Scientists assess SARS-CoV-2 mutants in ferret hosts

By Michael Greenwood, M.Sc.Aug 26 2021

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been documented in several mammalian species besides humans, including cats, dogs, and mink. A form of the angiotensin-converting enzyme 2 (ACE2) receptor that is responsible for interaction with the SARS-CoV-2 spike protein prior to host-cell entry is present in a wide variety of animals, and it is the affinity between the spike protein and this receptor that determines the risk of infection upon viral challenge.

For example, mice are inefficiently infected with human SARS-CoV-2 unless firstly humanized and made to express the human ACE2 receptor, or alternatively, SARS-CoV-2 is made to adapt to murine ACE2 by serial passage.

The COVID-19 outbreak reported amongst farmed minks in the Netherlands and several other counties during 2020 was seen to be a result of human to mink transmission, and once adapted to the population, mink-SARS-CoV-2 was noted to bear several mutations that enhance affinity towards the mink ACE2 receptor, namely Y453F or N501T to the spike protein.

Subsequently, farmworkers and those living in the local community were reported to be infected with SARS-CoV-2 bearing the Y453F mutation, raising concerns related to the development of novel variants of concern that may escape immunity induced by vaccination or past exposure.

In a paper recently uploaded to the preprint server bioRxiv* by the cause of SARS-CoV-2 mutations observed in mink and ferrets is investigated, and the danger posed by such mutations to humans assessed.

Study: Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway. Image Credit: Harald Schmidt / Shutterstock

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Ferrets are closely related to mink and have been extensively utilized in SARS-CoV-2 experiments regarding infection and transmission owing to their susceptibility and similarity to humans. Several studies have reported the same Y453F or N501T mutations to the SARS-CoV-2 spike protein as seen in infected mink. The mink and ferret ACE2 receptors are almost identical, potentially explaining why these mutations to the virus are common, though interestingly, these mutations have also been associated with an enhanced affinity towards the human ACE2 receptor.

The very similar N501Y mutation to the spike protein known to have developed in human SARS-CoV-2 during the pandemic also enhances affinity towards ACE2. Thus these mutations may instead non-specifically increase affinity towards a wide variety of mammalian ACE2 receptors.

Consistent spike mutations in ferrets

The group firstly demonstrated that the Y453F or N501T mutations develop in ferrets by inoculation with wildtype SARS-CoV-2, where N501T with Y453F is a minor variant were seen to appear just two days post-infection, with both mutations becoming equal in number by day 5. In initially uninfected ferrets that came into contact with the infected, transmitted virus population included a mixture of Y453F with a smaller proportion bearing the N501T mutation, also evening out after several days.

Viral load in Y453F ferrets was found to be higher than in those infected with wildtype SARS-CoV-2 two days post-infection. However, titers between the groups were equal by day five as the initially wildtype virus had since adopted these mutations. The Y453F mutation was maintained amongst those ferrets inoculated with this strain throughout the course of the experiment.

The group noted the development of several other mutations, including D614N to the spike protein, again very similar to the D614G mutation seen to have occurred in humans that is thought to enhance transmission by encouraging the adoption of a more frequently open spike protein state.

Ferret SARS-CoV-2 affinity towards ACE2

A library of mutated spike proteins was generated by the group to create lentivirus-based pseudovirus constructs, allowing the ability of the particles to gain cell entry using human, ferret, or rat ACE2 to be assessed. The group found that Y453F, N501T or F486L mutations enhanced the affinity of the spike protein towards both ferret and human ACE2, but not rat ACE2.

Upon closely examining the structure of the spike protein receptor-binding domain and ACE2 interface, the group note that these mutations are mainly located around regions of the ACE2 receptor that differ between humans and ferrets.

Primary human bronchial cells were then infected with either wildtype or SARS-CoV-2 bearing the Y453F mutation. In this case, the group note that wildtype virus significantly outcompeted the mutated version, with less than 5% of virus 48 hours post-infection still bearing the mutation. Incorporation of other mutations such as D614G, both of which are present in the mink-to-human SARS-CoV-2 strain recorded in the Netherlands in 2020, also did not induce greater viral loads in human cells, again being outcompeted by wildtype in mixed inoculation.

Ferret SARS-CoV-2 escape mutants

To investigate whether mutations adopted in mink hosts better facilitate escape from neutralizing antibodies induced by past infection in humans’ convalescent sera collected from individuals infected with wildtype SARS-CoV-2 was collected and applied to the virus bearing the Y453F mutation. The group found that the Y453F virus was, in fact, more easily neutralized than wildtype, only requiring around 60% as much sera for the same 50% rate of neutralization.

Against sera collected from individuals having received the Pfizer-BioNTech vaccine, the difference was less notable, however, with 7 of the 10 sera collected being slightly better at neutralizing the Y453F virus than wildtype.

Finally, the group demonstrated that mutations present in many of the currently circulating variants of concern could also enhance affinity to ferret ACE2, as well as human ACE2. Variants of concern investigated include alpha (B.1.1.7), iota (B.1.525), and beta (B.1.351), which each bear mutations such as L452R, E484K and N501Y.

Each of these strains showed significantly greater affinity towards ferret ACE2 than wildtype SARS-CoV-2 pseudovirus, as observed towards human ACE2, while the gamma (P.1) variant, which instead bears the K417N/T mutation, showed a reduction in affinity towards ferret ACE2 compared to human ACE2.

In conclusion, this study has demonstrated that ferrets may be a poor model for representing COVID-19 in humans. The ferret ACE2 receptor is not well utilized by human SARS-CoV-2 without mutations that enhance affinity. Therefore, any adaptations that enhance transmissibility in either species may not apply to the other. However, mutations that alter regions other than the receptor-binding domain (RBD), which interacts with ACE2, may still be applicable.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources