Host barriers to SARS-CoV-2 demonstrated by ferrets in a high-exposure domestic setting Kaitlin Sawatzkia,1, Nichola J. Hilla, Wendy B. Puryeara, Alexa D. Fossa, Jonathon J. Stonea, and Jonathan A. Runstadlera aDepartment of Infectious Disease and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA 01536 Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved March 23, 2021 (received for review December 12, 2020) Ferrets (Mustela putorius furo) are mustelids of special relevance been proposed as an animal model to study SARS-CoV-2 to laboratory studies of respiratory viruses and have been shown transmission. Based on in vivo data, we expect all naïve ferrets in to be susceptible to severe acute respiratory syndrome coronavirus 2 direct contact with an infected ferret will 1) become infected, 2) (SARS-CoV-2) infection and onward transmission. Here, we report the have measurable viral shedding or RNA via oral swabs up to 19 d results of a natural experiment where 29 ferrets in one home had postinfection, and 3) seroconvert with measurable antibodies prolonged, direct contact and constant environmental exposure to against SARS-CoV-2 receptor binding domain (RBD) (18, 19). two humans with symptomatic disease, one of whom was confirmed In March 2020, during the first wave of the SARS-CoV-2/ positive for SARS-CoV-2. We observed no evidence of SARS-CoV-2 transmission from humans to ferrets based on viral and antibody COVID-19 pandemic in the New England area, we developed a assays. To better understand this discrepancy in experimental and rapid response study to investigate the potential for human-to- natural infection in ferrets, we compared SARS-CoV-2 sequences animal spillover and onward transmission in domestic, farm, and from natural and experimental mustelid infections and identified wildlife species (CoVERS: Coronavirus Epidemiological Re- two surface glycoprotein Spike (S) mutations associated with mustel- sponse and Surveillance). The goal of CoVERS is to understand ids. While we found evidence that angiotensin-converting enzyme II whether and how SARS-CoV-2 transmission is occurring at these provides a weak host barrier, one mutation only seen in ferrets is interfaces, to refine public health guidelines, investigate whether located in the novel S1/S2 cleavage site and is computationally pre- there are additional risks to animal or human health associated dicted to decrease furin cleavage efficiency. These data support the with spillover, and evaluate the potential for establishment of MICROBIOLOGY idea that host factors interacting with the novel S1/S2 cleavage site endemic reservoirs. In the CoVERS in-home study, participants may be a barrier in ferret SARS-CoV-2 susceptibility and that domestic “ ” ferrets are at low risk of natural infection from currently circulating are sent a swab and send kit, which provides materials and SARS-CoV-2. We propose two mechanistically grounded hypotheses instructions to safely take longitudinal nasal and oral samples for mustelid host adaptation of SARS-CoV-2, with possible effects from their animals, store them in their freezers, and send them that require additional investigation. back for viral screening. This community science approach allows wide surveillance with no risk of human transmission, as kits are SARS-CoV-2 | virology | transmission | genetics | coronavirus decontaminated and opened in biosafety cabinets. Here, we highlight one enrolled household that created an exceptional evere acute respiratory syndrome coronavirus 2 (SARS-CoV- natural experiment with direct relevance to our understanding of S2), the virus that causes COVID-19, is a zoonotic member of SARS-CoV-2 reverse zoonosis and animal models of disease. Coronaviridae that emerged in 2019 as a major viral pandemic ∼ (1). As of February 2021, there have been 102 million confirmed Significance COVID-19 cases globally and ∼2.2 million deaths (2). SARS-CoV- 2 uses angiotensin I converting enzyme-2 (ACE2) as its primary Ferrets have been demonstrated to be susceptible to labora- cellular receptor for host entry and infection (3–5). In silico anal- tory infection of SARS-CoV-2, raising the possibility of natural yses of ACE2 genes in diverse mammalian species show that res- transmission from humans into their pets in domestic settings. idues important to viral binding are moderately conserved between We demonstrate that ferrets may have host barriers that limit humans and several domestic animals, and a broad range of species natural infection and transmission. First, we find no evidence have been demonstrated to be permissive to infection in vitro and of infection in 29 ferrets from a home with constant exposure in vivo (6–10). to two adults with one confirmed and one suspected case of It is not yet known whether natural infection of animals plays a symptomatic COVID-19. Second, we analyze genetic sequences role in public health epidemiology or has the potential to es- from viruses and hosts and demonstrate that ferrets have ge- tablish endemic reservoirs and threaten wildlife. SARS-CoV-2 netic factors that confer resistance to natural SARS-CoV-2 in- has been observed to be capable of natural human-to-animal re- fection. These data suggest that ferret infection may require verse zoonoses, transmitting from infected individuals into mink – Neovison viral adaptation, and therefore ferrets may only be semi- (11), dogs (12), and felines (13 15). American mink ( permissive models of SARS-CoV-2 disease or transmission. vison) are currently the only species observed to have natural human-to-animal spillover and onward transmission (11). To date, Author contributions: K.S., N.J.H., W.B.P., and J.A.R. designed research; K.S. and A.D.F. at least 27 mink farms in The Netherlands, Spain, Denmark, and performed research; N.J.H. contributed new reagents/analytic tools; K.S. and J.J.S. ana- United States have reported outbreaks, including at least one lyzed data; and K.S. wrote the paper. probable case of mink-to-human transmission (16, 17). The authors declare no competing interest. SARS-CoV-2 has also been shown to productively infect sev- This article is a PNAS Direct Submission. eral species, including ferrets and domestic cats, in vivo (9, 10, This open access article is distributed under Creative Commons Attribution License 4.0 18, 19). Ferrets (Mustela putorius furo) are of special relevance to (CC BY). laboratory studies of respiratory viruses like Influenza A virus and 1To whom correspondence may be addressed. Email: [email protected]. recapitulate clinical pathophysiological aspects of human dis- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ease. Given their susceptibility to experimental infection and doi:10.1073/pnas.2025601118/-/DCSupplemental. onward transmission via direct and indirect contact, ferrets have Published April 15, 2021. PNAS 2021 Vol. 118 No. 18 e2025601118 https://doi.org/10.1073/pnas.2025601118 | 1of6 Downloaded by guest on September 25, 2021 Results All samples were confirmed to have viable RNA, by a pre- Absence of Natural SARS-CoV-2 Human-to-Ferret Transmission in a liminary screen for constitutively expressed β-actin (Table 1). High-Exposure Setting. A household with 29 free-roaming ferrets Each sample was then tested for evidence of active or recent SARS- cared for by two related, adult housemates was enrolled in the CoV-2 infection, with three established primer sets: Open Reading CoVERS study. Individual 1 experienced fever and fatigue from Frame 1b nonstructural protein 14 (ORF1b) (20), Nucleocapsid (N) March 25 to April 6, and individual 2 experienced a sore throat, (14), and RNA-dependent RNA polymerase (RdRP) (21). All were anosmia, migraine, and fatigue from March 28 to April 13 (Fig. below the limit of detection and determined to be negative for 1A). Individual 2 tested positive for SARS-CoV-2/COVID-19 active or recent infection (Table 1). infection by nasopharyngeal swab and RT-PCR on April 1. In- We further took advantage of salivary immunoglobulin G dividual 1 is a suspected positive due to the timing and symptoms (IgG), which has been shown to be highly sensitive and specific but was not tested. Neither person was hospitalized, and both for SARS-CoV-2 testing (22). We tested samples for evidence of cared for the ferrets during the entirety of their disease courses. antibodies against SARS-CoV-2 surface glycoprotein RBD. Twenty- A 2-wk, in-home sample collection scheme was designed to two ferrets (23 total samples) were confirmed to have measurable begin during the household quarantine period (Fig. 1B). The total IgG via binding to recombinant protein A/G but were all ferrets were free to move in all spaces of the home during this negative for binding to RBD (Table 1). Therefore, there is no evi- period and handled as usual, including daily petting, feeding, and dence of viral infection or seroconversion in 29 ferrets living with grooming. Individual 1 described daily close contact with hold- twopeoplewithCOVID-19. ing, laying, and/or cuddling with all ferrets, as well as daily cage cleaning. The ferrets ranged in age from 8 mo to 7.5 y over 21 Identification of Two Mustelid-Associated Mutations in SARS-CoV-2 females and 8 males. A home sampling kit was sent to the par- Surface Glycoprotein. Our observed household data support the ticipants, including material to safely collect and store ferret oral idea that there may be important barriers to natural infection in swabs. One participant had significant animal handling experience ferrets; however, ferrets have been shown to be susceptible to and performed all sample collection to standardize sampling pro- infection and onward transmission in experimental laboratory cedures. Thirty oral swabs were collected and held in viral trans- infections (9, 10, 18, 19).