key: cord-0968224-0770q3i8
authors: Touret, Franck; Luciani, Léa; Baronti, Cécile; Cochin, Maxime; Driouich, Jean-Sélim; Gilles, Magali; Thirion, Laurence; Nougairède, Antoine; de Lamballerie, Xavier
title: Replicative Fitness of a SARS-CoV-2 20I/501Y.V1 Variant from Lineage B.1.1.7 in Human Reconstituted Bronchial Epithelium
date: 2021-07-06
journal: mBio
DOI: 10.1128/mbio.00850-21
sha: 9b91b59e4dfacadbae163c5f7a074bb15d97f266
doc_id: 968224
cord_uid: 0770q3i8

Since its emergence in 2019, circulating populations of the new coronavirus (CoV) continuously acquired genetic diversity. At the end of 2020, a variant named 20I/501Y.V1 (lineage B.1.1.7) emerged and replaced other circulating strains in several regions. This phenomenon has been poorly associated with biological evidence that this variant and the original strain exhibit different phenotypic characteristics. Here, we analyze the replication ability of this new variant in different cellular models using for comparison an ancestral D614G European strain (lineage B1). Results from comparative replication kinetics experiments in vitro and in a human reconstituted bronchial epithelium showed no difference. However, when both viruses were put in competition in human reconstituted bronchial epithelium, the 20I/501Y.V1 variant outcompeted the ancestral strain. All together, these findings demonstrate that this new variant replicates more efficiently and may contribute to a better understanding of the progressive replacement of circulating strains by the severe acute respiratory CoV-2 (SARS-CoV-2) 20I/501Y.V1 variant.

IMPORTANCE The emergence of several SARS-CoV-2 variants raised numerous questions concerning the future course of the pandemic. We are currently observing a replacement of the circulating viruses by the variant from the United Kingdom known as 20I/501Y.V1, from the B.1.1.7 lineage, but there is little biological evidence that this new variant exhibits a different phenotype. In the present study, we used different cellular models to assess the replication ability of the 20I/501Y.V1 variant. Our results showed that this variant replicates more efficiently in human reconstituted bronchial epithelium, which may explain why it spreads so rapidly in human populations.

KEYWORDS 20I/501Y.V1, B.1.1.7, SARS-CoV-2, ex vivo, in vitro, replicative fitness, variant N ovel severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) emerged in China by the end of 2019 and rapidly spread worldwide. In a few months, the D614G spike mutation was rapidly fixed in almost all circulating SARS-CoV-2 populations, without evidence of higher CoV disease 2019 (COVID-19) mortality or clinical severity (1). It is still being debated whether it is due to a random founder effect (1) or, more probably, whether the mutation enhances viral loads in the upper respiratory tract, increasing the infectivity and stability of virions (2) (3) (4) .

In September 2020, a variant named 20I/501Y.V1 from lineage B.1.1.7 (initially named VOC 2 2020212/01) emerged in the United Kingdom. It spread rapidly and is becoming dominant in Western Europe (5) and the United States (6) . There is consistent epidemiological evidence that this so-called "UK variant" is more efficiently transmitted (7) than the preexisting European strains, in particular in young patients. Moreover, this variant has also been associated in some studies with an increased risk of mortality (8-10), without any differences in symptomatology (11) .

Here, we present a comprehensive analysis of the replication ability in vitro and ex vivo of the 20I/501Y.V1 variant (strain UVE/SARS-CoV-2/2021/FR/7b isolated in February 2021 in Marseille, France; GISAID accession no. EPI_ISL_918165), using for comparison the lineage B.1 BavPat D614G strain that circulated in Europe in February/ March of 2020.

The first experiments were performed in two cell lines: VeroE6/TMPRSS2 cells, commonly used for SARS-Cov-2 isolation and propagation (12) , and Caco-2 cells, which endogenously express the ACE2 receptor and TMPRSS2 coreceptor at levels similar to those in Calu-3 cells (13) . Results of these experiments revealed highly similar replication kinetics, supporting the results of complete genome sequencing of both viral strains with regard to the integrity of the multibasic cleavage site in the spike protein ( Fig. 1A and B and see Table S1 in the supplemental material) (14) .

We then assessed the replicative fitness of both strains using a previously described model of reconstituted human airway epithelium (HAE) of bronchial origin (15) . Following the inoculation of the epithelia through their apical side at a multiplicity of infection (MOI) of 0.1 in order to mimic the natural route of infection, we monitored the excretion of new virions at the apical side between 2 and 4 days postinfection (dpi) and measured the intracellular viral RNA yields at 4 dpi. Infectious titers (Fig. 1D ) and viral RNA yields (Fig. 1E ) at the apical side at 3 and 4 dpi, as well as intracellular viral RNA yields at 4 dpi (Fig. 1G) , were slightly higher for the B.1.1.7 variant. However, differences were not significant, and estimated relative virion infectivities (i.e., the ratio of the number of infectious particles to the number of viral RNA particles) were similar for the two viruses at all sampling times (Fig. 1F) . All together, these results are in line with our findings for common cell lines and with a recent report (16) .

Based on these results, we performed competition experiments, which have previously been demonstrated to be effective to detect moderate replicative fitness differences (2, 17) . Accordingly, we inoculated epithelia with both viruses simultaneously as described above, sampled the apical side between 2 and 4 dpi, and extracted intracellular viral RNA yields at 4 dpi. Three infection inoculum ratios (B.1.1.7/B1 ratios, 70/30, 50/50, and 30/70) were used. Using two specific reverse transcription-quantitative PCR (RT-qPCR) assays (Fig. S1) , we estimated the proportion of each viral genome in the viral population (expressed as the B.1.1.7/B1 ratio in Fig. 1H and I) . Regardless of the initial ratio, we observed similar patterns in which B1 was outcompeted by the B.1.1.7 variant; all B.1.1.7/B1 ratio values estimated from apical-side washes were above 1 and over 57, 22, and 8 at 4 dpi for epithelia inoculated at the initial ratios of 70/30, 50/50 and 30/70, respectively (Fig. 1H) . Notably, B.1.1.7/B1 ratios measured at 4 dpi were significantly higher than the initial 50/50 and 30/70 inoculum ratios (P = 0.0475 and P = 0.0082, respectively, with the Kruskal-Wallis test with an uncorrected Dunn post hoc analysis). Similar results were observed when estimating the B.1.1.7/B1 ratios from intracellular viral RNAs (Fig. 1I) ; B.1.1.7/B1 ratios measured at 4 dpi were significantly higher than the initial 50/50 and 30/70 inoculum ratios (P = 0.0208, P = 0.0082, and P = 0.0475 with the 70/30, 50/50, and 30/70 inoculum ratios, respectively, as determined by the Kruskal-Wallis test with an uncorrected Dunn post hoc analysis).

Our results demonstrated that the 20I/501Y.V1 (B.1.1.7) variant is more fit than the lineage B.1 BavPat D614G strain in reconstituted bronchial human epithelium. This may be explained by the presence of the N501Y mutation in the receptor binding domain (RBD) of the spike protein, which enhances viral particle binding to the ACE2 receptor (18) . This may translate into a fitness advantage, as demonstrated in a recent study with engineered viral strains (19) . Similar observations have been made with the D614G mutation, with which the new G614 strains overcame the original D614 strains when put in competition (2) . All together, these findings may contribute to a better understanding of the progressive replacement of circulating strains by the SARS-CoV-2 20I/501Y.V1 variant (20) .

Supplemental material is available online only. TEXT S1, DOCX file, 0.02 MB. FIG S1, DOCX file, 0.2 MB. TABLE S1, DOCX file, 0.01 MB. DATA SET S1, DOCX file, 0.04 MB.

We thank C. Drosten for providing the SARS-CoV-2 BavPat strain through European Virus Archive Global (EVA Global). We thank Geraldine Piorkowski for the sequencing.

This work was supported by Inserm through the REACTing (Research and Action Targeting Emerging Infectious Diseases) initiative and by EVA Global, funded by the European Union's Horizon 2020 research and innovation program under grant agreement 871029. This work was also supported by the Fondation de France call for projects FLASH COVID-19, project TAMAC.

We declare that we have no conflict of interest.

Evaluating the effects of SARS-CoV-2 spike mutation D614G on transmissibility and pathogenicity

Spike mutation D614G alters SARS-CoV-2 fitness

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo

on behalf of COVID-19 Genomics Consortium UK. 2020. Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations -SARS-CoV-2 coronavirus/nCoV-2019 Genomic Epidemiology

Emergence of SARS-CoV-2 B.1.1.7 lineage -United States

Emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States

Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7

Case fatality risk of the SARS-CoV-2 variant of concern B.1.1.7 in England

Risk of mortality in patients infected with SARS-CoV-2 variant of concern 202012/1: matched cohort study

Genomics UK (COG-UK) Consortium. 2021. Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Robust and persistent SARS-CoV-2 infection in the human intestinal brush border expressing cells

Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia

Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape

Attenuation of tick-borne encephalitis virus using large-scale random codon re-encoding

SARS-CoV-2 RBD in vitro evolution follows contagious mutation spread, yet generates an able infection inhibitor

The N501Y spike substitution enhances SARS-CoV-2 transmission

Weekly epidemiological update-23