AK, Koundouno, FR, Faye, M, Düx, A, Hinzmann, J, Diallo, H, Ayouba, A, Le Keita

F, Soropogui, B, Ifono, K, Diagne, MM, Sow, MS Marcis

Alpha Kabinet Keita

Fara R Koundouno

Martin Faye

Ariane Düx

Julia Hinzmann

Haby Diallo

Ahidjo Ayouba

Frederic Le Marcis

Barré Soropogui

Kékoura Ifono

Moussa M Diagne

Mamadou S Sow

Joseph A Bore

Sebastien Calvignac-Spencer

Nicole Vidal

Jacob Camara

Mamadou B Keita

Annick Renevey4

Amadou Diallo

Abdoul K Soumah

Saa L Millimono

Almudena Mari-Saez

Mamadou Diop

Ahmadou Doré

Fodé Y Soumah

Kaka Kourouma

Nathalie J Vielle

Cheikh Loucoubar

Ibrahima Camara

Karifa Kourouma

Giuditta Annibaldis

Assaïtou Bah

Anke Thielebein

Meike Pahlmann

Steven T Pullan

Miles W Carroll

Joshua 13

Quick

Pierre Formenty

Anais Legand

Karla Pietro

Michael R Wiley

Noel Tordo

Christophe Peyrefitte

John T Mccrone

Andrew Rambaut

Youssouf Sidibé

Mamadou D Barry

Madeleine Kourouma

Cé D Saouromou

Mamadou Condé

Moussa Baldé

Moriba Povogui

Sakoba Keita

Mandiou Diakite

Mamadou S Bah

Amadou Sidibe

Dembo Diakite

Fodé B Sako

Fodé A Traore

Georges A Ki-Zerbo

Philippe Lemey

Stephan Günther

Liana E Kafetzopoulou

Amadou A Sall

Eric Delaporte

Sophie Duraffour

Ousmane Faye

Fabian Leendertz

Martine Peeters

Abdoulaye Toure

Faly N'

Magassouba

Resurgence of Ebola virus in 2021 in Guinea suggests a new paradigm for outbreaks

Seven years after the declaration of the first Ebola virus disease (EVD) epidemic in Guinea, the country faced a new outbreak in 2021 (February 14 th to June 19 th ) near the epicenter of the previous epidemic 1,2 . Complete or near-complete Ebolavirus genomes were generated from samples from 12 different patients using next generation sequencing. The new Zaire Ebolavirus (EBOV) genomes formed a well-supported phylogenetic cluster with genomes from the previous outbreak, indicating that the new outbreak was not the result of a new spillover event from an animal reservoir. The 2021 lineage shows considerably lower divergence than expected during sustained human-to-human transmission, suggesting a persistent infection with reduced replication or a period of latency. The resurgence of Ebola from humans five years after the end of the previous EVD outbreak reinforces the need for longterm medical and social care for survivors to reduce the risk of disease re-emergence and prevent further stigmatization.

Main text

 Guinea faced a new EVD outbreak in 2021, which started in Gouéké, a town about 200 km away from the epicenter of the 2013-2016 outbreak. The probable index case was a 51-yearold nurse, assistant of the hospital midwife in Gouéké. On the 21 st of January 2021, she was admitted to the hospital of Gouéké suffering from headache, asthenia, nausea, anorexia, vertigo, and abdominal pain. She was diagnosed with malaria and salmonellosis and released two days later. Feeling ill again once at home, she attended a private clinic in N'zérékoré, at 40 km, and visited a traditional healer, but died three days later. In the week following her death, her husband and other family members, who attended her funeral, fell ill and four of 2014

them died. They were reported as the first suspect cases by the national epidemic alert system on 11 th February. On February 12 th , blood was taken from two suspect cases admitted at the hospital in N'zérékoré. On February 13 th , both were confirmed with EVD by the laboratory in Guéckédou using a commercial real-time reverse transcription Polymerase Chain Reaction (RT-PCR) assay (RealStar Filovirus Screen Kit, Altona Diagnostics). On February 13 th , the husband of the index case, who travelled for treatment from Gouéké to Conakry, the capital city of Guinea (>700 km distance), was admitted to the Centre de Traitement Epidémiologique (CTEpi) in Nongo, Ratoma Commune. He presented with fever, nausea, asthenia of abdominal and lumbar pain and was considered highly suspicious for EVD. A blood sample was analyzed on the same day and found positive for Ebola Zaire (Zaire ebolavirus; EBOV) on the GeneXpert molecular diagnostic platform (Xpert Ebola test, Cepheid) and by an in house qRT-PCR. The laboratory confirmation of EVD in the three suspect cases led to the official declaration of the epidemic on February 14 th . At 5 th March, 14 confirmed cases and 4 probable cases of EVD have been identified, leading to 9 deaths including five confirmed cases as reported by the Agence Nationale de la Sécurité Sanitaire (ANSS) of Guinea. After a period of 25 days without new cases, two new cases have been reported around N'zérékoré on April 1 st and 3 th and on 19 th June 2021, the outbreak was declared over. Overall, 16 confirmed cases were reported, among them 12 people died.

Genomic characterization of the virus causing the 2021 Guinean EVD epidemic was of immediate public health importance. First, because diagnostic tools, therapeutics and vaccines, with proven effectiveness in recent EVD outbreaks, i.e. in Guinea (2013-2016) and in the Equateur and North-Kivu/Ituri provinces of the Democratic Republic of Congo (2018-2020), have primarily been developed for EBOV. [3][START_REF] Mbala-Kingebeni | Rapid Confirmation of the Zaire Ebola Virus in the Outbreak of the Equateur Province in the Democratic Republic of Congo: Implications for Public Health Interventions[END_REF][START_REF] Mbala-Kingebeni | Ebola virus disease outbreak in Equateur Province, Democratic Republic of the Congo: a retrospective genomic characterization[END_REF] Secondly, to identify whether the outbreak resulted from a new zoonotic transmission event or from the resurgence of a viral strain that had circulated in a previous EBOV outbreak -EBOV can persist in body fluids of EVD survivors and be at the origin of new transmission chains. [START_REF] Pigott | Mapping the zoonotic niche of Ebola virus disease in Africa[END_REF][7][START_REF] Sissoko | Ebola Virus Persistence in Breast Milk After No Reported Illness: A Likely Source of Virus Transmission From Mother to Child[END_REF] Although the Xpert Ebola test has been developed to detect only EBOV strains and the in-house qRT-PCR assay uses a probe specifically designed to detect EBOV 9 , additional confirmation by sequence analysis was sought by targeting a short fragment in the viral protein (VP) 35 region on the sample from the patient hospitalized in Conakry. The phylogenetic tree (Supplementary Figure 1) underscores that this highly conserved region can discriminate between Ebola virus species and confirmed that the new strain belongs to the species Zaire ebolavirus (EBOV). This confirmed that available vaccines and the vast majority of molecular diagnostics tools and therapeutics could be immediately applied.

To gain further insight into the genomic make-up of the viruses causing this outbreak, eleven complete or near-complete (>95% recovery), eight partial (>65% recovery) genomic sequences from 12 of all 14 confirmed cases were obtained by three different laboratories using different next generation sequencing technologies ( MinIon (Oxford Nanopore Technologies, Oxford, UK) and a hybrid-capture based approach using a probe panel that included Ebola virus (EBOV) specific targets followed by TruSeq Exome Enrichment, as previously described. [START_REF] Mbala-Kingebeni | Ebola virus disease outbreak in Equateur Province, Democratic Republic of the Congo: a retrospective genomic characterization[END_REF] Data generated between the three groups were pooled and the sequence with the highest quality was chosen for each patient. This allowed us to reconstruct twelve high quality EBOV genomes which cover 82.9%-99.9% of the reference genome (KR534588) (Table 1). The consensus EBOV sequences with the highest genome recovery (>82.9%) from 12 different patients were used in further analyses.

Maximum likelihood phylogenetic reconstruction places the 12 genomes from the 2021 Guinea outbreak as a single cluster among the EBOV viruses responsible for the 2013-2016 EVD outbreak in West Africa (Figure 1 and Figure 2). The 2021 genomes share 10 substitutions accumulated during the 2013-2016 outbreak (compared to KJ660346), including the A82V marker mutation for human adaptation in the glycoprotein that arose when the virus spread to Sierra Leone. [START_REF] Diehl | Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013-2016 Epidemic[END_REF][START_REF] Urbanowicz | Human Adaptation of Ebola Virus during the West African Outbreak[END_REF] These patterns provide strong evidence for direct linkage to human cases from the 2013-2016 outbreak rather than a new spillover from an animal reservoir. The 2021 lineage is nested within a clade that predominantly consists of genomes sampled from Guinea in 2014 (Figure 2). The branch by which the 2021 cluster diverges from the previous outbreak exhibits only 12 substitutions, which is far fewer than expected from EBOV evolution during 6 years of sustained human-to-human transmission (Figure 3). Using a local molecular clock analysis, we estimate a 6.4-fold (95% Highest Posterior Density interval (HPD): 3.3-fold,10.1-fold) lower rate along this branch. For comparison, we also estimate a 5.5-fold (1.6-fold,10.8-fold) lower rate along the branch leading to the 2016 flareup that was linked to a survivor with virus persistence for more than 500 days. 7,[START_REF] Blackley | Reduced evolutionary rate in reemerged Ebola virus transmission chains[END_REF] Rather than a constant long-term low evolutionary rate, some degree of latency or dormancy during persistent infection seems a more likely explanation for the low divergence of the 2021 genomes. We tested whether the 12 genomes from 2021, sampled over a time period of less than one month, contained sufficient temporal signal to estimate the time to most recent common ancestor (tMRCA) (Supplementary Figure 2), but did not identify statistical support for sufficient divergence accumulation over this short time scale. We therefore calibrated our analysis using an evolutionary rate that reflects EBOV evolution under sustained human-to- These results open a new perspective on the relatively rare observation of EBOV reemergence. It is assumed that all known filovirus outbreaks in humans are the result of independent zoonotic transmission events from bat reservoir species or from intermediate or amplifying hosts like apes and duikers. [START_REF] Pigott | Mapping the zoonotic niche of Ebola virus disease in Africa[END_REF] Here we clearly show that, even almost five years after the declaration of the end of an epidemic, new outbreaks could also be the result of transmission from humans infected during a previous epidemic. The viruses from the 2021 outbreak fall within the lineage of EBOV viruses obtained from humans during the 2014-2016 outbreak, therefore it is thus very unlikely that this new outbreak has an animal origin or is the result from a new cross-species transmission with the same lineage that remained latent in this natural host which in that scenario would be at the basis of the west african cluster. The limited genomic divergence between 2014-2015 and 2021 is compatible with a slow longterm evolutionary rate. However, a relatively long phase of latency may be more likely than continuous slow replication. Independent of the mechanistic explanation, the virus most likely persisted at low level in a human survivor. Plausible scenarios of EBOV transmission to the index case include (i) sexual transmission by exposure to EBOV in semen from a male survivor, (ii) contact to body fluids from a survivor with relapse to symptomatic EVD, for example during health care -the index case was a healthcare worker, or (iii) relapse of EVD disease in the index case, although she was not a known survivor, she may have had an asymptomatic or pauci-symptomatic EBOV infection during the previous outbreak. Detailed investigation by anthropologists on the family of the index case revealed that she was not known as an EVD survivor, nor her husband or close relatives. However, among more distantly related family, 25 individuals had EVD during the previous outbreak. Only five survived, but the index case apparently had no recent contacts with this part of the family.

Consultation of the hospital registers in Gouécké, showed that all patients seen by the index case in January 2021 were in good health and were still in good health in March 2021.

However, the index case also performed informal consultations outside the hospital environment which could not be verified. Alternatively, the nurse was not the actual index case but part of a small unrecognized chain of human-to-human transmission in this area of Guinea. However, the diversity of the currently available genomes is limited and molecular clock analysis suggests a recent time to the most recent common ancestor, with a mean estimate close to the time the nurse was first hospitalized and 95% HPD boundary around the turn of the year. This provides some reassurance that the outbreak was detected early.

The 2013-2016 outbreak in West Africa was the largest and most complex outbreak of EBOV with more than 28,000 cases, 11,000 deaths and an estimated 17,000 survivors, notably in Guinea, Liberia and Sierra Leone. [START_REF]Situation Report: Ebola Virus Disease[END_REF] The large outbreak provided new information about the disease itself as well as about the medical, social and psychological implications for EVD survivors. [START_REF] Diallo | Understanding the long-term evolution and predictors of sequelae of Ebola virus disease survivors in Guinea: A 48-month prospective, longitudinal cohort study (PostEboGui)[END_REF][START_REF] Etard | Multidisciplinary assessment of post-Ebola sequelae in Guinea (Postebogui): an observational cohort study[END_REF][START_REF]A Longitudinal Study of Ebola Sequelae in Liberia[END_REF] It was also possible to estimate to some extent the proportions of asymptomatic or pauci-symptomatic infections and to identify their role in specific unusual transmission chains. [START_REF] Diallo | Prevalence of infection among asymptomatic and paucisymptomatic contact persons exposed to Ebola virus in Guinea: a retrospective, cross-sectional observational study[END_REF][START_REF] Glynn | Asymptomatic infection and unrecognised Ebola virus disease in Ebolaaffected households in Sierra Leone: a cross-sectional study using a new non-invasive assay for antibodies to Ebola virus[END_REF][START_REF] Camara | Unrecognized Ebola virus infection in Guinea: complexity of surveillance in a health crisis situation: case report[END_REF] While the main route of human-to-human EBOV transmission is direct contact with infected body fluids from symptomatic or deceased patients, some transmission chains were associated with viral persistence in semen. 3 Several studies demonstrated viral persistence in more than 50% of male survivors at 6 months after discharge from Ebola Treatment Units (ETU) and the maximal duration of persistence in semen has been reported

to last up to 500 -700 days post ETU discharge in a handful of male EVD survivors. 9,[START_REF] Thorson | Persistence of Ebola virus in semen among Ebola virus disease survivors in Sierra Leone: A cohort study of frequency, duration, and risk factors[END_REF][START_REF] Fischer | Ebola Virus Ribonucleic Acid Detection in Semen More Than Two Years After Resolution of Acute Ebola Virus Infection[END_REF][START_REF] Sissoko | Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study[END_REF] Transmission through other body fluids (breast milk, cervicovaginal fluids) is also suspected. [START_REF] Sissoko | Ebola Virus Persistence in Breast Milk After No Reported Illness: A Likely Source of Virus Transmission From Mother to Child[END_REF][START_REF] Mate | Molecular Evidence of Sexual Transmission of Ebola Virus[END_REF][START_REF] Dokubo | Persistence of Ebola virus after the end of widespread transmission in Liberia: an outbreak report[END_REF][START_REF] Liu | Comprehensive Clinical and Laboratory Follow-up of a Female Patient With Ebola Virus Disease: Sierra Leone Ebola Virus Persistence Study[END_REF] Furthermore, some immunological studies among survivors suggest a continuous or intermittent EBOV antigenic stimulation due to persistence of an EBOV reservoir in some survivors [START_REF] Wiedemann | Long-lasting severe immune dysfunction in Ebola virus disease survivors[END_REF][START_REF] Adaken | Ebola virus antibody decay-stimulation in a high proportion of survivors[END_REF] , although this was not confirmed in another study. [START_REF] Thom | Longitudinal antibody and T cell responses in Ebola virus disease survivors and contacts: an observational cohort study[END_REF] Cases of relapse of EVD have also been sporadically reported and can be at the origin of large transmission chains as recently reported in the North-Kivu outbreak in DRC. [START_REF] Mbala Kingebeni | Ebola Virus Transmission Initiated by Relapse of Systemic Ebola Virus Disease[END_REF] For example, we recently reported presence of EBOV RNA in breast milk 500 days after ETU discharge in a woman who was not pregnant when she developed EVD. She attended the hospital due to complications at 8 months of pregnancy and a breast milk sample taken 1 month after delivery tested positive for EBOV RNA. 9 These examples illustrate that health care workers can be exposed to EBOV when taking care of patients who survived EVD, but have an unrecognized relapse of their infection. The 2021 outbreak now highlights that viral persistence and reactivation is not limited to a two-year period but can also occur much longer with late reactivation.

Active genomic surveillance already showed resurgence of previous strains in other outbreaks. For example, two EBOV variants circulated simultaneously within the same region during the recent 2020 outbreak in the Equateur Province, DRC. [START_REF] Muyembe-Tamfum | Two Ebola virus variants circulating during the 2020 Equateur Province outbreak[END_REF] Moreover, strains from the two consecutive outbreaks in Luebo, Democratic Republic of Congo (DRC), in 2007 and 2008, are also so closely related that it now appears difficult to exclude that the epidemic observed in 2008 was possibly due to a resurgence event from a EVD survivor from the 2007 outbreak. [START_REF] Leroy | Human Ebola outbreak resulting from direct exposure to fruit bats in Luebo, Democratic Republic of Congo, 2007[END_REF][START_REF] Grard | Emergence of divergent Zaire ebola virus strains in Democratic Republic of the Congo in 2007 and 2008[END_REF] However, the limited genomic sampling does not allow for a formal test of this hypothesis.

While the majority of EVD outbreaks remained limited both in number of cases and geographic spread, the two largest outbreaks in West Africa (December 2013 to June 2016) and in Eastern DRC (August 2018 to June 2020) infected thousands of individuals over wide geographic areas leading to large numbers of EVD survivors. This means that the risk of resurgence is higher than ever before. Continued surveillance of EVD survivors is therefore warranted to monitor reactivation and relapse of EVD infection and potential presence of virus in body fluids. This work and associated communications have to be conducted with the utmost care towards the well-being of EVD survivors. During the 2013-2016 Ebola outbreak in Guinea, Ebola survivors had a mixed experience after discharge from ETUs. On the one hand, they were considered as heroes by NGOs and became living testimonies of a possible recovery. [START_REF] Enria | What Crisis Produces: Dangerous Bodies, Ebola Heroes and Resistance in Sierra Leone[END_REF][START_REF] Ronse | What motivates Ebola survivors to donate plasma during an emergency clinical trial? The case of Ebola-Tx in Guinea[END_REF] On the other hand, they experienced different forms of stigmatization such as rejection by family and friends, refusal to be involved in collective work, loss of jobs and housing and sometimes self-isolation from social life and workplaces. [START_REF] Sow | Ebola en Guinée : formes de la stigmatisation des acteurs de Sante survivants[END_REF] The human origin of the current EVD outbreak and the associated shift in our perception of EBOV emergence, call for careful attention to survivors. The concrete danger that survivors will be stigmatized as a source of danger should be a matter of scrupulous attention. [START_REF] Farmer | Aids and Accusation. Haiti and the Geography of Blame[END_REF] This is especially true for the area of Gouécké which is only 9 km away from Womey, a village emblematic of the violent reaction of the population toward the EVD response team during the 2013-2016 epidemic. [START_REF] Wilkinson | Comparison of social resistance to Ebola response in Sierra Leone and Guinea suggests explanations lie in political configurations not culture[END_REF] Since the 2013-2016 EVD outbreak in Western Africa, genome sequencing became a major component of the outbreak response. [START_REF] Quick | Real-time, portable genome sequencing for Ebola surveillance[END_REF][START_REF] Gire | Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak[END_REF][START_REF] Arias | Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases[END_REF][START_REF] Pini | Field investigation with real-time virus genetic characterisation support of a cluster of Ebola virus disease cases in Dubréka, Guinea, April to June 2015[END_REF][START_REF] Mbala-Kingebeni | Medical countermeasures during the 2018 Ebola virus disease outbreak in the North Kivu and Ituri Provinces of the Democratic Republic of the Congo: a rapid genomic assessment[END_REF] Establishment of in-country sequencing and capacity building allowed for a timely characterization of EBOV strains in this new outbreak in Guinea. In addition to the importance of appropriate health care measures focused on survivors, late resurgence also highlights the urgent need for further research on potent antiviral agents that can eradicate the latent virus reservoir in EVD patients and on efficient vaccines with long-term protection. In parallel, vaccination could also be considered to boost protective antibody responses in survivors. [START_REF] Adaken | Ebola virus antibody decay-stimulation in a high proportion of survivors[END_REF] Vaccination of populations in areas with previous EBOV outbreaks could also be promoted to prevent secondary cases. All patient samples were whole EDTA blood except for patient 12 for whom a swab was used 547 support. [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF][START_REF] Nguyen | IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF] The GTR model plus a discrete gamma distribution were used as nucleotide substitution models. Sequencing reads were filtered (adapter removal and quality filtering) with Trimmomatic (Bolger, 2014) (settings: LEADING:30 TRAILING:30 SLIDINGWINDOW:4:30 MINLEN:40), merged with ClipAndMerge (https://github.com/apeltzer/ClipAndMerge), and mapped to the Zaire ebolavirus RefSeq genome (NC_002549) using BWA-MEM. [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF] Mapped reads were sorted and deduplicated with SortSam and MarkDuplicates from the Picard suite (Broad Institute, Picard; http://broadinstitute.github.io/picard). We generated consensus sequences using Geneious Prime 2020.2.3 (https://www.geneious.com) where unambiguous bases were called when at least 90% of at least 20 unique reads were in agreement (20x, 90%). For samples with few mapped reads (0001, 0002, 0010, 0030), we also called a consensus at 2X, 90% and 5X, 90%.

Full

Genome sequencing at PFHG

Sequencing at PFHG was performed using a mobile MinION facility deployed by BNITM to Guinea beginning of March 2021. A total of 13 EBOV positive initial diagnostic samples processed at the "Laboratoire des Fièvres Hémorragiques Virales de Gueckédou", the "Laboratoire Régional de l'Hôpital de N'Zérékoré" were used for sequencing. If RNAs from diagnostic procedures performed by the peripheral laboratories was not sent to PFHG, samples were inactivated and RNA was extracted from 50 µl for whole blood EDTA, 70 μl of plasma from EDTA blood or from 140 µl of wet swabs using the QIAamp Viral RNA Mini Kit (Qiagen) following the manufacturer's instructions. Tiled primers generating overlapping products combined with a highly multiplexed PCR protocol were used for the amplicon generation. [START_REF] Quick | Real-time, portable genome sequencing for Ebola surveillance[END_REF] At start of deployment, three different primers pools (V3 or pan_10_EBOV, V4 or pan_EBOV and Zaire-PHE or EBOV-Zaire-PHE) were tested and results were combined for optimal recovery of consensus. A new primer pool V5 (EBOV-Makona-V5) was further designed and implemented to increase consensus recovery. Primer pools V3, V4 and V5 were pipeline was used to obtain the consensus sequences. The data was normalized to 200 and using the --scheme-directory option the pipeline was directed to the respective primer scheme used for each barcode. Reads were aligned to the NCBI reference KJ660347 (Zaire ebolavirus isolate H.sapiens-wt/GIN/2014/Makona-Gueckedou-C07) for data generated using V3, V4, and V5 primer pools and to NC_002549.1 (Zaire ebolavirus isolate Ebola virus/H.sapienstc/COD/1976/Yambuku-Mayinga) for data generated using Zaire-PHE primer pools.

Sequencing at IPD

Viral RNA was extracted from 140 µl of whole blood samples using the QIAamp Viral RNA Mini Kit (Qiagen, Heiden, Germany) according to manufacturer's instructions and eluted in nuclease-free water for a final volume of 60 µl. Extracted RNA was tested by real-time reverse transcription-polymerase chain reaction (RT-PCR) as previously described. [START_REF] Weidmann | Rapid detection protocol for filoviruses[END_REF] Briefly, the DNA library were prepared and enriched using the Illumina RNA Prep with enrichment, (L) Tagmentation kit (Illumina, San Diego, CA, USA) according to the manufacturer's recommendations with a pan viral probe panel that included EBOV specific targets. [START_REF] Mbala-Kingebeni | Ebola virus disease outbreak in Equateur Province, Democratic Republic of the Congo: a retrospective genomic characterization[END_REF] The purified libraries were pooled and sequenced on the Illumina MiSeq platform using the Miseq reagents kit v3 (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.

Illumina sequence reads were quality trimmed by Prinseq-lite and consensus EBOV genome sequences were generated using an in-house de novo genome assembly pipeline.

Phylogenetic analysis of full-length genome sequences

Phylogenetic inference

The new EBOV genome sequences were embedded in different data sets for subsequent analyses. For phylogenetic reconstruction, we use a Zaire Ebola virus data set consisting of 55 representative genomes from previous outbreaks and a Makona virus data set consisting of

Figure 1 .

 1 Figure 1. Maximum likelihood phylogenetic reconstruction for 55 representative

Figure 2 .

 2 Figure 2. Maximum likelihood phylogenetic reconstruction for 1065 genomes sampled

Figure 3 . 2 .

 32 Figure 3. Temporal divergence plot of genetic divergence from the root against time of sampling for the tree shown in Figure 2. The regression is exclusively fitted to genomes sampled between 2014 and 2015. The same colors are used for the data points as in Figure 2. The dashed yellow lines highlight how the 2021 data points deviate from the relationship between sampling time and sequence divergence. According to this relationship, about 95 substitutions (95% prediction interval: 88-101) are expected on the branch ancestral to the 2021 cluster, whereas only 12 are inferred on this branch.

  -length genome sequencing of the new Ebola viruses Genome sequencing at CERFIG Whole genome sequencing was attempted on viral extracts for samples that were positive for EBOV NP and GP on the GeneXpert molecular diagnostic platform (Xpert Ebola Assay) with the glycoprotein (GP) and the Nucleoprotein (NP) of the Zaire Ebola virus. We extracted full nucleic acid using the QIAamp® Viral RNA Mini Kit (Qiagen). After DNase treatment with TURBO DNA-free™ Kit (Ambion) and clean-up with RNA Clean & Concentrator Kit (Zymo Research), RNA was converted to double-stranded cDNA (ds-cDNA) using the SuperScript™ IV First-Strand Synthesis System (Invitrogen) and NEBNEXT® mRNA Second Strand Synthesis Module (New England Biolabs). Resulting ds-cDNA was enzymatically fragmented with NEBNext® dsDNA Fragmentase® (New England Biolabs) and converted to dual indexed libraries with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs). To enrich EBOV in the libraries, we performed two rounds of hybridization capture (16 hours at 65°C) with custom made biotinylated RNA baits (120 nucleoties, 2-fold tiling; Arbor Biosciences) covering representative genomes for Zaire ebolavirus (KC242801), Sudan ebolavirus (KC242783), Reston ebolavirus (NC_004161), Taï Forest ebolavirus (NC_014372), Bundibugyo ebolavirus (KC545395) and Marburg marburgvirus (FJ750956), following the myBaits Hybridization Capture for Targeted NGS protocol (Version 4.01). After the second round, capture products were quantified using the Qubit 3.0 Fluorometer with Qubit™ dsDNA HS Assay Kit (Invitrogen), and pooled equimolarly for sequencing on an Illumina iSeq using iSeq 100 i1 Reagents (2 x 150-cycle).

Table

  human transmission (as estimated by the local molecular clock analysis). This resulted in a tMRCA estimate of January 22 nd 2021 [95% Highest Posterior Density interval: December 29 th , 2020; February 10 th , 2021].

Table 1 . Patient and sample characteristics and sequencing results obtained by the 538 laboratories involved in the study.

 1

	539
	540
						CERFIG	PFHG		IPD
							%		%	%
		Patient Sex	Age (years)	Date of sampling	Ct value 1	genome recovery	Ct value 1	genome recovery	Ct value 1	genome recovery
		1	F		12-Feb-2021 a	-2	-	22.4	87.8	29.3	99.6
					19-Feb-2021 b 33.1	0.3	-	-	-	-
		2	F		12-Feb-2021 a -	-	25.9	67.8	37.1	98.7
		3	M		13-Feb-2021 a 29.4	5.0	neg.	7.5	neg.	2.5
		4	M		20-Feb-2021 b 24.3	12.6	-	-	-	-
		5	M		22-Feb-2021 a 32.5	99.4	23.2	93.3	-	-
		6	M		23-Feb-2021 b -	-	20.5	97.3	-	-
		7	F		26-Feb-2021 a -	-	19.5	95.5	-	-
		8	M		26-Feb-2021 a -	-	18.8	98.1	-	-
		9	M		26-Feb-2021 a 24.6	99.9	27.8	77.6	-	-
		10	F		26-Feb-2021 a 26.0	99.7	23.0	82.2	-	-
		11	F		26-Feb-2021 a 36.4	75.4	28.8	82.9	-	-
		12	M		26-Feb-2021 a -	-	20.5	96.1	-	-
		13	M		26-Feb-2021 a 25.0	99.9	29.3	70.4	-	-
		14	F		4-Mar-2021 a	-	-	22.0	97.5	-	-
	541	1 Ct value was measured in the sequencing laboratory before starting the sequencing process.
	542	2 Sample was not tested in this laboratory
	543	The values differ between the laboratories due to possible degradation of the sample or the
	544	RNA during transport and storage.
	545 546	a patient samples at diagnosis b follow-up samples from patients

Acknowledgements

The authors thank the Agence Nationale de Sécurité Sanitaire (ANSS) and the Ministry of Health of the Republic of Guinea, the healthcare workers (medical doctors, nurses and laboratory technicians) from the treatment centers in Nzérékoré and Nongo, Conakry; the laboratory personnel in Guékédou, Institut National de Santé Publique (INSP, Conakry) and Centre de Recherche et Formation en Infectiologie de Guinéé (CERFIG, Conakry). CERFIG also thanks Jan Gogarten and the bioinformatics support of RKI. UK Health Security Agency would like to thank Oxford Nanopore for the donation of reagents and equipment to support the setting up of the sequencing capacity at PFHG. BNITM thanks the ARTIC network (https://artic.network/). ZMV I1-2517GHP-704 and ZMVI1-2519GHP704), and by the Coalition for Epidemic Preparedness Innovations (CEPI). Work of BNITM and the UK Health Security Agency has been further supported by the Research and Innovation Programme of the European Union under Horizon 2020 grant agreement no. 871029-EVA-GLOBAL. The European Mobile Laboratory (EMLab) coordinated by BNITM is a technical partner of the WHO Global Outbreak Alert and Response Network (GOARN) and the deployment of EMLab experts and sequencing capacities to Guinea has been coordinated and supported by the GOARN Operational Support Team at WHO/HQ and the WHO country office in Guinea. The research leading to these results has received funding from the European Research Council under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 725422-ReservoirDOCS). The Artic Network receives funding from the Wellcome Trust through project 206298/Z/17/Z. PL acknowledges support by the Research Foundation --Flanders (`Fonds voor Wetenschappelijk Onderzoek --Vlaanderen', G066215N, G0D5117N and G0B9317N).

0.004 DRC 1976 DRC 2017 DRC 2018 DRC 2018 DRC 1995 DRC 2007 Gabon 1994 Gabon 2001 Guinea Guinea Sierra Leone Liberia DRC 2014

Funding

The work of CERFIG and TransVIHMI was supported in part by grants EBO-SURSY project funded by European Union, International Mixt Laboratory "RESPIRE" of IRD (Institut de Recherche pour le Developpement), Montpellier Université d'Excellence (EBOHEALTH; I-Site MUSE, ANR-16-IDEX-0006) and Institut National de la Santé et de la Recherche Médicale (INSERM)/REACTing (REsearch and ACTion targeting emerging infectious diseases). The work of RKI was partly funded by the German Ministry of Health "Global Protection Program" project TRICE. Frédéric Le Marcis received funding through the program « EBOVAC3 Bringing a prophylactic Ebola vaccine to licensure » funded by IMI (grant agreement number 800176) and run by LSTHM and INSERM. The work of PFHG and BNITM was supported by the German Federal Ministry of Health through support of the WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Viruses at the Bernhard-Nocht-Institute for Tropical Medicine (agreement ZMV I1-2517WHO005), and through the Global Health Protection Programme (GHPP, agreements Funding acquisition: E.D., F.H.L, M.Pe., M.W.C, S.D., S.G. Provided reagents: F.H.L., K.P., M.R.W, S.C-S. Project implementation: A.K.K., A.L., A.Th., A.To., C.P, E.D., F.H.L, G.A.K, J.Q., M.Dia., M.Pa., M.Pe, M.S.B, M.W.C, N.M., N.T, P.F., S.D., S.G., S.K., S.T.P. Wrote the first draft of the manuscript: A.Du, A.K.K, M.Pe, S.C-S. wrote and edited the manuscript: A.A.S, A.Du., A.K.K, A.M.S., A.Re., A.Ra, A.To., E.D., F.H.L, F.L, L.E.K., M.F, M.M.D, M.Pe, N.M., O.F, P.L., S.C-S, S.D., S.G.

All authors read and approved the contents of the manuscript.

Data availability

Sequencing results were made publicly available on March 12 th through joint posting on https://virological.org/c/ebolavirus/guinea-2021/44. The sequences generated at CERFIG have been deposited under GitHub project link: https://github.com/kabinet1980/Ebov_Guinea2021/blob/main/EBOV_Guinea_2021_genomes _CERFIG.fasta and The European Nucleotide Archive (ENA) project number : PRJEB43650 (https://www.ebi.ac.uk/ena/browser/view/PRJEB43650); The sequences generated at PFHVG have been deposited under GitHub project link: https://github.com/PFHVG/EBOVsequencing and the genome sequences for the two samples at IPD are available under link: https://drive.google.com/drive/folders/14dfGdNjWw17TkjrEQKLCrwlJ4WBBHI6K .

Genome sequences are also available on NCBI GenBank with the following accession numbers: ERX5245591 to ERX5245598; MZ424849 to MZ424862; MZ605320 and

MZ605321

Code availability:

All the codes for the analyses presented in this paper, including the analysis pipeline is described in detail in methods and is available in published papers, public websites or for inhouse pipelines available upon request.

Author contributions

Conceived and designed the study: A.A.S, A.K.K, A.To., E.D., F.H.L, F.R.K., L.E.K., M.Pe, N.M., O.F, S.D., S.G. collected data and/or performed and/or performed medical examinations and/or laboratory diagnostics: A.B., A.Do., A.K.K, A.K.S., A.M.S., A.S., B.S., C.D.S, D.D., F.L, F.R.K., F.Y.S., F.A.T, F.B.S, G.A., H.D., I.C., J.A.B., J.C. ,K.I. ,Kb.K., Kr.K., L.M., M.B., M.B.K., M.C., M.D.B., M.K., M.Po., M.S.S., N.V., N.J.V., S.D., Y.S.

Competing interests: MWC received materials for this study from Oxford Nanopore. All other authors declare no competing interests.

Ethics Statement

Diagnostic specimens were collected as part of the emergency response from the Ministry of public health from Guinea, and therefore consent for sample collection was waived. All preparation of samples for sequencing, genomic analysis and data analysis was performed on anonymized samples identifiable only by their laboratory or epidemiological identifier.

Confirmation of Ebola virus species by sequence analysis of VP 35 fragment at CERFIG

Viral RNA was extracted from 140 ul of whole blood collected from the samples from the patient hospitalized in Conakry, with the Nuclisens kit (Biomerieux, France) and following manufacturer's instructions. Amplification of a small fragment of VP35 region was attempted in a semi-nested PCR with a modified protocol as previously described. [START_REF] Mbala-Kingebeni | Rapid Confirmation of the Zaire Ebola Virus in the Outbreak of the Equateur Province in the Democratic Republic of Congo: Implications for Public Health Interventions[END_REF] First-round VP35 PCR-products from positive samples were barcoded and pooled using the Native Barcoding Kit EXP-NBD104 (Oxford Nanopore Technologies, Oxford, UK). Sequencing libraries were generated from the barcoded products using the Genomic DNA Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies) and were loaded onto a R9 flow cell on a MinIon (Oxford Nanopore Technologies). Genetic data were collected for one hour. Basecalling, adapter removal and demultiplexing of fastq files were performed with MinKNOW, version 4.1.22. Fastq reads >Q11 were used for mapping a virus database with the Genome Detective tool (https://www.genomedetective.com/app/typingtool/virus/). The generated consensus sequence was used for further analysis. For phylogenetic inference, we retrieved one sequence per outbreak from the Haemorhagic Fever Virus (HFV) database to which we added the newly generated VP35 sequence of the novel outbreak. Phylogenetic analyses were done using Maximum Likelihood methods using IQ-Tree with 1,000 bootstraps for branch designed by the ARTIC network and Zaire-PHE primer pools by Public Health England (PHE). For V3, 62 primers were used, while for V4 and V5, 61 primers pairs were used, to amplify products of ~400 nt length. For Zaire-PHE, 71 primer pairs were used to amplify products of ~350 nt length for the ~20 kb viral genome. All primer pools used can be found in Supplementary Table S1. The multiplex PCR was performed as described by the most up-todate ARTIC protocol for nCoV-2019 amplicon sequencing (nCoV-2019 sequencing protocol V3 (LoCost) V.3 (Artic Network. https://artic.network/ncov-2019), adapted to include the EBOV specific primer sets. Briefly, RNA was directly used for cDNA synthesis using the LunaScript RT SuperMix (New England Biolabs) and the cDNA generated was used as template in the multiplex PCR, which was performed in two reaction pools using Q5 Hot Start DNA Polymerase (New England Biolabs). The resulting amplicons from the two PCR pools were pooled in equal volumes and the pooled amplicons were diluted 1:10 with nuclease-free water.

Sequencing libraries were prepared, barcoded and multiplexed using the Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-LSK109) combined with the Native Expansion pack (EXP-NDB104, EXP-NBD114, EXP-NBD196) following the ARTIC Network's library preparation protocol (nCoV-2019 sequencing protocol v3 (LoCost) V.3 (Artic Network. https://artic.network/ncov-2019). For the preparation of less than 11 samples, each sample was prepared in multiples to achieve the library concentration required for sequencing. Briefly, the diluted pooled amplicons were end-repaired using the Ultra II End Prep Module (New England Biolabs) followed by barcode ligation using the Blunt/TA Ligase Master Mix and one unique barcode per sample. Equal volumes from each native barcoding reaction were pooled and subsequently bead cleaned-up using 0.4x AMPure beads. The pooled barcoded amplicons were quantified using the Qubit Fluorometer (Thermo Fisher Scientific) and AMII adapter ligation was performed using the Quick T4 DNA Ligase (New 29 England Biolabs) followed by an additional bead clean-up. The adaptor ligated barcoded amplicon pool was quantified using the Qubit Fluorometer (Thermo Fisher Scientific) aiming for a minimum recovery of 15 ng sequencing library to load onto the flow cell.

Sequencing libraries were sequenced using R9.4.1 Flow Cells (FLO-MIN106D, ONT) on the Mk1C device (ONT) using MinKNOW version 21.02.2 with real-time high accuracy basecalling and stringent demultiplexing (minimum barcoding score = 60). Within the barcoding options, barcoding on both ends and mid-read barcodes were both switched on.

Reads were demultiplexed and binned in a barcode specific folder only if a barcode above the minimum barcoding score was identified on both read ends and if mid-read barcodes were not identified. Sequencing runs were stopped after ~24hr and basecalling was allowed to finish prior to data handling.

Bioinformatics data analysis was done as per ARTIC protocol using a combination of the ARTIC EBOV (Artic Network. https://artic.network/ebov/ebov-bioinformatics-sop.html) and ARTIC SARS-CoV-2 (Artic Network. https://artic.network/ncov-2019/ncov2019bioinformatics-sop.html) pipelines. A few minor modifications to the ARTIC bioinformatics protocol were incorporated. The two initial steps described, basecalling with guppy and demultiplexing, were skipped as these were both done on the Mk1C device in real-time during the sequencing run, subsequently, the bioinformatics analysis was initiated from the read filtering step (artic guppyplex). Briefly, the artic guppyplex program was used to collect reads for each barcode into a single fastq file, in the presence of a length filter to remove chimeric reads. Reads were filtered based on length with a minimum (option: --min-length) and maximum (option: --max-length) length cut-off based on the amplicon size used (For V3, V4 and V5 primer pools: --min-length 400 and --max-length 700, for Zaire-PHE primer pool:

--min-length 350 and --max-length 650). The quality check was skipped since only reads above a quality score of 7 were processed. Following merging and filtering, the arctic minion 1065 genomes sampled from Guinea, Sierra Leone and Liberia between 2014 and 2015.

Multiple sequence alignment was performed using mafft. [START_REF] Katoh | Multiple alignment of DNA sequences with MAFFT[END_REF] We identified 6 T-to-C mutations in the genome from patient 11 that were indicative of mutations induced by adenosine deaminases acting on RNA (ADARs). According to the recommendations by Dudas et al. [START_REF] Dudas | Virus genomes reveal factors that spread and sustained the Ebola epidemic[END_REF] , we masked these positions in this genome in all further analyses. Maximum likelihood trees were reconstructed using IQ-tree under the general time-reversible (GTR) model with gamma (G) distributed rate variation among sites. [START_REF] Minh | IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era[END_REF] Temporal divergence plots of genetic divergence from the root of phylogenies against sampling time were constructed using TempEst. [START_REF] Rambaut | Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen)[END_REF] To construct the temporal divergence plot for the Guinean 2021 genome data, we used a tree reconstructed under an HKY+G model.

Local molecular clock model analysis

We used BEAST to fit a local molecular clock model to a data set consisting of 1020 dated Makona virus genomes and one of the 2021 genomes (patient 1). [START_REF] Suchard | Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10[END_REF][START_REF] Worobey | Synchronized global sweep of the internal genes of modern avian influenza virus[END_REF] We specified a separate rate on the tip branch for this genome as well as on the tip branch for a genome in a 2016 flare-up. We used the skygrid coalescent model as a flexible nonparametric tree prior and an HKY+G substitution model. [START_REF] Gill | Improving Bayesian population dynamics inference: a coalescent-based model for multiple loci[END_REF]

Guinea 2021 tMRCA estimation

Temporal signal was evaluated using the BETS procedure. [START_REF] Duchene | Temporal signal and the phylodynamic threshold of SARS-CoV-2[END_REF] We estimated a slightly lower log marginal likelihood for a model that uses tip dates (-26063.6) compared to a model that assumes sequences are sampled at the same time (-26062.1). These BEAST analyses were performed using an exponential growth model, a strict molecular clock model and an HKY+G substitution model. We specified a lognormal prior with mean of 1 and standard deviation of 5 on the population size and a Laplace prior with a scale of 100 on the growth rate. Default 32 priors were used for all other parameters. For the divergence time estimation, we used a normal prior on the substitution rate with a mean of 0.001 and a standard deviation of 0.00004 based on the background EBOV rate estimated by the local molecular clock analysis.


Inappropriate intimacies: two performances by Silvia Calderoni and Lucia Calamaro

Joëlle Chambon

International Colloquium "Performing intimacy" Grupo de Investigação em Estudos Performativos, 4-6 octobre 2018, Centro Internacional de Artes José de Guimarães, Portugal I shall begin with some preliminary comments:

First about the title: in French, I would have given the title "Intimités déplacées". For the word "déplacé" in French has the litteral meaning of "which has been moved, dislodged", and the figurative meaning of "uncalled for, shocking, rude". I could not find an English word that conveyed both meanings, and I have chosen the word "inappropriate" because the word "propriety" (which refers to social conventions) is close to the word "property" (which means "ownership"), and my paper aims to underline the fact that, in the two performances I shall talk about, a certain kind of unconventional experience is proposed to the audience by disconnecting intimacy from identity (and ownership) -as I hope to explain later.

My second comment is on the topicality of the concept of intimacy, which has become in the last two decades an important issue for philosophy and sociology. What is at stake is the way contemporary life makes us conceive intimacy on the model of economy. After Bernard Stiegler, we can make the names of Zygmunt Bauman with his essay Liquid Love in 2003 or Eva Illouz who, in her 2007 book Cold Intimacies: The Making of Emotional Capitalism, explained the process by which emotional and economic relationships come to define and shape each other -a process she calls "emotional capitalism". All these reflections go back to the essays of Richard Sennett, The Fall of Public Man, in 1977, andAnthony Giddens, The Transformation of Intimacy, in 1992. These essays gave me keys for understanding the two performances I shall talk about, and I found particularly illuminating the way Michael Foessel (a French philosopher) defines intimacy on one hand as a relation and therefore a shared experience -as opposed to interiority (which cannot be shared) -and on the other hand as an experience set back from society and thus allowing us to look critically at it -as opposed to privacy, which is the area where individuals reach agreements and enter into contracts (the private refering to property and contract, whereas the intimate refers to ties and recognition).

Third and last preliminary comment: when it comes to performing intimacy, I would say that certainly there is no better example of it than the sweet conversation of Romeo and Juliet during their rare intimate moments. It could be interesting to analyse why contemporary theatre has become so sparing with love dialogues, why it cannot or doesn't want to make the audience witness two people connecting with each other, creating an intimate relationship by suspending temporarily the social realities, and commenting upon this unique and dazzling experience. In France, since Marguerite Duras, only Wajdi Mouawad has dared to write this kind of dialogue (for example in Scorched, Incendies) and most playwriters prefer to write about ex-lovers tearing each other to pieces (like Rambert in Love's end, Clôture de l'amour), or to propose a series of variations on the impossibility of writing love dialogues (like Pommerat in Reunification of the two Koreas, La Réunification des deux Corées). But this is not my point. I shall only emphasize that this scarcity corroborates my third and last preliminary comment: intimacy is threatened by modernity, and particularly by its obsession with control and measureable things.

I would like to focus on two recent performances which explore intimacy, and deal with emotional relationships without ever talking of love, either out of modesty, or because it is considered impossible : Tumore (Tumor) is a play written and directed in 2007 by the young italian playwright Lucia Calamaro, and MDLSX is a performance of 2015 by Silvia Calderoni, who wrote it in collaboration with Daniela Nicolo, who directed it with Enrico Casagrande -the three of them belonging to the Italian company Motus.

Each one of the two shows explores intimacy in a specific way, but in both cases in the light of extreme situations: terminal illness for Tumore and gender ambiguity for MDLSX. Tumore, subtitled "a desolate show", tells the last days of a young woman with a tumor, through the monologues and dialogues of her two companions, her Doctor and her Mother. The stage is bare, because, says Calamaro, « the location must remain undefined: [...] to be born, to die, maybe to love, all these things happen in those mysterious transition areas where matter changes its state ». The play tells how the two women, though helpless, get to share an intimacy with each other and with the dying one, and it comes as close as possible to the very intimate moment of dying.

It does so in different ways, the first and most striking one being the preparation of a lastchance surgical procedure, then the medical search inside the body and the failure of it. After this failure, there is a mock-religious re-enactment of the moment of death, with Monika-the Doctor, becoming a very clumsy angel who has taken away the wrong person, and Benedetta-the-Mother becoming her dying but very cynical daughter. And finally a third and last re-enactment of death takes place at the end of the play, with the lights being turned off one by one, while the voice of the young woman fades out.

To explore intimacy, Tumore relies mainly on the powers of language and on the old magic of character playing: it only needs the complementary energies of two actresses (Monika Mariotti's comic and almost grotesque talent versus the moody poetry of Benedetta Cesqui), and sometimes the disappearance of anything visible in order to make room for the voice only.

By contrast, MDLSX is a kind of psychedelic audio-visual DJ set; Silvia Calderoni has created a soundtrack linked to emotional moments in her own life, and her physicality and energy remind us of rock-stars like David Bowie or Iggy Pop. There are simple scenic devices like a DJ table, a triangular rug, a circular screen on which we can see Calderoni's evolution via some of her family's home videos, but also close-ups of her face as she delivers monologues into a small camera.

Most of all, her body is the story: she moves it, uncovers it, flaunts it, adorns it with false bodyhair, highlights it with laser lights. And the musical quality of all her transformations seems to epitomize the word which defines her: "genderfluid".

These two shows offer very different aesthetics, and two opposite ways of performing intimacy. Tumore refuses any of the usual accessories of the contemporary stage such as screens, music, dance, technology, whereas MDLSX uses all of them. Tumore distrusts the images and favours hearing over seeing; whereas MDLSX is obsessed with seeing, and constantly plays with light and dark, cameras, screens, and costume changes, in order to sharpen and then deceive our voyeuristic desire.

And yet, there is a certain likeness in the way both performances explore an intimacy disconnected from identity, and in the way the text is used to this effect.

When Calamaro begins to write Tumore, her impulse is autobiographic (it is about the real story of her best friend's death) and the form is monologic: it is a flow of words, a rambling on, half stream of consciousness and half comment on ordinary life. When meeting the actresses, the flow divides itself, words are distributed, characters appear: Calamaro's monologue becomes a play with dialogues. But when directing, Calamaro goes backwards and says to the actresses: "I want to hear you think, not talk". Even the roles are unsteady: Benedetta, who plays the Mother, only needs to turn her back to the audience and take down her hair bun to become and/or bring back her dead Daughter. Therefore, even though fiction seems to be maintained, the people onstage finally talk to us not really as characters do, but from a place both closer and less definite: as voices which could become ours, after having been those of Lucia, Monika, Benedetta -unstable embodiments of our intimate feelings and thoughts.

On the other hand, MDLSX may look like a performance of Silvia Calderoni, but it was actually (and unusually for the Motus company) written before the rehearsals, being largely based on the novel Middlesex, by Jeffrey Eugenides (the Greek-American author of Virgin Suicides). The novel is supposed to be the autobiography of an hermaphrodite, who is first raised as a girl named Calliope, then decides at the age of 16 to live as a man and becomes Cal. The director Daniela Nicolo says: "we like to define MDLSX as 'post-biographic'. The limit between Silvia's biography and Calliope's is vague, confused. The re-writing we made plays exactly on this. The audience wonders how much of it is fiction and how much reality ». Indeed, the power and appeal of the show, which has toured the world, seems to hinge on how the audience is constantly fluctuating between Calliope-Cal and Calderoni, novel and autobiography, story and performance -as between man and woman. We are invited to share an intimacy that we cannot assign to somebody.

But are we invited to share an intimacy? What are we really invited to do? This question brings me to the last point I would like to raise, which is the political meaning of intimacy in the two shows.

We could underline the fact that both shows criticize the medical power; but it is only a superficial way of being political. According to Michael Foessel, intimacy is politically important because it provides the experience of an authentic relation, where one learns the frailty and precariousness that is always involved in the fact of really being with the other; it is a sort of education for democracy, an ethical experience, set back from society but entangled with the history of individuals1 .

At one point in the novel Middlesex, the supposed author Calliope-Cal writes: "I feel you out there, reader. This is the only kind of intimacy I'm comfortable with. Just the two of us, here in the dark." Of course, a show can never establish with its audience the kind of intimacy a novel does with its reader; but the aesthetics of the show can aim at it, or not. And I would like to stress a sort of paradox: with MDLSX the Motus company wants to make a political statement about (I quote) "the human necessity to be in movement, (...) to begin new existential adventures (...) in opposition to rigid identity models"; and the show finally appears as a sort of celebration of an exceptional body, a hymn in praise of an exceptional freedom. But it is also a very solitary body, and a very solitary freedom, so that the show is hardly an invitation to share any intimacy with Calliope-Cal-Calderoni. On the other hand, Tumore has no overt political ambitions, but it does give the opportunity for the audience to share an intimate experience of human frailty and also of the power of ties. Of course, I shall not decide which of the two shows is the more political, because the connection between politics and aesthetics would require maybe another conference.

In the novel Middlesex, the exceptional story of Calliope-Cal is a consequence of the traumatic history of the greek community expelled from Asia Minor at the beginning of the

20th century.

My ambition was simply to show the very different meanings that can be conveyed by the performance of intimacy, through the very different ways texts and bodies are brought together on the contemporary stage.