPRSS2 expressing cells being detected in the nasal brushing of older people when compared to young individuals 20, 21 .

Here we assessed the effect of age on SARS-CoV-2 infection in ferrets. We compared the susceptibility of young (6-month-old) and aged (18-to 39-month-old) ferrets to four different doses of SARS-CoV-2. Viral replication, viral load and shedding in respiratory secretions and feces were monitored by rRT-PCR, virus isolation and titrations for 14 days post-inoculation (pi), while seroconversion was assessed by virus neutralization assays. The virological and serological findings were used to estimate the median infectious dose (ID50) of SARS-CoV-2 in young and aged ferrets. Additionally, expression of ACE2 and TMPRSS2 were assessed in upper and lower respiratory tract of ferrets and correlated with outcomes of SARS-CoV-2 infection and replication.

ferrets. To assess the susceptibility of ferrets to SARS-CoV-2, a total of 40 (20 young [6-monthold] 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. While control animals (four young and four aged animals) were kept under ABLS-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, c) . The body weight had slight variation in all groups, but no difference was noticed between SARS-CoV-2-inculated groups to the mock-control ferrets (normalized, day 0 represent 100%), in both young and aged animals (Fig. 1d, e) . No marked differences in daily activity of mock-control-or SARS-CoV-2-inoculated animals was noticed. It is important to note that, young and aged ferrets naturally exhibit differences in behavior 22 . While young animals are more active and curious, aged ferrets are less active and they are more restful and calmer than young animals, and this 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 mockinoculated ferrets throughout the experimental period.

Virus shedding in respiratory secretions and feces following SARS-CoV-2 inoculation. 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 swab (RS) samples were collected on days 0, 1, 3, 5, 7, 10 and 14 post-inoculation (pi) (Fig. 1a ). Viral RNA was detected throughout the experiment in young and aged ferrets in SARS-CoV-2-inoculated animals in varying levels until day 14 pi. Higher levels of viral RNA were detected in OPS when compared to NS and RS (Fig. 2) . Detection of 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, c) . In the groups inoculated with 10 2 PFU, 2/4 young ferrets tested positive by rRT-PCR on days 1-5 pi, while 4/4 aged animals were positive on days 1-7 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; p<0.02) (Fig. 2d, 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 1-5 pi and viral RNA loads were similar between young and aged animals (Fig. 2g ). Viral RNA was detected in RS samples in 2-3/4 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 1-5 pi in both young and aged animals ( Fig.   2j , k, l). Interestingly, marked differences in SARS-CoV-2 RNA load were observed in NS on day 7 pi (p<0.02), and on OPS and RS samples on day 10 pi (p<0.0001; 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, b) .

Higher frequency of infectious virus shedding was detected between days 3-5 pi (Fig. 3a, b) . In the group inoculated with 10 3 PFU, infectious virus shedding was detected in OPS of all 4/4 young and aged ferrets (Fig. 3c, d) . No infectious virus was detected in NS or RS samples in the groups inoculated with 10 2 or 10 3 PFU (Fig. 3a , b, c, d). In the group inoculated with 10 6 PFU, SARS-CoV-2 was isolated in all 4/4 young and aged ferrets, with all animals in the aged group shedding infectious virus between days 1-7 pi (Fig. 3f) . Infectious SARS-CoV-2 was also isolated from NS samples from 2/4 young-and 4/4 aged animals (Fig. 3e, 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 3-5 pi (Fig. 4) . In general, viral titers and the frequency of animals shedding detectable infectious SARS-CoV-2 were higher in the aged group, when compared to young animals (Fig. 4) . In animals inoculated with 10 2 PFU, statistically significant differences in virus shedding between young and aged ferrets was 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 log10TCID50.mL -1 [50% tissue culture infectious dose per milliliter]), only 1/4 young ferret shed 1.0 log10TCID50.mL -1 (Fig. 4a) . In animals inoculated with 10 3 PFU, all 4/4 young and aged animals shed infectious virus (Fig. 3c , d) on day 3 pi. Viral titers were significantly higher in aged ferrets when compared to 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 ( 

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 were also required to define an animal as infected. Given the consistency of virus shedding detected in oropharyngeal swabs ( Fig. 3; Fig. 4 ), the frequency of young and aged ferrets shedding virus in oropharyngeal secretions that seroconverted to SARS-CoV-2 were then used to estimate the median infectious dose (ID50) of the virus using the three-parameter logistic dose response model.

Notably, all three parameters (rRT-PCR positive OPS, VI positive OPS and seroconversion) used to determine the infection status of young and aged ferrets provided consistent outcomes ( Table 1) were higher in the nasal turbinates of aged ferrets than in young animals (p<0.05) (Fig. 8a, b) .

While no differences in ACE2 and TMPRSS2 expression between age groups were observed in the lungs (Fig. 8c, d) . Additionally, ACE2 expression was higher in the upper respiratory tract (URT) when compared to the lower respiratory tract (LRT) in both young and aged animals (p<0.05 and p<0.0001, respectively) ( Fig. 8e, g) . Expression levels of TMPRSS2, on the other hand, was higher in the LRT when compared URT in both young and aged ferrets (p<0.0001) (Fig.   8f , 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 (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 ID50 estimated using the three-parameter logistic dose response model in aged animals was ~32 PFU, while in young animals it was ~100 PFU (~3X higher). Similarly, a recent study conducted with a French SARS-CoV-2 isolate (UCN19) demonstrated successful infection of 10-month old ferrets with 2 x 10 3 PFU of the virus 23 . Interestingly, when younger ferrets (7month old) and a lower dose (5 x 10 2 PFU per animal) were used, only 1 of 6 inoculated ferrets was infected after intranasal inoculation with of a SARS-CoV-2 isolate from Australia (Victoria/1/2020) 24 . Despite inherent experimental differences, observations from these earlier studies are consistent with the results presented 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 humans and animal models [25] [26] [27] [28] [29] .

Differences in viral RNA load between age groups inoculated with 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 when compared to NS and RS. Higher viral loads in OPS or nasopharyngeal swab (NPS) samples have also been described in humans when compared to sputum or anterior nares samples (ANS) [30] [31] [32] [33] . Most importantly, shedding of infectious virus in aged ferrets inoculated with SARS-CoV-2 was prolonged when compared to 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 when 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 when compared to 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 when compared to 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 10-14 pi. These results corroborate virus shedding patterns observed in humans, in which the infectious period was shown to last 7-to-10 days following infection 34 . Importantly, in humans a decrease in SARS-CoV-2 infectivity parallels increased levels of neutralizing antibodies in serum 8, 34 . This was also observed here ferrets, which suggests that antibody responses could 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 mirror clinical observations in humans, which point to increased susceptibility and higher levels of virus replication in the respiratory tract of older people when compared to young children 12, 14 . Additionally, results presented here corroborate findings of a recent study by Kim and collaborators 35 , 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 35 . These changes are likely due to enhanced immune responses following higher viral replication in aged animals. Based on our ID50 estimates indicating that the infectious dose of SARS-CoV-2 is ~3X higher in young ferrets (~100 PFU) when compared to 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 expression of both ACE2 and TMPRSS2 were lower in nasal turbinates (primary site of SARS-CoV-2 replication in the URT) of young ferrets when compared to expression levels in aged animals. Additionally, expression of ACE2 was higher in the URT when compared to the lung (LRT). These observations corroborate findings in humans 20 and suggest that differences in expression of ACE2 and TMPRSS2 in the respiratory tract may contribute to agerelated susceptibility to SARS-CoV-2. The spectrum of factors that can contribute to SARS-CoV- 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 dose of the virus when compared to young animals. Additionally, SARS-CoV-2 replication in the URT and shedding in respiratory secretions is enhanced in aged ferrets when compared to young animals. We also showed that similar to what has been described in humans 20 , aged ferrets express higher levels of ACE2 and TMPRSS2 -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 expression levels of ACE2 and TMPRSS2 at the sites of virus entry. 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), inoculated with 150 µl of each sample and the inoculum adsorbed for 1 h at 37 °C with 5% CO2. Mock-inoculated cells were used as negative controls. After adsorption, replacement cell culture media supplemented with FBS as described above was added, and cells were incubated at 37 °C with 5% CO2 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 36, 37 . 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 cells cultures were subjected to IFA 36, 37 . OPS were subjected to end point titrations. For this, the original sample was subjected to limiting dilutions and inoculated into Vero E6/TMPRSS2 cells cultures in 96-well plates. At 48 h postinoculation, cells were fixed with 3.7% formaldehyde for 30 min at room temperature (rt), permeabilized with 0.2% Triton X-100 for 10 min at rt (in Phosphate Buffered Saline [PBS]) and subjected to an immunofluorescence assay (IFA) using a rabbit polyclonal antibody (pAb) specific for the SARS-CoV-2 nucleoprotein (N) (produced in Dr. Diel's laboratory) followed by incubation with a goat anti-rabbit IgG (goat anti-rabbit IgG, DyLight ® 594 Conjugate, Immunoreagent Inc.).

Karber's method and expressed as TCID50.ml -1 . Fig. 1 Experimental design, body weight and temperature following SARS-CoV-2inoculation. Forty ferrets (Mustela putorius furo) -Twenty young (6-month-old) and twenty aged (18 to 39-month-old [30.5±5.3 -average±SD]) were allocated to ten 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 post-inoculation (pi). Oropharyngeal-(OPS), nasal-(NS), rectal swab (RS), and blood were collected on various time points pi. Animals were humanely euthanized on day 14 pi. Following necropsy, tissues were collected and processed for rRT-PCR and virus isolation. Black hatched squares represent actual collection/measure time points for each sample type/parameter described in the figure (a). Body temperature following intranasal SARS-CoV-2 inoculation recorded throughout the experimental period in young (b) or aged ferrets (c). Body weight measurements throughout the experimental period in young (d) or aged ferrets (e) expressed as per cent from weight on day 0 (individual ferret weight was normalized to day 0, which represents 100%). The levels of ACE2 and TMPRSS2 expression in the upper (nasal turbinates) and lower (lungs) respiratory tract of all ferrets in the study were assessed by qRT-PCR. Expression levels of ACE2 and TMPRSS2 (a and b, respectively) in nasal turbinates of young and aged ferrets. Expression levels of ACE2 and TMPRSS2 (c and d, respectively) in the lungs of young and aged ferrets. Expression levels of ACE2 and TMPRSS2 in nasal turbinates and lung of young (e and f, respectively), and aged (g and h, respectively) ferrets. **** = p<0.0001; *** = p<0.001* = p<0.05 Fig. 9 Age-related differential susceptibility to SARS-CoV-2 infection. Aged ferrets were more likely to get infected when exposed to lower infectious dose of the virus when compared to young animals. SARS-CoV-2 replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets when 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.

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

The authors declare no competing interests.