key: cord-0294401-myohjke7 authors: Zhou, Jie; Peacock, Thomas P.; Brown, Jonathan C.; Goldhill, Daniel H.; Elrefaey, Ahmed M.E.; Penrice-Randal, Rebekah; Cowton, Vanessa M.; Lorenzo, Giuditta De; Furnon, Wilhelm; Harvey, William T.; Kugathasan, Ruthiran; Frise, Rebecca; Baillon, Laury; Lassaunière, Ria; Thakur, Nazia; Gallo, Giulia; Goldswain, Hannah; Donovan-Banfield, I’ah; Dong, Xiaofeng; Randle, Nadine P.; Sweeney, Fiachra; Glynn, Martha C.; Quantrill, Jessica L.; McKay, Paul F.; Patel, Arvind H.; Palmarini, Massimo; Hiscox, Julian A.; Bailey, Dalan; Barclay, Wendy S. title: Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway date: 2021-08-20 journal: bioRxiv DOI: 10.1101/2021.08.20.456972 sha: f3937bfa012d71e3e86df8b00d7d78d8cf685f62 doc_id: 294401 cord_uid: myohjke7 SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections. Introduction 32 SARS-CoV-2 is a betacoronavirus that is thought to have emerged from an animal source in 33 2019 and rapidly spread by human-to-human transmission across the globe causing the COVID-19 34 pandemic. SARS-CoV-2 is transmitted efficiently by the airborne route due to its ability to efficiently 35 enter cells in the upper respiratory tract. The spike glycoprotein is responsible for host receptor 36 binding and membrane fusion of coronaviruses. SARS-CoV-2 spike binds to host angiotensin-37 converting enzyme 2 (ACE2) via the receptor binding domain (RBD) and is activated by TMPRSS2 38 protease expressed at the apical surface of the airway epithelium to mediate fusion 1 . In addition, 39 compared to closely related coronaviruses, SARS-CoV-2 spike contains a tract of basic amino acids at 40 the S1/S2 cleavage site that can be recognised by furin, enabling spike to be efficiently primed for 41 fusion by TMPRSS2. This allows rapid fusion of spike at the cell surface and avoids restriction factors 42 present in the late endosome and endolysosome 2,3 . 43 A series of molecular interactions between amino acids in the spike RBD and the interacting 44 surface of ACE2 result in SARS-CoV-2 binding to human ACE2 with high affinity 4,5 . SARS-CoV-2 shows 45 a broad host tropism and can experimentally infect many animal species, largely determined by the 46 efficiency with which spike can interact with the animal ACE2 orthologues 5,6 . For example, mice are 47 not dependent on the presence or absence of D614G, as Y453F in a 614D background showed a similar 155 effect ( Figure 3C , Extended data Figure 2B ). Consistent results were also seen using a cell-cell fusion 156 assay (Extended data Figure 2C ,D). Examining the structure of the spike RBD/ACE2 interface, each of 157 these mink/ferret-adaptations is close to residues that differ between human and mustelid ACE2, as 158 others have previously modelled 29 . For example, Y453F lies close to H34Y (histidine in human ACE2, 159 tyrosine in mustelid), N501T lies close to G354R, and F486L lies between ACE2 residues L79H, M82T 160 and Q24L ( Figure 3D ). Although the Y453F containing virus is highly similar to that which circulated in mink early in 170 the pandemic, the most prominent zoonotic spillover from mink was the Cluster 5 virus, which further 171 contained D614G and Δ69-70. D614G and Δ69-70 are thought to potentially enhance virus infectivity 172 in some backgrounds 30 . Therefore, we performed a similar competition experiment between a mixed 173 inoculum of 40% Cluster 5 isolate and 60% early B.1 lineage, D614G containing virus ('WT'; IC19). 174 Again, we observed that the Y453F-containing Cluster 5 was outcompeted, constituting only ~10% of 175 reads by 24 hours post-infection ( Figure 4B ). 176 Finally, to further confirm that the attenuation of the Y453F containing viruses, particularly 177 the ferret-adapted strain, wasn't due to other changes in the genome (such as E S6L described above) 178 we generated by reverse genetics (RG) two viruses on a Wuhan-hu-1, both carrying the D614G mutation in spike, WT (D614G), while the other additionally contained Y453F (D614G + Y453F). As with 180 the ferret adapted P2 virus and Cluster 5 isolate we saw that the Y453F + D614G RG virus produced 181 less infectious virus upon replication in the primary airway cells as compared to the otherwise isogenic 182 WT (D614G) virus, significantly so at 24 hours post-infection ( Figure 4C ). 183 To investigate whether a mustelid-adapted SARS-CoV-2 crossing back into the human 185 population would have a large impact on re-infections or vaccine-breakthrough we next tested 186 whether the mutation at Y453F facilitated escape from antibody neutralization. Surprisingly, Y453F-187 containing 'Ferret P2' virus was significantly more easily neutralised by convalescent first wave 188 antisera than wild type requiring only 0.6 as much antisera for a 50% neutralisation titre ( Figure 5A ). 189 We further investigated the relative antigenicity of Y453F, this time using the above-described RG 190 viruses and antisera from health care workers who had received two doses the of Pfizer-BioNtech-191 BNT162b2 vaccine. Again, we saw the Y453F-containing virus was more readily neutralised by 7 of the 192 10 vaccinee sera, although the difference was not significant ( Figure 5B ). 193 We next performed pseudovirus neutralisation assays with the previously described first wave 194 convalescent antisera against pseudoviruses expressing the common mustelid adaptations or with full 195 Cluster 5 spike. The B.1.351 (Beta) spike showed a significant, ~5-fold drop in mean NT50 ( Figure 5C ), 196 consistent with this virus being more difficult to neutralise with first wave antisera 31 . None of the 197 tested mink/ferret adaptations had any significant impact on antigenicity. 198 Many circulating variants of concern show a greater ability to enter via mustelid ACE2. 199 Following worldwide circulation of SARS-CoV-2, a number of 'variant of concern' and 'variant 200 of interest' lineages have arisen associated with properties such as increased transmissibility, higher 201 pathogenicity, and antigenic escape 32 . These generally locally, or globally, outcompeted other 202 lineages to become predominant, including the Alpha variant (B.1.1.7), first associated with infections the UK 23 . A number of these variants have RBD mutations such as L452R, E484K and/or N501Y which 204 are thought to promote humans ACE2 binding 22 . 205 To investigate whether these variants may be more able to infect mink or ferrets than the 206 progenitor lineage B or B.1 viruses through better use of mustelid ACE2, we again used pseudoviruses 207 expressing these variant spike proteins and normalised entry to human ACE2 ( Figure 6 ). We found 208 that nearly all variants of concern tested could better utilise mink ACE2 than WT (D614G only) Brazil) showed no improved usage of ferret ACE2. It appears L452R, E484K and N501Y may promote 216 use of ferret ACE2, while K417N/T may result in a greater reduction in ferret ACE2 usage relative to 217 human ACE2. Overall, these data suggest multiple circulating variants of concern may be able to infect 218 mustelid hosts with only minimal, or indeed without, further adaptation. 219 In this study, we have performed a full risk assessment of mustelid hosts, such as mink and 221 ferrets, as reservoirs for the emergence of antigenic variants or new variant of concern. We have 222 shown SARS-CoV-2 is poorly adapted to mustelid ACE2 and therefore quickly gains adaptations, such 223 as Y453F, N501T or F486L to utilise mustelid ACE2. However, Y453F in particular, negatively impacts 224 replication kinetics of SARS-CoV-2 in human cells, potentially explaining why the Danish mink-origin 225 outbreaks did not propagate further following the culling of the mink. Furthermore, in line with other 226 studies 16,17,31 , we found none of these mutations had a large antigenic impact, so vaccination is likely 227 to remain effective against mustelid-adapted strains. Finally, we have shown that several VOC strains, or VOC-associated mutations, partially adapt SARS-CoV-2 spike to mustelid ACE2. Therefore, it is likely 229 VOC lineages will continue to infect mink farms and risk spilling back over into humans. 230 Except for the Danish mink-adapted SARS-CoV-2 spillback, Y453F is found rarely in humans 231 with very few isolates reported in GISAID and only a single report of the mutation arising in 232 immunocompromised patients -this is despite Y453F having been shown in several studies to 233 enhance human ACE2 binding, in a similar manner to the VOC-associated mutations N501Y or L452R 234 22, 33, 34 . This would suggest that unlike the VOC-associated mutations such as N501Y, Y453F affects viral 235 fitness in human cells. We have shown that, even in the presence of the putative stabilising NTD 236 deletion, Δ69-70 30 , virus harbouring the Y453F substitution was outcompeted by a closely related 237 virus in human cells. 238 Here, we have demonstrated many VOCs, particularly Alpha/B.1.1.7 as well as those 239 containing L452R (such as Delta/B.1.617.2) could have a fundamental fitness advantage in mink by 240 increasing interaction with mustelid ACE2, compared to previous non-variant strains. At present 241 (August 2021), the vast majority of mink-origin SARS-CoV-2 sequences on GISAID are from the year 242 2020, even though there are a number of ongoing mink outbreaks reported in Europe 35,36 , suggesting 243 a significant reporting lag. None of the four WHO-designated variants of concern have yet been 244 associated with mink farm outbreaks. It remains to be seen whether these VOCs would replicate in 245 mink/ferrets without any further adaptation, but we have shown that the most common mustelid 246 adaptations would be unlikely to have a large effect on VOC antigenicity. It will be key in the coming 247 years to continue to closely survey farmed mink and to sequence and share any SARS-CoV-2 genomes 248 from these animals in a timely manner as SARS-CoV-2 could still adapt in unexpected ways in mink 37 . 249 This work also suggests that, particularly when investigating spike RBD mutants, ferrets (or 250 indeed mink) are poor models for humans, as mustelid ACE2 is poorly utilised by non-adapted SARS-251 CoV-2 spike. Thus, it is not a given that adaptation to human ACE2 will also result in increased 252 infectiousness, transmissibility or pathogenicity in the ferret model. However, ferrets remain a useful model for investigating non-RBD phenotypes though care should be taken to use previously ferret 254 adapted viruses to prevent rapid adaptation. 255 The early SARS-CoV-2 strain, England/2/2020 (VE6-T) was previously isolated by Public Health 281 England as previously described 40 . The D614G containing strain, SARS-CoV-2/England/IC19/2020, was 282 used as previously described 41 . The Cluster 5 isolate -SARS-CoV-2/hu/DK/CL-5/1 -was isolated as 283 previously described 16 Transformation-Associated Recombination (TAR) method in yeast was used to generate the 294 mutant viruses described in this study. We followed essentially previously described methods 43 with 295 some modifications. Briefly, a set of overlapping cDNA fragments representing the entire genome of 296 SARS-CoV-2 Wuhan isolate (GenBank: MN908947.3) were chemically synthesized and cloned into 297 pUC57-Kan (Bio Basic Canada Inc). Where appropriate the relevant synthetic cDNA fragment carried 298 the mutation D614G or Y453F + D614G in the viral S gene. The cDNA fragment representing the 5' 299 terminus of the viral genome contained the bacteriophage T7 RNA polymerase promoter preceded by 300 a short sequence stretch homologous to the XhoI-cut end of the TAR in yeast vector pEB2 44 . The 301 fragment representing the 3' terminus contained the T7 RNA polymerase termination sequences followed by a short segment homologous to the BamHI-cut end of pEB2. These cDNA fragments were 303 excised by restriction digestion and gel-extracted or PCR-amplified using appropriate primers. These 304 fragments were then pooled and co-transformed with XhoI-BamHI-cut pEB2 into the Saccharomyces 305 cerevisiae strain TYC1 (MATa, ura3-52, leu2Δ1, cyh2 r , containing a knockout of DNA Ligase 4) 44 Ferret (Mustela putorius furo) infection studies with SARS-CoV-2 virus were performed as 397 described previously 3 . All ferret studies were performed in a containment level 3 laboratory, using a 398 bespoke isolator system (Bell Isolation Systems). Outbred female ferrets (16-20 weeks old) weighing each virus while lightly anaesthetized with ketamine (22 mg kg−1) and xylazine (0.9 mg kg−1). 401 Prior to the start of the study ferrets were confirmed to be seronegative to SARS-CoV-2. All 402 animals were nasal-washed daily, while conscious, by instilling 2 ml of PBS into the nostrils; the 403 expectorate was collected into disposable 250-ml sample pots. Ferrets were weighed daily post 404 infection, and body temperature was measured daily via subcutaneous IPTT-300 transponder (Plexx 405 B.V). 406 For Sanger sequencing, RNA was extracted from nasal washes using QIAamp viral RNA mini 408 kit (Qiagen). RNA was reverse transcribed using Superscript IV (Invitrogen) and PCR of the spike was 409 performed using KOD polymerase (Merck). For next generation sequencing RNA from virus-containing 410 samples were extracted using the QIAsymphony DSP Virus/Pathogen mini kit (Qiagen). RNA was 411 DNase-treated using the TURBO-free Kit (Invitrogen; (AM1907). cDNA was synthesised using the 412 superscript IV reverse transcriptase (Invitrogen) and random primer mix (NEB) before amplification by 413 the ARTIC Network protocol using the multiplexed primer scheme version 3. Fast5 files were 414 basecalled with guppy (v.5.0.7) with high accuracy calling (hac). The fastq files produced by Nanopore 415 sequencing were filtered with lengths 400 and 700 using Artic-ncov2019 pipeline v1.2.1 416 (https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.htµl) by "artic guppyplex" function. 417 The other function of "artic minion" in the Artic-ncov2019 pipeline with "--medaka --medaka-model 418 r941_min_high_g360 --normalise 0" parameters was then used to process the filtered fastq files to 419 generate ARTIC V3 primer trimmed bam files and consensus genome sequences. These primer 420 trimmed bam files were further analysed using DiversiTools (http://josephhughes.github.io/btctools/) 421 with the "-orfs" function to generate the ratio of amino acid change in the reads and coverage at each 422 site of protein in comparison to the reference SARS-CoV-2 genome (MN908947.3) as we previous 423 description 53 . All sequences with host species labelled as Neovison vison were retrieved from the Global 426 Initiative on Sharing All Influenza Data (GISAID) database (sequences retrieved on 7 July 2021). A table 427 of accession IDs and acknowledgement is given in Supplementary Table S1 . 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Figure made using PyMOL (Schrödinger) and PDB: 671 7A94 60 The common mink and ferret adaptation, Y453F, attenuates virus replication in primary 674 human airway cells Human primary airway epithelial cells cultured at air-liquid interface were infected at an MOI 676 of approximately 0.1 with A) a mixture of parental and ferret-adapted England/2 virus B) A mixture of 677 Mink-adapted 'Cluster 5' virus and a D614G control or C) either isogenic WT (D614G) or D614G + Y453F 678 -containing reverse genetics-derived virus isolates. Virus titres were measured by TCID50 (C) E gene 679 Statistics for competition assays were determined by One-Way ANOVA with multiple 680 comparisons against time 0. Statistics for the head-to-head growth curve (C) were determined by 681 multiple unpaired T-tests on log-transformed data. All infections were performed on triplicate wells 682 from matched donors Figure 5. Mink and ferret associated mutations have a minimal impact on SARS-CoV-2 antigenicity Live virus neutralisation comparing WT or Y453F-containg ferret passage 2 (A) or the isogenic 685 reverse genetics-derived WT (D614G) and D614G + Y453F-containing SARS-CoV-2 isolates (B) using N=6 human convalescent antisera from the first UK wave (~April-June 2020; A) or N=10 double-dose 687 Fold differences annotated on graph 688 indicate differences in geometric means of NT50. Statistics were determined by two-tailed Wilcoxon 689 test with matched pairs C) Pseudovirus neutralisation of different mink-adaptations containing mutants using N=8 691 human convalescent antisera from the first UK wave Friedman non-parametric test with multiple comparisons against WT. *0.05 ≥ P Figure 6. Several variants of concern show enhanced entry into ferret ACE2 expressing cells A) Mutant SARS-CoV-2 spike-containing pseudovirus entry into HEK 293Ts expressing human 696 or ferret ACE2, or empty vector. Entry normalised to signals from human ACE2 expressing cells RBD mutational profile of the different spike proteins is shown below. Cells in orange 701 indicate changes from WT/D614G. Alpha also known as B.1.1.7; Beta also known as B.1.351; Gamma 702 also known as P.1; Eta also known as B.1.525 Extended data Figure legends 704 Extended data Figure 1. Amino acid differences between ferret and mink ACE2 Differences between ferret and mink ACE2 are shown on the structure of human ACE/Spike 706 structure PDB: 7A94 60 Extended data from Figure 3 A) Non-normalised data from Figure 3B B) Non-normalised data from extended data Figure 3C Cell-cell fusion assays of HEK 293Ts with rLUC-GFP1-7 transfected with the stated Spike 711 452 The authors declare no competing interests.