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SARS-CoV-2 transmission via apical syncytia release from primary bronchial epithelia and infectivity restriction in children epithelia

Guillaume Beucher, Marie-Lise Blondot1, A. Celle2, Noémie Pied1, Patricia Recordon-Pinson1, Pauline Esteves2, Muriel Faure1, Mathieu Métifiot1, Sabrina Lacomme3, Denis Dacheaux1, Derrick R. Robinson1, Gernot Längst4, Fabien Beaufils2, Marie-Edith Lafon1, Patrick Berger2, Marc Landry3, Denis Malvy3, Thomas Trian2, Marie-Line Andreola1, Harald Wodrich1 •
Centre national de la recherche scientifique1, French Institute of Health and Medical Research2, University of Bordeaux3, University of Regensburg4
28 May 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this paper, the authors used reconstituted primary bronchial epithelia from adult and children donors to follow the infection dynamic following infection with SARS-CoV-2.
Abstract: The beta-coronavirus SARS-CoV-2 is at the origin of a persistent worldwide pandemic. SARS-CoV-2 infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract eventually causing acute severe respiratory syndrome with a high degree of mortality in the elderly. Here we use reconstituted primary bronchial epithelia from adult and children donors to follow the infection dynamic following infection with SARS-CoV-2. We show that in bronchial epithelia derived from adult donors, infections initiate in multi-ciliated cells. Then, infection rapidly spread within 24-48h throughout the whole epithelia. Within 3-4 days, large apical syncytia form between multi-ciliated cells and basal cells, which dissipate into the apical lumen. We show that these syncytia are a significant source of the released infectious dose. In stark contrast to these findings, bronchial epithelia reconstituted from children donors are intrinsically more resistant to virus infection and show active restriction of virus spread. This restriction is paired with accelerated release of IFN compared to adult donors. Taken together our findings reveal apical syncytia formation as an underappreciated source of infectious virus for either local dissemination or release into the environment. Furthermore, we provide direct evidence that children bronchial epithelia are more resistant to infection with SARS-CoV-2 providing experimental support for epidemiological observations that SARS-CoV-2 cases’ fatality is linked to age. Significance Statement Bronchial epithelia are the primary target for SARS-CoV-2 infections. Our work uses reconstituted bronchial epithelia from adults and children. We show that infection of adult epithelia with SARS-CoV-2 is rapid and results in the synchronized release of large clusters of infected cells and syncytia into the apical lumen contributing to the released infectious virus dose. Infection of children derived bronchial epithelia revealed an intrinsic resistance to infection and virus spread, probably as a result of a faster onset of interferon secretion. Thus, our data provide direct evidence for the epidemiological observation that children are less susceptible to SARS-CoV-2.

Summary (4 min read)

Jump to: [Introduction] – [Generation of a fully differentiated bronchial epithelia model] – [SARS-CoV-2 monitoring and BE infection] – [Infection kinetic of epithelia from different adult donors] – [Infected multi-ciliated cells form syncytia with basal cells at the apical side of the BE] – [Infected cells and syncytia are released into the apical BE lumen and transmit infection] – [Epithelia from children partially restrict SARS-CoV-2 infection but not syncytia formation] – [Discussion] – [Monoclonal antibodies and ethics statement] – [Viral production] – [Culture of primary bronchial epithelia (BE) and ethics statement] – [Infection of epithelia] – [Quantification of SARS-CoV-2 RNA by qRT-PCR] – [Immunofluorescence detection, antibodies and confocal microscopy.] and [Electron microscopy]

Introduction

  • Coronaviruses with zoonotic origin have emerged as a new public health concern during the first decades of the 21th century.
  • The distal third encodes for the 4 structural proteins, Envelope (E), Membrane (M), Nucleocapsid (N) and Spike (S) and seven putative ORFs encoding accessory proteins and potential virulence factors (6) (7) (8) .
  • Epidemiological data have demonstrated that if all ages of the population are susceptible to SARS-CoV-2 infection, SARS-CoV-2 infection severity is different between children versus the adult populations and varies with age (23) .
  • Only limited data are available about the mechanism of viral spreading over time and how the virus is released from the epithelia and might participate in the transmission of the infection between individuals or within an individual.
  • Regarding the inflammatory response, interferon induction appears limited in the most severe clinical cases (29) (30) (31) .

Generation of a fully differentiated bronchial epithelia model

  • One of the major initial targets for SARS-CoV-2 is the respiratory tract.
  • Bronchial epithelia are pseudo-stratified cell layers with typical cell junctions, as well as a mucus layer and beating cilia on the lumen side (33, 34) .
  • Patients were between 46 and 63 years old with a normal body mass index [BMI] (Table1).
  • Basal cells were then seeded on cell culture insert and differentiated at the air-liquid interface for approximately 21 days (Fig. S1A ).
  • Moreover, their data showed a prominent exposure of ACE2 on individual cilia reaching into the apical lumen (orange arrows), which suggests facilitated access e.g. for virus coming in through the respiratory tract.

SARS-CoV-2 monitoring and BE infection

  • Apical and basolateral compartments were collected 3 days post-infection (dpi) and used to infect Vero E6 cells (Fig. 1A ).
  • To ascertain that this CPE is indeed due to viral replication and not a toxic effect from the inoculation, the authors extracted total RNAs from the Vero E6 supernatant on the next day (4 dpi) and quantified viral RNAs using in-house qRT-PCR targeting the N-gene region.
  • To investigate which cell type is the primary target during SARS-CoV-2 infection, fully differentiated epithelia were infected with SARS-CoV-2 at a MOI of 0.01 for 1 h from the apical side after which the viral suspension was removed.
  • Conversely, the signal arising for the N protein staining was systematically associated with strong labelling for acetylated tubulin, a specific marker for multi-ciliated cells (orange arrow, Fig. 1E , movie S6).
  • Infected cells were exclusively located at the apical surface of the BE (Fig. 1C-E ).

Infection kinetic of epithelia from different adult donors

  • To better understand how SARS-CoV-2 spreads in the epithelium after initial infection of multiciliated cells, the authors infected BEs from four individual adult donors (A1 to A4, Table 1 ) and monitored them over the course of 7 days.
  • Similar results were obtained with the other two donors suggesting rapid onset of viral replication and spread (not shown).
  • Therefore, the authors also quantified the size of the N protein associated signals over time (Fig. 2C ).
  • The analysis revealed a statistically significant average increase in signal size between the third and fourth day for all four donors.
  • Altogether, these data suggested that apical SARS-CoV-2 inoculation of BEs resulted in efficient infection and subsequent progeny production and release into the apical lumen.

Infected multi-ciliated cells form syncytia with basal cells at the apical side of the BE

  • Using high-resolution microscopy, the authors observed that larger N-positive signals corresponded to multinucleated cellular structures reminiscent of syncytia.
  • These syncytia could be found in all regions of the epithelia (Fig. 3 ) and their formation at day 4 was common to all four donors tested.
  • Unexpectedly, the N-positive syncytia forming on day three and four also stained positive for the basal cell marker cytokeratin 5 (Fig. 3A ).
  • Zooming in on different regions of the epithelia revealed that newly formed syncytia frequently lost their stain for acetylated tubulin (Fig. 3C side panel).
  • These structures harbored only reminiscent cilia structures in place of multi-ciliated cells in non-infected epithelia.

Infected cells and syncytia are released into the apical BE lumen and transmit infection

  • Because cells and syncytia were extruding from the epithelium, the authors wondered whether infected cells/syncytia could be released from the epithelium and account for the spreading of the infection.
  • Apical washes of epithelia were performed after three and four days of infection and concentrated on microscope slides via cytospin.
  • After 24h, cells were fixed and analyzed by IF microscopy.
  • Inoculation with the supernatant as well as the cellular fraction of the apical wash showed efficient Vero E6 cell infection (data not shown).
  • In parallel, inoculated Vero E6 cells were monitored for the appearance of a virus-induced CPE.

Epithelia from children partially restrict SARS-CoV-2 infection but not syncytia formation

  • In their experimental model, each epithelium can be traced to an individual donor, while generating enough individual inserts to allow biological repeats and kinetic studies.
  • Quantifying the total number of infected cells in each epithelium confirmed their observation (Fig. 5D ).
  • In contrast to the adult control, no significant differences in signal size was observed between day three and day four for either of the children derived epithelia (Fig. 5E ).
  • Taken together their analysis clearly demonstrated that epithelia from children were less susceptible to SARS-CoV-2 and exhibited an intrinsic resistance towards virus infection and/or spread.
  • The difference in the kinetic for Interferon l secretion between adults and children in response to SARS-CoV-2 infection may thus provide an explanation why in their model children derived BE resist better to SARS-CoV-2 infection.

Discussion

  • The authors used human reconstituted bronchial epithelia to investigate the onset of infection and replication of SARS-CoV-2 in BE.
  • This suggested that syncytia are formed by the fusion of basal cells with infected multi-ciliated cells.
  • Strikingly, the authors find very different spreading of SARS-CoV-2 in children BE versus adult BE.
  • Rather than rapidly spreading throughout the entire epithelia, as observed for adults, the infected cells in children form cluster or foci of infected cells.
  • Such an agerelated susceptibility of BE has been reported for other respiratory pathogens including respiratory viruses such as Rhinovirus-C, Adenovirus and RSV (Respiratory Syncytial Virus) (51, 52)(53) but also fungi (Aspergillus fumigatus) (54) and bacteria (Haemophilus influenzae) (55) .

Monoclonal antibodies and ethics statement

  • Monoclonal antibodies were raised against bacterially expressed and purified SARS-CoV-2 N protein in 3 mice using the protocol as previously described (59) .
  • Hybridomas were cloned by limiting dilution and screened by immunofluorescence on infected VERO cells.
  • Clone 3G9 was retained for this study and antibody was affinity purified from hybridoma supernatant prior to.

Viral production

  • The culture supernatant was clarified by centrifugation (5 minutes at 1500 rpm) and aliquots were stored at −80°C.
  • Plates were incubated at 37°C and examined for cytopathic effect.
  • Quantification of cytopathic effect was determined using the Cell tox TM green cytotoxicity assay according to manufacturer instructions and a Victor Nivo reader (Perkin Elmer).
  • PFU/ml was estimated from the TCID 50 determination.

Culture of primary bronchial epithelia (BE) and ethics statement

  • Bronchial epithelial cell culture was established from bronchial brushings or lung resection performed between the third and fifth bronchial generation from patients undergoing elective surgery as previously described (34) .
  • Then, 10 5 basal cells were grown on cell culture inserts (Corning, New York, NY) within the air-liquid interface for 21 days using PneumaCult ALI medium (Stemcell, Vancouver, Canada).
  • Such a culture allows the differentiation into pseudostratified muco-ciliary airway epithelium composed of ciliated cells, goblet cells, club cells and basal cells.
  • The complete differentiation was assessed by the capacity of cilia to beat and mucus production under light microscope.
  • The study received approval from the local and national ethics committee from the CNIL through the TUBE collections.

Infection of epithelia

  • Prior to infection, epithelia were washed three times with PBS to remove mucus and basal ALI medium was exchanged with 500 µL of fresh medium.
  • The inoculum containing 1200 PFU of virus or medium-only controls were added to the apical surface to a final volume of 100 µL.
  • Viral supernatant was removed after 1 hour incubation at 37°C and infection was followed for the indicated time points.
  • Viral production was then quantified by qRT-PCR using 3 consecutively collected apical washes of 100 µL PBS.

Quantification of SARS-CoV-2 RNA by qRT-PCR

  • For quantification of viral RNA by qRT-PCR, total RNA was isolated using the High Pure Viral RNA kit according to the manufacturer's instruction.
  • Viral RNA was quantified using GoTaq® 1- Step RT-qPCR kit .
  • Copy numbers were calculated from a standard curve produce with serial 10-fold dilutions of SARS-CoV-2-RNA.
  • The melting curve was obtained by temperature increment 0,5°C/s from 60°C to 95°C.

Immunofluorescence detection, antibodies and confocal microscopy.

  • For antigen detection, BE were washed repeatedly with PBS to remove mucus then fixed with 4% paraformaldehyde for 30min using complete insert immersion.
  • Epithelia were then washed and permeabilized with 0.5% TritonX-100 in PBS for 10min at room temperature and blocked in IF buffer (PBS containing 10% SVF and 0.5% saponin) for 1h at room temperature.
  • Primary antibody and fluorescently labeled phalloidin to stain the actin cytoskeleton was diluted in IF buffer and applied to inserts for 1h at room temperature.
  • High resolution analysis was performed on a SP8 confocal microscope (Leica Microsystems at the Bordeaux Imagery center) using maximal pixel resolution at 20x, 40x or 63x respectively and 0.3µm Z-stacks resolution.
  • Image processing was done using Image J software.

Electron microscopy

  • For electron microscopy, epitheliums were first washed in physiological serum and then fixed with 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1M phosphate buffer (pH 7.4) during 2h minimum at room temperature (RT).
  • The polymerization of the resin was carried out over a period between 24-48 hours at 60°C.
  • Ultrathin sections (70 nm) were picked up on copper grids and then stained with uranyless and lead citrate.
  • Grids were examined with a Transmission Electron Microscope (H7650, Hitachi, Tokyo, Japan) at 80kV.

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Figures (1)

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,!Sabrina!Lacomme
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Derrick!Robinson
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,!F a b i e n !Be a ufils!
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,!Marie-Edith!Lafon
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!Patrick!Berger
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Marie-Line!Andreola
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and!Harald!Wodrich
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a
Univ.! Bordeaux,! CNRS,! Microbiologie! Fondamentale! et! Pathogénicité,! UMR! 5234,! F-33000!9!
Bordeaux,!!France.!!10!
b
INSERM,! Centre! de! Recherche ! Cardio-thoracique! de! Bordeaux,! U1045,! CIC! 1401,! Bo rde au x,!11!
33000,!France.,!!12!
c
!University!of!Bordeaux,!CNRS,!Institute!of!Neurodegenerative!Diseases,!IINS,!UMR!5293,!13!
Bordeaux,!France,!University!of!Bordeaux,!CNRS,!INSERM,!Bordeaux!Imaging!Center,!BIC,!UMS!14!
3420,!US!4,!Bordeaux,!France!15!
d
Biochemistry!Center!Regensburg,!Universität!Regensburg,!Regensburg,!Germany!!16!
e
CHU!de!Bordeaux,!Service!d’exploration!fonctionnelle!respiratoire,!service!de!pédiatrie!médicale,!17!
CIC!1401,!33000,!Bordeaux,!France,!!18!
f
!CHU!de!Bordeaux,!Laboratoire!de!Virologie,!33000!Bordeaux,!France!19!
g
Department!for!infectious!and!tropical!d’idéales,!University!Hospital!center!Pellegrin,!20!
Bordeaux,!&!Inserm!1219,!University!of!Bordeaux,!Bordeaux,!France.!!21!
22!
Author Contributions: *These!authors!contributed!equally,!#Thes e !a u thors!co n t rib uted!eq ually!23!
Competing Interest Statement: The authors have no competing interest 24!
Keywords: SARS-CoV-2, human bronchial epithelia, Age, Syncytia formation, IFN response 25!
This PDF file includes: 26!
Main Text 27!
Figures 1 to 5 28!
Tables 1 29!
Supplemental Figures S1 to S3 30!
31!
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 28, 2021. ; https://doi.org/10.1101/2021.05.28.446159doi: bioRxiv preprint
!
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2!
!
Abstract 32!
The!beta-coronavirus!SARS-CoV-2!is!at!the!origin!of!a!persistent!worldwide!pandemic.!SARS-CoV-33!
2!infections!initiate!in!the!bronchi!of!the!upper!respiratory!tract!and!are!able!to!disseminate!to!34!
the! lower! respiratory! tract! eventu ally! causing! acute! severe! respiratory! syndrome! with! a! high!35!
degree!of!mortality!in!the!elderly.!Here!we!use!reconstituted!primary!bronchial!epithelia!from!36!
adult!and!children!donors!to!follow!the!infection!dynamic!following!infection!with!SARS-CoV -2.!37!
We!show!that!in !bronch ia l!epithelia !derived!from!adult!donors,!infectio n s!initiate!in!multi-ciliated!38!
cells.!Then,!infection!rapidly!spread!within!24-48h!throughout!the! whole!epithelia.!Within! 3-4!39!
days,!la rg e!ap ica l!syn cy tia !form!betwee n !multi-ciliated! cells!and!ba sal!cells,! wh ich!d issipate!into!40!
the!a pica l!lumen .!We!show !that!these !syncytia!are!a! sign ifican t!source!of!the!released !infectious!41!
dose.!In!stark!contrast!to!these !findings,!bronchial!epithelia!reconstituted!from !children!d onors!42!
are!intrinsically!more! resistant!to !virus!infe ction !and !show !ac tive!restriction !of!virus!sp read .!This!43!
restriction!is!pa ired!with!accelerate d!release!of!IFN!compared !to!adult!donors.!Taken!together!our!44!
findings! reveal! apical! sync ytia! form atio n! as! an! und era pp rec iated ! sourc e! of! infectiou s! viru s! for!45!
either! local! dissemination! or! release! into! the! environment.! Furthermore,! we! provide! direct!46!
evidence! that! children! bronchial! epithelia! are! more! resistant! to! infe ctio n! w ith! S AR S- CoV -2!47!
providing!experimental!support! for! epidemiological!observations!that! SARS-CoV-2!cases’!fatality!48!
is!linked!to !a g e.!49!
Significance Statement 50!
Bronchial!epithelia!are!the!primary!target!for!SARS-CoV-2!infections.!Our!work!uses!reconstituted!51!
bronchial!epithelia!from!adults!and!children.!We!show!that!infection!of!adult!epithelia!with!SARS-52!
CoV-2! is! rapid! and! results! in! the! synchronized! release! of! large! clusters! of! infected! cells! and!53!
syncytia! into! the! apical! lum en ! contributing! to ! the! relea sed ! infectio us! virus! do se .! Infection ! of!54!
children!derived!bronchial!epithelia!revealed!an!intrinsic!resistance!to!infection!and!virus!spread,!55!
probably! as! a! result! of! a! faster! onset! of! interferon! secretion.! Thus,! our! data! provide! direct!56!
evidence!for!the!epidemiological!observation!that!children!are!less!susceptible!to!SARS-CoV-2.! 57!
58!
Main Text 59!
60!
Introduction 61!
62!
Coronaviruses!with!zoonotic!origin!have!emerged!as!a!new!public!health!concern!during!the!first!63!
decades! of! the! 21th! century.! Two! highly! pathogenic! coronaviruses,! severe! acute! respiratory!64!
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 28, 2021. ; https://doi.org/10.1101/2021.05.28.446159doi: bioRxiv preprint
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syndrome! coronavirus! (SARS-CoV)! and! Middle-East! respiratory! syndrome! coronavirus! (MERS-65!
CoV),! cause d ! se v e re ! re s p ir a to r y! in f ec t io n s ! in ! hu mans! during! regionally! confined! epidemics! in!66!
2002!(1)!and!between!2010-15!(2),!respectively.!In!late!2019,!clusters!of!patients!with!pneumonia!67!
in! Wuhan !in!the !H ub e i!pro v in ce !in!Ch in a !were!shown!to!be!infected!with!the!novel!severe!acute!68!
respiratory! syndrome! coronavirus! 2 ! (SARS-CoV-2)! (3-5).! SARS-CoV-2! infections! are! associated!69!
with!acute!respiratory!illness!referred!to!as!Coronavirus!disease !(COVID-19).!Since!its!description,!70!
SARS-CoV-2!infections!are!at!the!root!of!an!enduring!worldwide!pandemic,!having!cau sed!as!of!71!
May!2021! over!3!million!deaths !and!m ore!than!148!million! confirmed!infections!(data!from!the!72!
John!Hopkin s!un ivers ity!coronavirus!resource!center,!https://coronavirus.jhu.edu/ ).!SAR S-CoV-2!73!
is!an!e n ve lo p e d!virus!w ith !a!posit ive !single-stran ded!RNA!of!around !30 !kb.!The!5’!proximal!two!74!
thirds!o f!the!polyadenylated!genome!encodes!ORF1a!and!ORF1b,!which!are!autoproteolytically!75!
processed! into! several! no n-structural! proteins! required! for! replication ! and! transc riptio n.! The!76!
distal!third!encod es!for! the ! 4! structural!proteins,!Envelope!(E),!Mem brane!(M ),!Nucleocapsid!(N )!77!
and!Spike!(S)!and!seven!putative!ORF s!encoding!acce ssory!proteins!and !potential!virulence!factors!78!
(6-8).! The! surface! e xpose d! Spike!protein!gives! the! virus! its! crown-like! a p p ea ra n ce ! in! e lec tro n !79!
microscopy! and!mediates! the! attachment! to!the! main!cellular!recepto r!ACE2!(9).!Coronaviruses!80!
can!cau se!a! wide!range! of!respirato ry!illnesses,!from !mild!upper!respiratory!tract!infection!up! to !81!
a! severe! acute! respiratory! syndrome! (10).! The! latter! is! characterized! by! excessive! cytological!82!
damage!and!infla m mation .!Post!mortem!biopsies!in!patients!that!died!from!COVID-19!point!to!83!
airways! and! lungs! as! primary! targets! of! the! disease! (11,! 12)! with! advanced! diffus e! a lve o lar !84!
damage,!pulmonary!thrombosis!and!abnormal!syncytia!formation!(13,!14).!Se ve ra l!stu d ie s!su g ge st!85!
that!cytokine!storm!and!inflammato ry!infiltrates!in!the!alveolar!space!are!associated!with!disease!86!
severity!and!death!in!COVID-19! (15,!16).!While!SARS-CoV-2!is!genetically!close!to!SARS-CoV,!it!87!
shows!much!higher!effective!transmissibility!(17,!18) .!One! reason! for!this! higher!contagiousness!88!
is!an!active!virus!replication!in!tissues!of!the!upper!respiratory!tract!at!an!early!stage!of!infection,!89!
with!a!high!number! of!virus! cop ies!produced! fou r!da ys!after!the!beginning! of!symptoms,!and!an!90!
active!replication!in!th e!th roa t!(19)!(20).!Furthermore,! Zou!et!al!(21)!reporte d!that!the!viral!load!91!
detected!in!asymptomatic!patients!was!similar!to!that!of!sym pto ma tic!patients!on!day!4!after!92!
symptoms! onset,! suggesting! equal! transmission! potential! of! asymptomatic! or! minimally!93!
symptomatic! patients! at! very! early! stages! of! infection! (22).! Epidemiological! data! have!94!
demonstrated!that!if!all!ages!o f!the!p opulation!are!susc ep tible!to!SAR S-CoV-2!infection,!SARS-95!
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 28, 2021. ; https://doi.org/10.1101/2021.05.28.446159doi: bioRxiv preprint
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CoV-2!infection!severity!is!different!between!children!!"#$%$!the!adult!populations!and!varies!with!96!
age!(23).!A!recent! m u lti-national!epidemiologic!study!found!that!children!under!9!years!old!have!97!
very!low!case-fatality!rates!of!SARS - CoV-2!infection!com pared!to!older!patients!(24).!M o reo v er ,!98!
these! studies! consolidate! a! large! dis cre p a nc y ! in! dea th ! rates! of! SARS-CoV-2! infected! patients!99!
associated!with!age.!Death!rate!in!children! (<9! years)!is!under!0.001%!incre a sin g ! t o ! 8 % !in! e ld erly !100!
patients!(>80!years).!A! re ce n t!m eta d a ta!an aly sis !of!seve ral!stu d ies!came!to!the!sam e!correlation!101!
between!age!and!severity!(25).!The!reason!for!this!age-related!discrepancy!is!not!clear!and!could!102!
be!linked!to!a!decreased!transmission!and/or!viral!load!with!SARS-CoV-2!in!children!compared!to!103!
adults.!Only!limited!data!are!available!about!the!mechanism!of!viral!spreading!over!time!and!how!104!
the!viru s!is!re lease d!from !the! ep ithe lia!and!might!participate !in!th e!transm ission !of! th e!infection!105!
between!individuals!or!within!an!in div id u al .!Over!the!course!of!a!51!days!period,!infectio n !of!a!106!
reconstituted !human!airway!epithelium!infected !with!SARS-CoV-2!showed!multiple!waves!of!viral!107!
replication!associated !with!a!degradation!of!tight!junction!and!a!decrease!in!ciliary!expression!(26,!108!
27).!In !this!model,!plaque-like!cytopathic!effects!could!be! ob served!with!the!formation!of! multi-109!
nucleated!cells!(28).!Regarding! the!inflammatory!response,!interferon! induction!appears!limited!110!
in! th e !most! sev ere!clinical! cases!(29-31).!In!con tra st,!re leas e!of! INF-l!was!induced!at!day!4! post-111!
infection!of! bronchial!epithelia! (BE).!Notew or th y,!viral!RNA!production!in!BE!incre as e d!at!day!2,!112!
suggesting!a!delay!in!the !induction!of!the!cellular!antiviral!response.!A!very!recent!report!studying!113!
cell-intrinsic! chan ge s ! occur rin g! in! differen tia te d ! hum a n ! nasal! epithelial!cultures!from! children,!114!
adults!and!elderly,!have!show n!that!ageing!contributed!to!viral!lo ad ,!tran s c rip t io n a l!re sponse s,!115!
IFN! signa lin g ! an d ! a n tiv ira l! res p o n s es! (32).! Yet,!such!data!using! a!model!mimicking!the! human!116!
bronchial!epithelium!are!still! missing.! Here,!we! developed! a! model! of!reconstituted!bronchial!117!
epithelium!(BE)!in!air-liquid!inte rfa c e!der ive d !from !bro n chial!epithe liu m !samples !of!adult!donors,!118!
which! is!the!primary!site!of!SARS-CoV-2!infection.!We!monitored! the !replication!of!SARS-CoV-2!119!
over!several!days!and!followe d!virus !sprea d!in!the!ep ithe lia.!Using!high-resolution !im a g in g,!w e !120!
observed!the!mas sive!fo rm atio n !and!apical!release! of! syncytia!occurrin g!between!day! three! and!121!
four! post-infect ion .! W e ! sho wed! that! syncytia! and ! cells! released! into ! the! apical! lum en ! are!122!
infectiou s,! sugg es tin g ! they! contribute! to! the! spreading! of! the! virus! in! the! epithelium,! and! by!123!
extension,! may! transmit! v irus! within! th e! patient! to! the ! lower! respiratory! tract! o r! into! th e!124!
environment.!Furthermore,!using!reconstituted!BE!derived!from !children,!we!show ed!that!viral!125!
production!in!children!epithelia!is!very!low!compared!to!adults,!and!that!viral!spread!is!restricted.!126!
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 28, 2021. ; https://doi.org/10.1101/2021.05.28.446159doi: bioRxiv preprint
!
!
5!
!
These!re sults!may!explain!the!clinical!and !epidemiological! obse rvations!that! S AR S-CoV-2!is! m ore!127!
likely! to! infec t! older! patients!than!children!and!that! older! patients! sh ow ! more! severe! clinical!128!
manifestations.! 129!
130!
Results 131!
132!
;6-6+,*0&-)&7),)7<445):0776+6-*0,*6:)8+&-390,4)620*9640,)/&:64)133!
One!of!the!major! in itia l!targets!for!SARS-CoV-2!is!the!respiratory!tract.!Primary!infections!often!134!
initiate!in!the!upper!respiratory !tract!from!which!they!can!spread!to!the!lower!respiratory!tract!to!135!
cause! severe!disease! (18).! B ro n chia l! epith elia! are! pseudo-stratified! cell! layers! with!typical!cell!136!
junction s,!as!well!a s !a!mucus!layer!and !beating!cilia!on!the!lumen!side!(33,!34).!To!study!the!SARS-137!
CoV-2! infectio n ! process! in! a ! p h y sio lo g ica lly! relevant! mo d e l,! we! established! a! cellular! &'( !&)#*!138!
model!of!bronchial!epithelia!differentiated!in!air-liquid!interfa ce !from! in divid ua l! do n ors ! (Fig .!S 1).!139!
Primary!bronchial!epithelial!cells!were!collected!from!surgical!bronchial!resection!or!fibroscopy!140!
from!in divid ua l!adult!donors!at!the!Bordeaux!university!hospital.! P atie n ts!were!betw ee n !46!an d !141!
63!years! old!with! a!normal! body! m ass! index![BM I]!(Tab le1).!B a sa l!ep ith elia l!ce lls!w e re!expanded !142!
&'(!&) #* !in!culture !flask!until!conflu e n ce .!Basal!cells!were!then!seeded !on!cell!culture!insert!and!143!
differentiated! at! the! air-liquid! interface ! for! a pp rox im ate ly! 21! days! (Fig.! S1A).! ! Using ! this!144!
differentiation!protocol,!we!were!able!to!generate!between!12-24!individual!inserts!from!a!single!145!
donor!allowing!comparative!analysis.!Immuno-fluores cen ce !(IF)!analys is!confirmed!the!presence!146!
of!differentiated!cell!types.!Specific!antibodies!allowed!the!detection!of!acetylated!tubulin!an d !147!
mucin,!characteristic!of!multi-ciliated!cells!and!go blet!cells!resp ec tively! (Fig.!S1B,!movie!S1)!or!148!
acetylated!tubulin! and!cytokeratin! 5!(multi-ciliated! ce lls!and!basal!cells,!Fig.!S1C ,! m ovie!S2).! This!149!
analysis! confirmed! the! pseudostratified! apical-to-basolateral! organizational! integrity! of! the!150!
epithelia,!"+,+!a!single!cell!layer!of!apical!multi-ciliated!cells!covering!a!layer!of!basal!cells!and!was!151!
further! confirme d ! by! electron! microscopy! (Fig.! S1D).! The! presence! of! well! differentiated! cilia!152!
structures!and!tight!junctions!was!also!confirmed!(Fig.!S1D).!Next,!w e!determined!the!localization!153!
of!ACE2,!the!primary!receptor!for!SARS-CoV-2! in!o ur!m odel! using!IF! analysis!(Fig.!S1E,!m ovie! S3).!154!
Co-label!with!antibodies!against!ACE2!and!acetylated!tubulin!confirmed!that!ACE2!was!expressed!155!
in! apical! multi-ciliated! cells! as! previously! reported! (4,! 35).! M or eo v er,! o u r! d a ta! s h owed! a!156!
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 28, 2021. ; https://doi.org/10.1101/2021.05.28.446159doi: bioRxiv preprint
Citations
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Journal Article•DOI•
SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation.

[...]

Maaran Michael Rajah1, Maaran Michael Rajah2, Mathieu Hubert2, Elodie Bishop1, Elodie Bishop2, Nell Saunders2, Rémy Robinot2, Ludivine Grzelak2, Ludivine Grzelak1, Delphine Planas2, Jérémy Dufloo2, Jérémy Dufloo1, Stacy Gellenoncourt1, Stacy Gellenoncourt2, Alice Bongers2, Alice Bongers1, Marija Zivaljic1, Marija Zivaljic2, Cyril Planchais2, Florence Guivel-Benhassine2, Françoise Porrot2, Hugo Mouquet2, Lisa A. Chakrabarti2, Julian Buchrieser2, Olivier Schwartz2, Olivier Schwartz1 •
University of Paris1, Pasteur Institute2
25 Oct 2021-The EMBO Journal
TL;DR: In this paper, the SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells, and the syncytia forming potential of spike variant proteins remain poorly characterized.
Abstract: Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.

90 citations

Journal Article•DOI•
The Mechanism and Consequences of SARS-CoV-2 Spike-Mediated Fusion and Syncytia Formation.

[...]

Maaran Michael Rajah1, Ana Cristina Gallego-Hernández2, Maaran Michael Rajah3, Annie Bernier4, Julian Buchrieser1, Olivier Schwartz3, Olivier Schwartz1 •
Pasteur Institute1, Vaccine Research Institute2, University of Paris3, Curie Institute4
01 Oct 2021-Journal of Molecular Biology
TL;DR: In this article, the authors discuss the molecular determinants of S mediated fusion and the antiviral innate immunity components that counteract syncytia formation in SARS-CoV-2 infection and pathology.

59 citations

Posted Content•DOI•
B.1.1.7 and B.1.351 SARS-CoV-2 variants display enhanced Spike-mediated fusion

[...]

Maaran Michael Rajah1, Maaran Michael Rajah2, Mathieu Hubert1, Elodie Bishop2, Elodie Bishop1, Nell Saunders1, Rémy Robinot1, Ludivine Grzelak1, Ludivine Grzelak2, Delphine Planas1, Marija Zivaljic2, Marija Zivaljic1, Cyril Planchais1, Florence Guivel-Benhassine1, Françoise Porrot1, Hugo Mouquet1, Lisa A. Chakrabarti1, Julian Buchrieser1, Olivier Schwartz2, Olivier Schwartz1 •
Pasteur Institute1, University of Paris2
11 Jun 2021-bioRxiv
TL;DR: In this paper, the SARS-CoV-2 emerging variants display enhanced syncytia formation, and the spike protein of Alpha, Beta and Delta, in the absence of other viral proteins, induce more syncytsia than D614G, while antibody escape mutation E484K, K417N and Delta242-244 hamper it.
Abstract: Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighbouring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha and Beta spread and fusion in cell cultures. Alpha and Beta replicated similarly to D614G reference strain in Vero, Caco-2, Calu-3 and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Alpha, Beta and D614G fusion was similarly inhibited by interferon induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes differentially modified fusogenicity, binding to ACE2 and recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation. SynopsisThe Spike protein of the novel SARS-CoV-2 variants are comparative more fusogenic than the earlier strains. The mutations in the variant spike protein differential modulate syncytia formation, ACE2 binding, and antibody escape. O_LIThe spike protein of Alpha, Beta and Delta, in the absence of other viral proteins, induce more syncytia than D614G C_LIO_LIThe ACE2 affinity of the variant spike proteins correlates to their fusogenicity C_LIO_LIVariant associated mutations P681H, D1118H, and D215G augment cell-cell fusion, while antibody escape mutation E484K, K417N and {Delta}242-244 hamper it. C_LIO_LIVariant spike-mediated syncytia formation is effectively restricted by IFITMs C_LI

43 citations

Posted Content•DOI•
SARS-CoV-2 Alpha, Beta and Delta variants display enhanced Spike-mediated Syncytia Formation

[...]

Maaran Michael Rajah1, Maaran Michael Rajah2, Mathieu Hubert1, Elodie Bishop1, Elodie Bishop2, Nell Saunders1, Rémy Robinot1, Ludivine Grzelak1, Ludivine Grzelak2, Delphine Planas1, Jérémy Dufloo2, Jérémy Dufloo1, Stacy Gellenoncourt1, Stacy Gellenoncourt2, Alice Bongers2, Alice Bongers1, Marija Zivaljic1, Marija Zivaljic2, Cyril Planchais1, Florence Guivel-Benhassine1, Françoise Porrot1, Hugo Mouquet1, Lisa A. Chakrabarti1, Julian Buchrieser1, Olivier Schwartz1, Olivier Schwartz2 •
Pasteur Institute1, University of Paris2
02 Aug 2021-bioRxiv
TL;DR: In this paper, the SARS-CoV-2 emerging variants display enhanced syncytia formation, and the spike protein of Alpha, Beta, and Delta, in the absence of other viral proteins, induce more synytia than D614G, while antibody escape mutation E484K, K417N and Δ242-244 hamper it.
Abstract: Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighbouring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha and Beta spread and fusion in cell cultures. Alpha and Beta replicated similarly to D614G reference strain in Vero, Caco-2, Calu-3 and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Alpha, Beta and D614G fusion was similarly inhibited by interferon induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes differentially modified fusogenicity, binding to ACE2 and recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation. Synopsis The Spike protein of the novel SARS-CoV-2 variants are comparative more fusogenic than the earlier strains. The mutations in the variant spike protein differential modulate syncytia formation, ACE2 binding, and antibody escape. The spike protein of Alpha, Beta and Delta, in the absence of other viral proteins, induce more syncytia than D614G The ACE2 affinity of the variant spike proteins correlates to their fusogenicity Variant associated mutations P681H, D1118H, and D215G augment cell-cell fusion, while antibody escape mutation E484K, K417N and Δ242-244 hamper it. Variant spike-mediated syncytia formation is effectively restricted by IFITMs

41 citations

Journal Article•DOI•
A Simple and Fast Method to Sequence the Full-Length Spike Gene for SARS-CoV-2 Variant Identification from Patient Samples

[...]

Patricia Recordon-Pinson, Marie-Lise Blondot, Pantxika Bellecave, Marie-Edith Lafon, Camille Tumiotto, Mathieu Métifiot, Marie-Line Andreola 
03 Sep 2021
TL;DR: The present work describes a procedure based on the widely available Sanger technology to produce a full-length sequence of the spike gene from patient-derived samples, and describes the challenge to efficiently monitor the appearance of spike mutations in the population.
Abstract: Since the beginning of the pandemic, a race has been underway to detect SARS-CoV-2 virus infection (PCR screening, serological diagnostic kits), treat patients (drug repurposing, standard care) and develop a vaccine. After almost a year of active circulation worldwide, SARS-CoV-2 variants have appeared in different countries. Those variants include mutations in multiple regions of the genome, particularly in the spike gene. Because this surface protein is a key player in both the spread of the virus and the efficacy of vaccine strategies, the challenge is to efficiently monitor the appearance of spike mutations in the population. The present work describes a procedure based on the widely available Sanger technology to produce a full-length sequence of the spike gene from patient-derived samples.

5 citations

References
More filters
Journal Article•DOI•
Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

[...]

Chaolin Huang1, Yeming Wang2, Xingwang Li3, Lili Ren4, Jianping Zhao5, Yi Hu5, Li Zhang1, Guohui Fan2, Jiuyang Xu6, Xiaoying Gu2, Zhenshun Cheng7, Ting Yu1, Jia'an Xia1, Yuan Wei1, Wenjuan Wu1, Xuelei Xie1, Wen Yin5, Li Hui2, Min Liu2, Yan Xiao4, Hong Gao4, Li Guo4, Jungang Xie5, Guang-Fa Wang8, Rongmeng Jiang3, Zhancheng Gao8, Qi Jin4, Jianwei Wang4, Bin Cao2 •
Wuhan Jinyintan Hospital1, China-Japan Friendship Hospital2, Capital Medical University3, Peking Union Medical College4, Huazhong University of Science and Technology5, Tsinghua University6, Wuhan University7, Peking University8
24 Jan 2020-The Lancet
TL;DR: The epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of patients with laboratory-confirmed 2019-nCoV infection in Wuhan, China, were reported.

36,578 citations

Journal Article•DOI•
A Novel Coronavirus from Patients with Pneumonia in China, 2019.

[...]

Na Zhu1, Dingyu Zhang, Wenling Wang1, Xingwang Li2, Bo Yang1, Jingdong Song1, Xiang Zhao1, Baoying Huang1, Weifeng Shi, Roujian Lu1, Peihua Niu1, Faxian Zhan1, Xuejun Ma1, Dayan Wang1, Wenbo Xu3, Wenbo Xu1, Guizhen Wu1, George F. Gao, Wenjie Tan1 •
Chinese Center for Disease Control and Prevention1, Capital Medical University2, Anhui University of Science and Technology3
24 Jan 2020-The New England Journal of Medicine
TL;DR: Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily, which is the seventh member of the family of coronaviruses that infect humans.
Abstract: In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).

21,455 citations

Journal Article•DOI•
A simple method of estimating fifty per cent endpoints

[...]

Lowell J. Reed, H. Muench
01 May 1938-American Journal of Epidemiology

17,196 citations

Journal Article•DOI•
A pneumonia outbreak associated with a new coronavirus of probable bat origin

[...]

Peng Zhou1, Xing-Lou Yang1, Xian Guang Wang2, Ben Hu1, Lei Zhang1, Wei Zhang1, Hao Rui Si1, Yan Zhu1, Bei Li1, Chao Lin Huang2, Hui-Dong Chen2, Jing Chen1, Yun Luo1, Hua Guo1, Ren Di Jiang1, Meiqin Liu1, Ying Chen1, Xu Rui Shen1, Xi Wang1, Xiao Shuang Zheng1, Kai Zhao1, Quanjiao Chen1, Fei Deng1, Lin Lin Liu3, Bing Yan1, Fa Xian Zhan3, Yan-Yi Wang1, Gengfu Xiao1, Zhengli Shi1 •
Chinese Academy of Sciences1, Wuhan Jinyintan Hospital2, Centers for Disease Control and Prevention3
03 Feb 2020-Nature
TL;DR: Identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China, and it is shown that this virus belongs to the species of SARSr-CoV, indicates that the virus is related to a bat coronav virus.
Abstract: Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1–4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5–7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor—angiotensin converting enzyme II (ACE2)—as SARS-CoV. Characterization of full-length genome sequences from patients infected with a new coronavirus (2019-nCoV) shows that the sequences are nearly identical and indicates that the virus is related to a bat coronavirus.

16,857 citations

Journal Article•DOI•
SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

[...]

Markus Hoffmann1, Hannah Kleine-Weber1, Simon Schroeder2, Nadine Krüger3, Tanja Herrler, Sandra Erichsen4, Tobias S. Schiergens5, Georg Herrler3, Nai Huei Wu3, Andreas Nitsche6, Marcel A. Müller2, Christian Drosten7, Christian Drosten2, Stefan Pöhlmann1 •
German Primate Center1, Humboldt University of Berlin2, University of Veterinary Medicine Vienna3, Paracelsus Private Medical University of Salzburg4, Ludwig Maximilian University of Munich5, Robert Koch Institute6, Charité7
16 Apr 2020-Cell
TL;DR: It is demonstrated that SARS-CoV-2 uses the SARS -CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming, and it is shown that the sera from convalescent SARS patients cross-neutralized Sars-2-S-driven entry.

15,362 citations

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Frequently Asked Questions (1)
Q1. What are the contributions mentioned in the paper "Sars-cov-2 transmission via apical syncytia release from primary bronchial epithelia and infectivity restriction in children epithelia" ?

In this paper, the authors proposed a method to detect the presence of infectious and tropical diseases in Bordeaux, France.