Host barriers to SARS-CoV-2 demonstrated by ferrets in a high-exposure domestic setting

Ferrets (Mustela putorius furo) are mustelids of special relevance to laboratory studies of respiratory viruses and have been shown to be susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and onward transmission. Here, we report the results of a natural experiment where 29 ferrets in one home had prolonged, direct contact and constant environmental exposure to two humans with symptomatic disease, one of whom was confirmed positive for SARS-CoV-2. We observed no evidence of SARS-CoV-2 transmission from humans to ferrets based on viral and antibody assays. To better understand this discrepancy in experimental and natural infection in ferrets, we compared SARS-CoV-2 sequences from natural and experimental mustelid infections and identified two surface glycoprotein Spike (S) mutations associated with mustelids. While we found evidence that angiotensin-converting enzyme II provides a weak host barrier, one mutation only seen in ferrets is located in the novel S1/S2 cleavage site and is computationally predicted to decrease furin cleavage efficiency. These data support the idea that host factors interacting with the novel S1/S2 cleavage site may be a barrier in ferret SARS-CoV-2 susceptibility and that domestic ferrets are at low risk of natural infection from currently circulating SARS-CoV-2. We propose two mechanistically grounded hypotheses for mustelid host adaptation of SARS-CoV-2, with possible effects that require additional investigation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is a zoonotic member of Coronaviridae that emerged in 2019 as a major viral pandemic (1). As of February 2021, there have been ∼102 million confirmed COVID-19 cases globally and ∼2.2 million deaths (2). SARS-CoV-2 uses angiotensin I converting enzyme-2 (ACE2) as its primary cellular receptor for host entry and infection (3–5). In silico analyses of ACE2 genes in diverse mammalian species show that residues important to viral binding are moderately conserved between humans and several domestic animals, and a broad range of species have been demonstrated to be permissive to infection in vitro and in vivo (6–10).It is not yet known whether natural infection of animals plays a role in public health epidemiology or has the potential to establish endemic reservoirs and threaten wildlife. SARS-CoV-2 has been observed to be capable of natural human-to-animal reverse zoonoses, transmitting from infected individuals into mink (11), dogs (12), and felines (13–15). American mink (Neovison vison) are currently the only species observed to have natural human-to-animal spillover and onward transmission (11). To date, at least 27 mink farms in The Netherlands, Spain, Denmark, and United States have reported outbreaks, including at least one probable case of mink-to-human transmission (16, 17).SARS-CoV-2 has also been shown to productively infect several species, including ferrets and domestic cats, in vivo (9, 10, 18, 19). Ferrets (Mustela putorius furo) are of special relevance to laboratory studies of respiratory viruses like Influenza A virus and recapitulate clinical pathophysiological aspects of human disease. Given their susceptibility to experimental infection and onward transmission via direct and indirect contact, ferrets have been proposed as an animal model to study SARS-CoV-2 transmission. Based on in vivo data, we expect all naïve ferrets in direct contact with an infected ferret will 1) become infected, 2) have measurable viral shedding or RNA via oral swabs up to 19 d postinfection, and 3) seroconvert with measurable antibodies against SARS-CoV-2 receptor binding domain (RBD) (18, 19).In March 2020, during the first wave of the SARS-CoV-2/COVID-19 pandemic in the New England area, we developed a rapid response study to investigate the potential for human-to-animal spillover and onward transmission in domestic, farm, and wildlife species (CoVERS: Coronavirus Epidemiological Response and Surveillance). The goal of CoVERS is to understand whether and how SARS-CoV-2 transmission is occurring at these interfaces, to refine public health guidelines, investigate whether there are additional risks to animal or human health associated with spillover, and evaluate the potential for establishment of endemic reservoirs. In the CoVERS in-home study, participants are sent a “swab and send” kit, which provides materials and instructions to safely take longitudinal nasal and oral samples from their animals, store them in their freezers, and send them back for viral screening. This community science approach allows wide surveillance with no risk of human transmission, as kits are decontaminated and opened in biosafety cabinets. Here, we highlight one enrolled household that created an exceptional natural experiment with direct relevance to our understanding of SARS-CoV-2 reverse zoonosis and animal models of disease.