SARS-CoV-2 variants bearing the N501Y mutation have extended host range creating more concerning variants that can then be passed back to humans

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cientists from Institut Pasteur, Université de Paris-France have in a new study discovered the SARS-CoV-2 variants bearing the N501Y mutation have extended host range to include mice and warns that mice getting infected with the SARS-CoV-2 coronavirus from humans could potentially help create more concerning variants that can then be passed back to humans.

Receptor recognition is a major determinant of viral host range, infectivity and pathogenesis. Emergences have been associated with serendipitous events of adaptation upon encounters with novel hosts, and the high mutation rate of RNA viruses may explain their frequent host shifts.

SARS-CoV-2 extensive circulation in humans results in the emergence of variants, including variants of concern (VOCs) with diverse mutations notably in the spike, and increased transmissibility or immune escape.

The study findings were published on a preprint server and are currently being peer reviewed.

 https://www.biorxiv.org/content/10.1101/2021.03.18.436013v2

Host range expansion or switch to other species has been prevalent in the course of coronaviruses evolutionary history (Cui et al., 2019; Woolhouse et al., 2005). Understanding the host range and how it is modified as the pathogen evolves is critical to estimate the emergence risk and determine the reservoirs to monitor.

In the case of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, animals such as non-human primates, hamsters, ferrets, minks and cats were shown to be permissive (Johansen et al., 2020).

By contrast, the zoonotic virus was shown to not replicate in mice and rats due to poor binding of the virus spike on the rodent cellular receptor angiotensin-converting enzyme 2 (ACE2) (Zhou et al., 2020).

At the end of 2020, the emergence of variants of concern (VOCs) was noted in different parts of the world (WHO, 2021). The Alpha variant (Pango lineage B.1.1.7 (Rambaut et al., 2020a)), was noted for its rapid spread in the UK (Rambaut et al., 2020b), while the Beta variant (Pango lineage B.1.351) expanded in multiple regions of South Africa (Tegally et al., 2021), and the Gamma variant (Pango lineage P.1) emerged in Manaus, Brazil (Faria et al., 2021).

The global circulation and spread of these variants have led to concerns about increased transmission and their potential to evade immunity elicited by vaccination or naturally acquired. More recently, the Delta variant (Pango lineages B.1.617.x and AY.x), initially described in India, has rapidly expanded worldwide and became dominant in many countries (Mlcochova et al., 2021).

Alpha, Beta and Gamma SARS-CoV-2 VOCs all harbor the N501Y change in the spike glycoprotein which belongs to a set of 6 key amino acid residues critical for the tight interaction of the receptor binding domain (RBD) with hACE2 (Yi et al., 2020).

Strikingly, this mutation was also noted, among others, in independently generated mouse-adapted SARS-CoV-2 strains (Gu et al., 2020; Rathnasinghe et al., 2021). Here, we assessed the replication potential in cells and in mice of low-passage clinical SARS-CoV-2 VOCs isolates as well as their transmission potential by direct contact between mice.

DISCUSSION

The worldwide dissemination of SARS-CoV-2 with over 250 million confirmed cases, has been accompanied with the emergence of a multitude of variants bearing a constellation of changes in the ~30 kb viral genome. Some of these variants carry changes, often in the spike, which have been associated with increase in transmissibility or pathogenicity in humans (Barton et al., 2021; Harvey et al., 2021; Volz et al., 2021).

Among those, the N501Y mutation has repeatedly appeared independently in the pandemic, and is notably shared by the three first VOCs. This change has been shown to increase the affinity of the spike protein for hACE2 (Starr et al., 2020; Tian et al., 2021; Zahradnik et al., 2021), which could increase transmissibility (Liu et al., 2021).

Our results, both in vitro and in vivo, demonstrate that SARS-CoV-2 variants carrying the N501Y mutation can efficiently replicate in mice. This host range expansion appears to be determined by N501Y, alone or in combination with additional mutations in the spike, such as those characteristic of the VOCs, but independently from the D614G mutation which is absent in the A.27 lineage.

However, we noted that the viral titers of Alpha in the mice lungs were lower than those measured with other N501Y variants, suggesting that one or more mutations in the Alpha constellation negatively impact the virus fitness in mice. Interestingly, an Alpha isolate carrying the E484K substitution, present in Beta in Gamma, yielded lung viral titers comparable to other N501Y variants.

This result is consistent with our structural modeling data indicating that E484K further increases the compatibility of the RBD with mACE2, and with recent results obtained with pseudotyped viruses showing that the N501Y, E484K and their combination increase entry in mACE2 expressing cells (Li et al., 2021).

Further in-depth studies will be needed to characterize the pathological consequences of infection with these variants. Notably, a mouse adapted strain carrying N501Y (Gu et al., 2020), in combination with two other substitutions in the spike (Sun et al., 2021) was reported to induce respiratory symptoms and lung inflammation with age-related mortality, while the young adult mice infected with our variants showed no signs of disease, except BALB/c mice infected with Beta which developed transient body weight loss.

Thus, other changes among the constellations defining the VOC lineages might also play a role in the resulting in vivo phenotype. Indeed, most of the reported mouse-adapted variants also contain genetic changes outside of the spike. Further studies are needed to dissect the combinatory role of the mutations defining the SARS-CoV-2 VOCs, as well as their pathogenicity in older mice, or in other host genetic backgrounds.

The ability of viruses of the N501Y-bearing lineages to replicate in common laboratory mice will facilitate in vivo studies in this species, to evaluate countermeasures (vaccines or therapeutic interventions), to assess antibody cross-reactivity and vaccine cross-protection (Martinez et al., 2021), and for functional studies using genetically altered mouse strains.

It has been proposed that persistence can select for host range expansion of animal viruses, by selecting for virus variants that recognize phylogenetic homologues of the receptor (Baric et al., 1999; Schickli et al., 1997). Interestingly, there is still speculation on the mechanism of emergence of the VOCs, which are all characterized by an unusually large number of mutations compared to their last common ancestor, including a number of changes (substitution and deletions) in the spike protein.

While the genomic surveillance could have missed evolutionary intermediates leading to these lineages, similar patterns of accumulated changes were noted in some longitudinal studies of immunocompromised individuals infected by SARS-CoV-2 for extended periods of time (Choi et al., 2020; Kemp et al., 2021), leading to the hypothesis of a role of such long-term infections in their emergence.

As mutations in positions of interest in the spike RBD (417, 484 and 501), alone or in combination, have been noted in other lineages during the extensive circulation of SARS-CoV-2 in human populations (2021), and associated in vitro with modification of the affinity for human ACE2, the observed host range expansion nevertheless likely represents only a serendipitous by-product of selection for increased transmissibility of SARS-CoV-2 in its current host.

To evaluate the consequence of host range expansion for wild mice, we assessed the possibility of transmission by close contact. We did not test indirect transmission by aerosols since mice, unlike ferrets or minks, do not efficiently transmit respiratory viruses by this route. We demonstrated that the Beta VOC could be transmitted not only from an infected mouse to a first-level contact individual, but also from first-level to second-level contact mice.

Almost all published studies have considered only transmission from an experimentally infected individual to a first contact, which does not reflect the situation which may occur in wild rodent groups since the infectious dose used for intranasal inoculation is likely much higher that the dose an animal may be accidentally exposed to.

One exception is a study on deer mice (Peromyscus maniculatus) which reported inconsistent virus detection in oral swabs after two passages (Griffin et al., 2021). Due to the low reliability of oral-pharyngeal swabs in mice for assessing the presence of the virus (which may reside mostly in the lower respiratory tract), we used antibody titers against the spike as an indicator of viral exposure. Although not proportional to viral load, antibody levels reflect the intensity of viral exposure.

As expected, infected mice showed very high levels of anti-spike antibodies. The observation that Contact 2 mice had similar levels as Contact 1 mice suggests that they had been exposed to virus doses in the same range of magnitude. Although not yet established, it is likely that the infectious dose for mice of these new variants is low. Moreover, SARS-CoV-2 infection from exposed mice (Contact 1 to Contact 2) occurred in the absence of any clinical signs of illness, consistent with the observation of frequent inter-human transmission from asymptomatic infected individuals (Muller, 2021).

Our results clearly demonstrate that, once they have been exposed to the virus, mice can transmit the virus to others, which raises major questions on the risk of mice or other rodents living in very large numbers in proximity to humans, of becoming secondary reservoirs for SARS-CoV-2 in regions where the N501Y-carrying variants circulate, from where they could evolve separately and potentially spillback to humans. Indeed, rodents have been hypothesized as the ancestral host of some betacoronaviruses (lineage A, which includes the seasonal human coronaviruses OC43 and HKU1 (Lau et al., 2015; Tsoleridis et al., 2019)).

Similar and actionable concerns were raised upon the detection of SARS-CoV-2 in Mink farms in The Netherlands (Oreshkova et al., 2020) and in Denmark (Lassaunière R et al., 2020) due to the density of animals housed, and the detection of changes in the virus genome. While rodent densities are highly variable and more difficult to estimate, multiple spillovers have been detected in free-living White-tailed deer populations in North America (Kuchipudi et al., 2021). We posit that host range should be closely monitored along the continued evolution of SARS-CoV-2.

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