As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen from China continues to cause new infections worldwide, causing coronavirus disease 2019 (COVID-19), genomic surveillance has demonstrated the emergence of several new variants of the virus. These have, in some cases, been associated with increased transmissibility and/or immune evasion. Now, a new preprint suggests that they could also extend the range of hosts that can be infected by the virus.

Study: SARS-CoV-2 variants of concern Alpha, Beta, Gamma and Delta have extended ACE2 receptor host-ranges. Image Credit: CI Photos/Shutterstock

A preprint version of this study is available on the bioRxiv* server, while the article undergoes peer review.

Background

The beta-coronavirus SARS-CoV-2 is thought to have emerged from a bat reservoir. It has been found to engage mammalian cells via the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell membrane. An unknown intermediate host is probably involved in the species jump, according to many scientists.

The earlier SARS-CoV as well as closely related species like the RaTG13 also originated in bats, supporting this hypothesis. Moreover, recently identified Sarbecoviruses in bats have been found to be almost identical to this virus at the spike region, at the receptor-binding domain (RBD). These findings seem to make it more probable that other coronaviruses have a high affinity for human ACE2 molecules, currently circulating in wild animal populations such as bats.

In addition, it has been experimentally possible to infect ferrets, mice, bats, monkeys, chimpanzees, cats, dogs, and mink, with these viruses, showing that the host range for these viruses is indeed broader than just bats and humans. The implications of this finding are threefold.

One, this indicates an ongoing risk of transmission to humans from wild or domestic animals, such that these could act as reservoirs of the virus from where repeated waves of infection may arise. Second, the virus could spread between different animal species themselves. Thirdly, such animals may be of use as COVID-19 model animals to examine the development of therapeutic and vaccine platforms as well as to understand more about the disease itself.

Earlier experiments by the current authors, as well as other scientists, have shown that SARS-CoV-2 spike proteins are capable of binding to several different kinds of ACE2 molecules in mammals. Moreover, pseudovirus experiments have shown the ability of this spike protein to infect dogs, cats, pangolins, rabbits, and other mammals, though not rats, ferrets, and certain birds and bats, to the same extent.

As the virus spread and replicated rapidly in human populations the world over, hundreds of mutations have occurred in the ribonucleic acid (RNA) genome. Many of these have affected the RBD, changing the shape of the epitopes and thus altering its recognition by immune receptors as well as the host cell receptor ACE2..

Some of these have been clustered together in specific viral variants or lineages, forming some variants of concern (VOCs) such as the Alpha, Beta, Gamma, and Delta. The Alpha VOC was the first to rise to global prominence, with its markedly higher transmissibility, almost wiping out the previously dominant D614G strain.

Similarly, the Delta strain rapidly became dominant during the second quarter of 2021, replacing most of the strains that were circulating earlier. This type of replacement is attributed to the ongoing adaptation of the virus to the human host, as well as its increased ability to evade the immune responses of the host by slipping under the immune radar thanks to mutations at the right spots on the spike antigen and especially the RBD.

Spike mutations are also known to have favorably impacted the replication capacity of the virus, its infectivity, and its ability to antagonize the innate immune host response. The current preprint examined the binding of four VOC spike variants to ACE2 receptors from different species.

What did the study show?

The investigators used pseudoviral infection experiments, with the viral particles expressing four different spike antigens from the four VOCs mentioned above. They compared spike-ACE2 binding across multiple hosts to that of the original or wildtype SARS-CoV-2 spike. The spike proteins were engineered to enhance their incorporation into the viral particles, thus potentially improving the efficiency of infection.

Human, civet, ferret, mouse, hamster, rat, and pig ACE2 molecules were assessed for the tropism of the virus. While the first two were known hosts, in the current or earlier outbreaks, the rodents, except for the rat, had been experimentally infected and used as models for the virus. Rats may be a reservoir for human-derived SARS-CoV-2 due to their habitat, which brings them into close contact with the virus in human sewage. Pigs are known to be a reservoir for the Nipah and influenza viruses.

With human ACE2, the pseudotyped viruses showed only a small increase in binding with the Beta variant but not with any other, compared to the wild-type spike. In the civet, the Beta VOC was the only one to show a significant increase in the extent of viral cell entry.

Mouse ACE2 binding was significantly improved for all four VOCs, corroborating the difficulty in achieving infection with this receptor using the viral isolates from early in the pandemic. The Delta variant, the only one that did not show the presence of N501Y, showed a lower increase in mouse ACE2 binding than the other VOC spikes. In rat ACE2, too, the non-Delta VOCs showed notable increases in ACE2 receptor binding.

The changes for ferret ACE2 binding were seen in the form of increased binding with the Beta and Gamma VOCs, but no significant changes were observed with the hamster or pig ACE2. The latter had shown themselves to be readily infected by the wild-type isolates, unlike rats, mice, ferrets, and civets.

Thus, the VOCs that contain the N501Y mutation have a broader range of hosts, while the Delta variant shares a similar range to the wild-type virus containing D614G.

The role of the N501Y mutation in overcoming host receptor restrictions in mouse ACE2 expressing cells was clearly seen since its introduction to the wild-type virus allowed infection equivalent to that of the Alpha VOC that also possesses this mutation. Changes at other sites such as the furin cleavage site, P681H, such as that seen in the Alpha VOC, or the Δ69-70 deletions in the N-terminal domain (NTD) did not seem to change viral entry kinetics in mice or rat ACE2 expressing cells, though the deletions were not independently examined.

In civets, the N501Y and K417N mutations seemed to inhibit viral attachment, but E484K enhanced viral entry, and this may contribute to the small increase in ACE2 binding with the Beta VOC spike. When introduced into the Alpha spike variant, the E484K compensated for the inhibition caused by the former mutations.

What are the implications?

The study shows the importance of understanding the functional changes mediated by the spike protein, including viral entry and increased host receptor range, as well as immune evasion. These could lead to higher infectivity, transmissibility, and virulence. The findings of this paper draw attention to the potential for reverse zoonosis, with the SARS-CoV-2 replicating in a wild animal species, to eventually spill back into the human population.

The small number of mutations observed to underlie significant functional changes is proof of the importance of viral evolution between and within hosts.

As of now, the researchers say, “these VOCs have not yet been linked to any significant increase in spill-over back into livestock, companion animals or wildlife ACE2 proteins.”

This could be because of poor sampling of potential reservoir species or limitation of contact between humans and animals in situations of infection. Alternatively, the frequency at which spill-overs occur could be very low, or it could just be that animal infection with these VOCs does not cause any marked difference in disease characteristics.

All four VOCs were able to overcome their relative inability to engage the mouse ACE2, confirming that small spike protein changes are required to allow the spike to bind to cognate receptors in various species. This was seen in mink, which led to the mass destruction of these animals to prevent the spread of the virus among them, and potentially, to other animals. However, it is important to realize that the mink mutations like Y453F inhibit human ACE2 binding.

The demonstrable differences shown by individual VOCs binding to rat, ferret, or civet ACE2 receptors are attributable to N501Y and E484K substitutions within the RBD.

The animal models used to screen antivirals or vaccines against SARS-CoV-2 could yield different data on exposure-infection rates or pathogenicity, depending on whether they are exposed to the wild-type or VOC virus. This could cause confusion about the efficacy of such interventions.

Secondly, ACE2 restriction could drive viral adaptation within the animals, which could again breed confusion as to the actual transmissibility of the variant involved. Hamsters are an exception in this regard.

Finally, the ability of SARS-CoV-2 to infect a broad host range is extended by the VOCs, mostly without inhibiting its infectivity towards the original host species. How far this will continue and how it will affect the chances of reverse zoonosis cannot be predicted at present.

The researchers postulate,

The conceivable ‘worst case scenario’ for SARS-CoV-2 reverse zoonosis is that the virus establishes itself in a new reservoir and at such a level that antibody selection pressure takes place and/or prolonged antigenic drift leading to escape mutants that are relevant to immune human populations.”

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.