Ferrets (Mustela putorius furo) are mustelids of special relevance to laboratory studies of respiratory viruses and have been shown to be susceptible to 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 COVID-19. We observed no evidence of SARS-CoV-2 transmission into the household ferret population via RT-PCR and ELISA. 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 mutations associated with mustelids. While there is evidence that ACE2 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 activity. These data suggest that host factors interacting with the novel S1/S2 cleavage site are a barrier in ferret SARS-CoV-2 susceptibility and that domestic ferrets are at low risk of
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). European mink (Mustela lutreola) 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 and 2) have measurable viral shedding or RNA via oral swabs up to 19 days post-infection and 3) seroconvert with measurable antibodies against SARS-CoV-2 receptor binding domain (RBD) 54 (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 if and how SARS-CoV-2 transmission is occurring at these interfaces to refine public health guidelines, investigate if there are additional risks to animal or human health associated with spillover and evaluate the potential for establishment of endemic reservoirs. 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.
Absence of natural SARS-CoV-2 human-to-ferret transmission in a high exposure setting, A household with free-roaming ferrets cared for by two adults was enrolled in the CoVERS study. Individual 1 experienced fever and fatigue from March 25-April 6 and Individual 2 experienced a sore throat, anosmia, migraine and fatigue from March 28-April 13 (Fig. 1A). Individual 2 tested positive for SARS-CoV-2/COVID-19 infection by nasopharyngeal swab and RT-PCR on April 1. Individual 1 is a probable positive due to the timing and symptoms but was not tested. Neither person was hospitalized, and both cared for the ferrets during the entirety of their disease courses. A two-week, in-home sample collection scheme was designed to begin during the household quarantine period (Fig. 1B). The ferrets were free to move in all spaces of the home during this period and handled as usual, including regular petting, feeding and grooming. The ferrets ranged in age from 8 months to 7.5 years of age over 21 females and 8 males. A home sampling kit was sent to the participants including material to safely collect and store ferret oral swabs. One participant had significant animal handling experience and performed all sample collection to standardize sampling procedures. Thirty oral swabs were collected and held in viral transport media in the participants’ freezer until the end of the study period. Frozen samples were directly transferred to a lab member and processed.
All samples were confirmed to have viable RNA by a preliminary screen for constitutively expressed ß-actin (Table 1). Each sample was then tested for evidence of active or recent SARS-CoV-2 infection with three established primer sets: ORF1b-nsp14 (20), Nucleocapsid (N) (14) and RNA-dependent RNA polymerase (RdRP) (21). All were below the limit of detection and determined to be negative for active or recent infection (Table 1). We further took advantage of salivary immunoglobulin, which has been shown to be highly sensitive and specific for SARS-CoV-2 testing (22). We tested samples for evidence of antibodies against SARS-CoV-2 surface glycoprotein receptor binding domain (RBD). Twenty-two ferrets (23 total samples) were confirmed to have measurable total IgG via binding to recombinant protein A/G but were all negative for binding to RBD (Table 2). Therefore, there is no evidence of viral infection or seroconversion in 29 ferrets living with two people with COVID-19.
Reference & Source information: https://www.biorxiv.org/
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