- Email: [email protected]
Human pegivirus persistence in human blood virome after allogeneic haematopoietic stem-cell transplantation
Accepted Manuscript Human pegivirus persistence in the human blood virome after allogeneic haematopoietic stem cell transplantation Diem-Lan Vu, Samuel Cordey, Federico Simonetta, Francisco Brito, Mylène Docquier, Lara Turin, Christian van Delden, Elsa Boely, Carole Dantin, Amandine Pradier, Eddy Roosnek, Yves Chalandon, Evgeny M. Zdobnov, Stavroula MasouridiLevrat, Laurent Kaiser PII:
S1198-743X(18)30410-5
DOI:
10.1016/j.cmi.2018.05.004
Reference:
CMI 1316
To appear in:
Clinical Microbiology and Infection
Received Date: 9 January 2018 Revised Date:
11 April 2018
Accepted Date: 1 May 2018
Please cite this article as: Vu D-L, Cordey S, Simonetta F, Brito F, Docquier M, Turin L, van Delden C, Boely E, Dantin C, Pradier A, Roosnek E, Chalandon Y, Zdobnov EM, Masouridi-Levrat S, Kaiser L, Human pegivirus persistence in the human blood virome after allogeneic haematopoietic stem cell transplantation, Clinical Microbiology and Infection (2018), doi: 10.1016/j.cmi.2018.05.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED Human pegivirus persistence in the MANUSCRIPT human blood virome after allogeneic
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haematopoietic stem cell transplantation
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Diem-Lan Vu1,6*, Samuel Cordey2,3*, Federico Simonetta4*, Francisco Brito3,5, Mylène
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Docquier3, Lara Turin2,3, Christian van Delden1,3,6, Elsa Boely6, Carole Dantin4, Amandine
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Pradier4, Eddy Roosnek3, Yves Chalandon3,4, Evgeny M. Zdobnov3,5, Stavroula Masouridi-
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Levrat4, Laurent Kaiser1,2,3
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Division of Infectious diseases, University of Geneva Hospitals, Geneva, Switzerland
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Laboratory of virology, Division of Laboratory Medicine, University of Geneva Hospitals,
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Geneva, Switzerland
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Faculty of medicine, Geneva, Switzerland
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Division of Haematology, University of Geneva Hospitals, Geneva, Switzerland
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Swiss Institute of Bioinformatics, Faculty of medicine, Geneva, Switzerland
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Swiss transplant cohort study
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*equal contribution.
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Category: original article
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Abstract words count: 237; manuscript words count: 2496
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Running title: Pegivirus in allo-HSCT blood virome
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Corresponding author:
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Diem-Lan Vu
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Laboratory of enteric viruses, Department of Genetics, Microbiology and Statistics
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Faculty of Biology, University of Barcelona
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Av. Diagonal 643, 08028 Barcelona, Spain 1
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Tel: +34603361321
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Fax: +412203729830
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Email: [email protected]
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Abstract
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Objectives: As commensal viruses are defined by the immunological tolerance afforded to
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them, any immunomodulation, such as is received during haematopoietic stem cell
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transplantation, may shift the demarcation between innocuous viral resident and disease-
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causing pathogen.
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Methods: We analysed by deep-sequencing the plasma virome of 40 allogeneic
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haematopoietic stem cell transplant (allo-HSCT) patients 1 month post-transplantation. As
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human pegivirus (HPgV) was highly prevalent, we performed a one-year screening of 122
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plasma samples by specific rRT-PCR assay. We used log rank test and the Gray test to assess
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association with outcomes and Mann-Whitney test and multivariable linear regression model
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to assess association with T-cell reconstitution.
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Results: Polyomaviruses (PyV) (20/40 patients), anelloviruses (16/40), pegiviruses (14/40)
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and herpesviruses (14/40) were most frequently identified, including 10 cytomegalovirus, 3
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Epstein-Barr, 2 herpes simplex-1, 1 human herpesvirus (HHV)-6b and 1 HHV-7; 18 Merkel
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cell-PyV, 2 BK-PyV, 3 PyV-6 and 1 JC-PyV. Papillomavirus and adenovirus were identified
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in 11 and 2 patients, respectively. The HPgV-specific rRT-PCR screening identified 51/122
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positive samples, high viral loads and persistent infections up to one year post-transplantation.
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Comparison between patients with or without HPgV infection at time of transplantation didn’t
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reveal significant difference in infections, engraftment, survival, graft-versus-host disease,
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relapse or immune reconstitution.
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Conclusion: The blood virome after allo-HSCT includes several DNA viruses notably
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herpesviruses and polyomaviruses. Among RNA viruses, HPgV is highly prevalent, persisting
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for several months and thus may deserve special attention in further research on immune ACCEPTED MANUSCRIPT
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reconstitution.
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Introduction
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Throughout life, humans encounter countless viruses. While the majority do not cause overt
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disease, each leaves an indelible mark on the immune system and some may persist as
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commensal residents, tolerated under a tight immunological restraint [1]. Defining pathology
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as the binary presence/absence of a pathogen is then a reductive analysis of a more complex
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interaction, where immunological crosstalk from commensal tolerance is able to influence the
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immune response to pathogens, determine their pathology or have an indirect effect on host
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health [1]. Next-generation sequencing (NGS) can elucidate the true diversity of the human
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virome, providing a more holistic definition of viral pathogenicity in context of the greater
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microbiome. The human virome plays a diverse and important role in host biology, either by
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interacting with its immune system or with other components of its microbiome [2]. As
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virome is essentially comprised of a collection of antigens and pathogen-associated molecular
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patterns, it can influence the host response to other infections [3], or enhance susceptibility to
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inflammatory diseases [4].
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Allogeneic haematopoietic stem cell transplant (allo-HSCT) recipients’ virome is of particular
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interest, due to their high state of immunosuppression: the consequences of shifts in
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immunocompetence towards commensal viruses are still unknown. Investigating HSCT
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virome could change the shape of routine viral screening in transplant donors/recipients: there
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is already evidence that complications experienced by HSCT recipients such as graft-versus-
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host disease (GvHD) may be modulated by the presence of commensal viruses [5].
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Using NGS, we analysed the human blood virome of HSCT patients at the peak of
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immunosuppression and after multiple transfusions. According to the high prevalence of
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human pegivirus (HPgV) identified and its potential for immune modulation [6], we then
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more deeply investigated itsACCEPTED potential influence on immune reconstitution and patient MANUSCRIPT
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outcome.
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Methods
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Study design (Figure S1)
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The primary cohort, comprising 40 plasma specimens of allo-HSCT recipients (Table S1),
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was retrospectively analysed by NGS. Plasma specimens were collected at a median of 33
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days post-transplantation (range 26-40 days).
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A specific HPgV investigation included the first cohort plus 82 additional allo-HSCT patients:
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we performed an HPgV-specific real-time RT-PCR (rRT-PCR) assay on stored plasma at
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days 0 and 30 post-transplantation for all 122 samples, and at four additional time points
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(days before, +100, +180, +365 from transplantation), when specimens were available. For
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the immune reconstitution and outcome analyses, we selected patients transplanted for the
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first time (excluding 5/122 patients) and considered the time point d0 post-transplantation to
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categorize patients as infected (HPgV (+)) or non-infected (HPgV (-)).The study was
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approved by the cantonal ethic committee.
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NGS and sequence analysis
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Unbiased NGS (DNA and RNA-seq library preparation, paired-end sequencing by using the
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100-base-pair protocol with indexing on a HiSeq 2500 sequencer (Illumina, San Diego, CA,
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USA) was performed on the plasma samples. We analysed results using the ezVIR pipeline
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HPgV rRT-PCR assay
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190 ul of plasma were spiked with 10 µl of standardized canine distemper virus of known
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concentration and extracted with the NucliSENS easyMAG (bioMérieux, Geneva,
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Switzerland) nucleic acid kit in a 25 ul elution volume, according to the manufacturer’s
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instruction. Extracted RNA was used for HPgV-specific rRT-PCR screening analysis, using a
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previously published assay [8]. PCR assay reaction was performed using the QuantiTect 6
Probe RT-PCR Kit (Qiagen, ACCEPTED Valencia, CA. MANUSCRIPT USA) on a StepOnePlus instrument (Applied
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Biosystems, Rotkreuz, Switzerland) under the following cycling conditions: 50°C for 30 min;
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95°C for 15 min; 45 cycles of 15 s at 94°C; and 1 min at 55°C. Data were analysed with the
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StepOne software V.2 (Applied Biosystems). Analytical sensitivity was assessed with a
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plasmid-derived transcribed RNA including the target region (kindly provided by Professor
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JT Stapleton) and showed a 100% detection limit corresponding to 5 RNA copies/reaction
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(approximately 132 RNA copies/ml of plasma).
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Flow cytometry
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Anti-coagulated blood (0.5-1 ml) obtained at 1, 3, 6, 9 and 12 months post-transplantation,
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was lysed with ammonium chloride and stained with fluorochrome-conjugated antibodies
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directed against CD3, CD4, CD8, CD56, CCR7 and CD45RA. Samples were incubated 15
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minutes in the dark and FACS analysis was performed using a Navios flow cytometer
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(Beckman Coulter, Nyon, Switzerland). FACS data were analyzed using FlowJo software.
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NK cells were identified as CD3- CD56+ lymphocytes and further divided in CD56bright and
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CD56dim NK cells. CD3+ CD4+ and CD3+ CD8+ T cell subpopulations were further
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identified based on CCR7 and CD45RA expression.
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Statistical analyses
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X2 or Fischer exact tests were used for categorical variables. Mann-Whitney test or Wilcoxon
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matched pairs signed rank test were used for continuous variables. Log-rank test was used for
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survival analyses. We used multivariable regression models to adjust for confounding factors
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(Table 1). Cumulative incidence estimates of acute GvHD (aGvHD), relapse and infections
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were calculated using the Gray test with death from other causes as a competitive event. A P
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value < .05 was considered statistically significant. Statistics were performed using GraphPad
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Prism version 7, R version 3.2.0 with the EZR graphical user interface and Stata/IC 13.1.
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Results
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NGS allo-HSCT blood virome study
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Among the 40 allo-HSCT patients screened by NGS, three (7.5%) were negative for any viral
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sequences. Thirty-two (80%) and 19 (47.5 %) had at least one detectable DNA and RNA viral
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sequence, respectively. Among the 37 positive patients, the number of concomitantly detected
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viruses ranged from one to 5 viruses in a single patient. Figure 1A provides the virus families
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identified and the number of patients concerned. Among DNA viral sequences, the most
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frequent were polyomaviruses (PyV) (20/40 patients, 50%), followed by anelloviruses (40%),
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herpesviruses (35%), papillomaviruses (HPV) (27.5%) and adenoviruses (5%). For RNA viral
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sequences, the most frequent were HPgV (35%), followed by hepatitis C virus (HCV) (5%),
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and Rubella virus (2.5%).
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Figure 1B shows the specific viral sequences identified for each patient according to their
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respective number of reads. The anelloviruses were mostly Torque Teno (TT) virus (15/21);
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the other cases were TT Midi Virus and TT Mini Virus. Five patients had more than one
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species detected. Among herpesviruses, cytomegalovirus (CMV) was the most frequent
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(10/17), followed by Epstein-Barr virus (EBV), herpes simplex-1 (HSV-1), human
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herpesvirus (HHV)-6b and HHV-7. Two different types of herpesviruses were simultaneously
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detected in three patients. The polyomaviruses included Merkel cell (MCPyV) (18/24), BK
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virus, PyV-6 and JC virus. Four patients were positive for two different polyomaviruses. HPV
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matching to the 107 genotype was predominant among papillomaviruses (6/11), followed by
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genotypes 17, 4, 15, and FA75. No multiple infections were identified for HPV.
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There was no differences in the blood virome composition between patients receiving non-
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and myeloablative conditioning (Mann-Whitney test, data not shown).
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Confirmatory testing by r(RT-)PCR
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Our r(RT-)PCR specific assays for HPgV, HCV, adenovirus, BK, JC polyomavirus, CMV, ACCEPTED MANUSCRIPT
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EBV and HHV-6 confirmed all cases except two EBV, one BK polyomavirus and one
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adenovirus. Oppositely, three CMV and one EBV, with relatively low viral loads (< 3.2x10E3
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copies/ml) were detected by rPCR but missed by NGS. One BK virus was filtered by NGS as
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a contaminant although then confirmed by rPCR. The HSV-1 and the Rubella cases were not
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confirmed by r(RT-)PCR. We didn’t screen the HHV-7, the anelloviruses nor the
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papillomaviruses by rPCR. Figure S2 provides the HPgV-positive samples phylogenetic tree.
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HPgV investigation
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Given the high frequency of HPgV detection, the lack of knowledge on its infection kinetics
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and its potential immunomodulatory role, we performed an HPgV-specific rRT-PCR
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screening on 122 consecutive allo-HSCT patients with a mean follow-up of 339 days post-
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transplantation (Figure 2). Overall, 51 patients (41.8%) were positive for HPgV at least once
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during the follow-up period. 32% of patients were positive before transplantation, of whom all
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except one patient remained positive until the last time point (d180-365). In 7/51 positive
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patients, the infection appeared after transplantation; we screened the donor serum from 6 of
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these patients: one donor was positive for HPgV, whose recipient became viraemic at the time
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of transplantation. The median viral load of positive cases ranged between 1.7x104 and 5.2
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x105 copies/ml of plasma (Figure 2).
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HPgV and transplantation outcomes
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We next assessed the association between HPgV infection and transplantation outcomes.
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Table 1 presents the patients’ characteristics. HPgV(+) group included higher proportions of
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patients with myeloid malignancies (85% vs 68%; p=0.0072) and lower proportions of
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patients with lymphoid malignancies (13% vs 27%; p=0.0208). Accordingly, higher
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proportions of HPgV(+) patients received a myeloablative conditioning. No difference was 9
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observed in the time to neutrophil engraftment (Figure 3A), in overall survival, progressionACCEPTED MANUSCRIPT
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free survival or GRFS (Figure 3B a-c), nor in cumulative incidences of grades II–IV aGvHD
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(Figure 3C), disease relapse (Figure 3D) or infections (Figure S3A-D and Table S2 for
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microbial agents identified) between HPgV(+) and HPgV(-) patients.
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HPgV association with cellular and humoral immunity
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We compared cellular immune reconstitution in HPgV(+) and HPgV(-) patients over one year
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post-transplantation. At 1 and 12 months post-transplantation, we detected significantly lower
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numbers of CD4 and CD8, and higher NK cell counts in HPgV(+) patients, respectively
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(Figure 4A). However, multivariable linear regression analysis, taking into account clinical
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variables differing between both groups at the studied time points (i.e. sex and disease status
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at 1 month and sex, donor type and T-cell depletion at 12 months), did not support these
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differences. Similar analyses on cell subsets distributions among T and NK cells identified
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lower proportions of CD8 TEMRA T cells in HPgV(+) patients at 12 months, although this
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difference was not confirmed after adjustment (Figure 4B).
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Our analyses also failed to detect any significant impact of IVIG administration on HPgV
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titres (Figure S4).
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Discussion
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We reported all RNA and DNA viruses detectable by unbiased NGS following allo-HSCT.
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Our results reveal that so-called commensal viruses are a major component of the allo-HSCT
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blood virome, where HPgV was identified in more than 30% of patients.
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Overall, polyomavirus, anellovirus and pegivirus were most frequently identified. The median
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number of concomitant viral sequences was 2 per patient, with a maximum of 5 distinct
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sequences identified in four patients. According to a previous study, the DNA blood virome
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of solid organ transplant recipients (SOT) is composed of 70% of Anelloviridae, 13% of
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Herpesviridae and 5% of Polyomaviridae and 0% of Papillomaviridae [9]. The equivalent
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findings in our cohort is 28%, 22%, 32% and 14% respectively. This divergence could be
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partially explained by critical differences in the protocols used for genetic material extraction
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and bio-informatics pipelines. If comparing with the gut virome of allo-HSCT patients [5], the
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same persistent viruses than ours were identified (Anelloviridae, Polyomaviridae,
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Herpesviridae, and Papillomaviridae found in 70%, 34%, 27% and 18% of patients,
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respectively); differences between both studies mainly rely on the viruses’ tropism
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(Flaviviridae found exclusively in blood and exogenous enteric viruses in the gut).
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Anelloviruses are known to cause prolonged viremia and multiple co-infections. Two thirds of
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our study population were positive for at least one anellovirus, corresponding to previously
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reported prevalence [10]. While it has not been linked with overt organ disease, TTV has been
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proposed as a proxy of immune suppression in both SOT [11], and allo-HSCT recipients [12].
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MCPyV and PyV 6 are found on the skin of healthy individuals. While MCPyV has been
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associated with the Merkel cell carcinoma, PyV 6 is currently not associated with any disease.
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Peripheral blood mononuclear cells (PBMC) are a reservoir for many Polyomaviridae [13]; as
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we studied cell-free plasma samples, this could explain the relatively low number of reads in
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the PyV(+) samples. Similarly, papillomaviruses can persist in PBMCs, and HPV DNA is ACCEPTED MANUSCRIPT
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generally found in 8-15% of healthy blood donors [14], suggesting that HPV can circulate in
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blood and are not necessarily skin contaminants.
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The r(RT-)PCR assays couldn’t confirm the HSV-1 and the Rubella results. Clinical history
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didn’t indicate any HSV infection and the Rubella-positive patient was serologically positive
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before transplantation, as for measles and mumps, suggesting previous vaccination. Although
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the low number of reads detected in these cases suggests non-specific sequences, HSV
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viremia can occur in up to 26% of critically ill patients [15], including immunocompromised
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patients without organ disease [16]. Moreover, the cumulative burden of dsDNA viruses
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detected in plasma has been associated with an increased mortality in HSCT recipients [17],
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although they rarely caused end-organ disease and were infrequently the direct cause of death.
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Similarly, in children with primary immunodeficiencies, cases of chronic skin granuloma
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induced by persistent rubella vaccine strains exist [18]. Thus, commensal viruses may either
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have an indirect role in pathology or cause unexpected clinical syndrome.
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HPgV viremia is found in 1-5% of healthy blood donors in developed countries [6]. Viral load
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can reach 107 copies/ml and transmission through blood transfusion is well-described [19].
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HPgV primarily infects haematopoietic stem cells [20], but can also be found in T, B, NK
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cells and monocytes [6]. HPgV genome can persist in serum micro vesicles and be further
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transmitted to PBMCs in vitro, leading to active replication and production of infectious viral
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particles [8]. Our HPgV prevalence is similar to the one of other studies [21-26] (Table S3),
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but our investigation could suggest an HPgV transmission by the donor in one patient,
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although a pre-transplant undetectable but positive low viral load is not excluded and that firm
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evidence are lacking in absence of sequencing. Previous studies have demonstrated that HPgV
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can persist for up to 9 years [26]. Besides, weMANUSCRIPT provided detailed quantitative viremia kinetics, ACCEPTED
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definitely proving that HPgV is a persistent infection.
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Although
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immunomodulatory effect in HIV patients [6]. We assessed the potential impact of HPgV on
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T cell reconstitution. We identified a difference in the total CD4 and CD8 cell count at 1
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month and the rate of CD8 TEMRA at 12 months between HPgV positive and negative cases,
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which could be comparable to what is seen in HIV patients [27]. The effect did not persist
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after multivariable analyses, and our results can thus not identify any effect of HPgV on cell
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reconstitution; nevertheless, many confounders can attenuate the potential effect of HPgV, as
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this was the case in HPgV co-infected HIV patients who experienced immune reconstitution
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after HAART [28]. Similarly, our results did not uncover an association between HPgV
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infection and allo-HSCT outcomes, but we found a significant difference in the primary
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disease between HPgV(+) and HPgV(-) patients, potentially suggesting an association
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between HPgV and some haematological malignancies, similarly to what we recently reported
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for TT virus [12].
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Our virome NGS study focused on a single time point post-transplantation without
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comparison to the virome before transplantation and we focused our investigation on HPgV
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due to its immunomodulatory properties, of interest in the context of allo-HSCT. Yet, Legoff
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et al have recently demonstrated an increase in the proportion of the eukaryotic viruses in the
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gut virome after allo-HSCT [5]; in addition, picobirnavirus infection was specifically
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predictive of aGvHD, reinforcing the need to investigate the role of so-called orphan disease
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viruses in non-infectious complications.
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In conclusion, our study highlights that most of allo-HSCT recipients have at least one virus
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circulating in their blood after transplantation. Our observations that HPgV is predominant 13
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and persistent in allo-HSCT blood virome, should promote further research into the potential ACCEPTED MANUSCRIPT
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impact that commensal viruses can altogether have on immunosuppression.
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Fundings
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This work was supported by grants of the Ernst and Lucie Schmidheiny foundation to L.K.;
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the Henri-Dubois-Ferrière Dinu Lipatti Foundation to D-L.V.; and the Faculty of medicine of
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Geneva.
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Acknowledgments
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We thank Pr Jaques Schrenzel, Dr Vladimir Lazarevic and Dr Nadia Gaia for their
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contribution to the bio-informatics analyses and Mary-Anne Hartley for editorial assistance.
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Potential conflict of interest. All authors: No reported conflicts of interest.
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References
294
[1]
142-50.
Barton ES, White DW, Cathelyn JS, Brett-McClellan KA, Engle M, Diamond MS, et
AC C
[3]
al. Herpesvirus latency confers symbiotic protection from bacterial infection. Nature
299
2007; 447(7142): 326-9.
300 301
Virgin HW. The virome in mammalian physiology and disease. Cell 2014; 157(1):
EP
[2]
297 298
TE D
borders in clinical virology. Clin Microbiol Infect 2017; 23(10): 688-90.
295 296
Vu D-L, Kaiser L. The concept of commensal viruses almost 20 years later: redefining
[4]
Cadwell K, Patel KK, Maloney NS, Liu TC, Ng AC, Storer CE, et al. Virus-plus-
302
susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes
303
in intestine. Cell 2010; 141(7): 1135-45.
14
304
[5]
Legoff J, Resche-RigonACCEPTED M, Bouquet J,MANUSCRIPT Robin M, Naccache SN, Mercier-Delarue S, et
305
al. The eukaryotic gut virome in hematopoietic stem cell transplantation: new clues in
306
enteric graft-versus-host disease. Nat Med 2017; 23(9): 1080-85.
307
[6]
Chivero ET, Stapleton JT. Tropism of human pegivirus (formerly known as GB virus C/hepatitis G virus) and host immunomodulation: insights into a highly successful
309
viral infection. J Gen Virol 2015; 96(Pt 7): 1521-32.
310
[7]
RI PT
308
Petty TJ, Cordey S, Padioleau I, Docquier M, Turin L, Preynat-Seauve O, et al. Comprehensive human virus screening using high-throughput sequencing with a user-
312
friendly representation of bioinformatics analysis: a pilot study. J Clin Microbiol
313
2014; 52(9): 3351-61. [8]
Chivero ET, Bhattarai N, Rydze RT, Winters MA, Holodniy M, Stapleton JT. Human
M AN U
314
SC
311
315
pegivirus RNA is found in multiple blood mononuclear cells in vivo and serum-
316
derived viral RNA-containing particles are infectious in vitro. J Gen Virol 2014; 95(Pt
317
6): 1307-19. [9]
De Vlaminck I, Khush KK, Strehl C, Kohli B, Luikart H, Neff NF, et al. Temporal
TE D
318 319
response of the human virome to immunosuppression and antiviral therapy. Cell 2013;
320
155(5):1178-87.
Med Virol 2010; 20(6): 392-407.
322 323
[11]
Focosi D, Antonelli G, Pistello M, Maggi F. Torquetenovirus: the human virome from
bench to bedside. Clin Microbiol Infect 2016; 22(7): 589-93.
324 325
Maggi F, Bendinelli M. Human anelloviruses and the central nervous system. Rev
EP
[10]
AC C
321
[12]
Masouridi-Levrat S, Pradier A, Simonetta F, Kaiser L, Chalandon Y, Roosnek E.
326
Torque teno virus in patients undergoing allogeneic hematopoietic stem cell
327
transplantation for hematological malignancies. Bone Marrow Transplant 2016; 51(3):
328
440-2.
15
329
[13]
Toracchio S, Foyle A, ACCEPTED Sroller V, ReedMANUSCRIPT JA, Wu J, Kozinetz CA, et al. Lymphotropism
330
of Merkel cell polyomavirus infection, Nova Scotia, Canada. Emerg Infect Dis 2010;
331
16(11): 1702-9.
332
[14]
Chen AC, Keleher A, Kedda MA, Spurdle AB, McMillan NA, Antonsson A. Human papillomavirus DNA detected in peripheral blood samples from healthy Australian
334
male blood donors. J Med Virol 2009; 81(10): 1792-6.
335
[15]
RI PT
333
Ong DS, Bonten MJ, Spitoni C, Lunel FM, Frencken JF, Horn J, et al. Epidemiology of multiple herpes viremia in previously immunocompetent patients with septic shock.
337
Clin Infect Dis 2017; 64(9):1204-10.
338
[16]
SC
336
Lepiller Q, Sueur C, Solis M, Barth H, Glady L, Lefebvre F, et al. Clinical relevance of herpes simplex virus viremia in Intensive Care Unit patients. J Infect 2015; 71(1):
340
93-100.
341
[17]
M AN U
339
Hill JA, Mayer BT, Xie H, Leisenring WM, Huang ML, Stevens-Ayers T, et al. The cumulative burden of double-stranded DNA virus detection after allogeneic HCT is
343
associated with increased mortality. Blood 2017; 129(16): 2316-25
344
[18]
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342
Neven B, Perot P, Bruneau J, Pasquet M, Ramirez M, Diana JS, et al. Cutaneous and Visceral Chronic Granulomatous Disease Triggered by a Rubella Virus Vaccine Strain
346
in Children With Primary Immunodeficiencies. Clin Infect Dis 2017; 64(1): 83-6. [19]
of transfusion-associated hepatitis G virus infection and its relation to liver disease. N
348
Engl J Med 1997; 336(11): 747-54.
349 350
Alter HJ, Nakatsuji Y, Melpolder J, Wages J, Wesley R, Shih JW, et al. The incidence
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[20]
Bailey AL, Lauck M, Mohns M, Peterson EJ, Beheler K, Brunner KG, et al. Durable
351
sequence stability and bone marrow tropism in a macaque model of human pegivirus
352
infection. Sci Transl Med 2015; 7(305): 305ra144.
16
353
[21]
Skidmore SJ, Collingham KE, Harrison P, Neilson JR, Pillay D, Milligan DW. High ACCEPTED MANUSCRIPT
354
prevalence of hepatitis G virus in bone marrow transplant recipients and patients
355
treated for acute leukemia. Blood 1997; 89(10): 3853-6.
356
[22]
Corbi C, Traineau R, Esperou H, Ravera N, Portelette E, Benbunan M, et al. Prevalence and clinical features of hepatitis G virus infection in bone marrow allograft
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recipients. Bone Marrow Transplant 1997; 20(11): 965-8.
359
[23]
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Rodriguez-Inigo E, Tomas JF, Gomez-Garcia de Soria V, Bartolome J, Pinilla I, Amaro MJ, et al. Hepatitis C and G virus infection and liver dysfunction after
361
allogeneic bone marrow transplantation: results from a prospective study. Blood 1997;
362
90(3): 1326-31. [24]
Akiyama H, Nakamura N, Tanikawa S, Sakamaki H, Onozawa Y, Shibayama T, et al.
M AN U
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SC
360
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Incidence and influence of GB virus C and hepatitis C virus infection in patients
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undergoing bone marrow transplantation. Bone Marrow Transplant 1998; 21(11):
366
1131-5. [25]
Ljungman P, Halasz R, Hagglund H, Sonnerborg A, Ringden O. Detection of hepatitis
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367 368
G virus/GB virus C after allogeneic bone marrow transplantation. Bone Marrow
369
Transplant 1998; 22(5): 499-501.
liver function of recipients with hepatitis G virus infection after bone marrow
371
transplantation. Bone Marrow Transplant 1999; 24(4): 359-63.
372 373
[27]
Rydze RT, Bhattarai N, Stapleton JT. GB virus C infection is associated with a
reduced rate of reactivation of latent HIV and protection against activation-induced T-
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cell death. Antivir Ther 2012; 17(7): 1271-9.
375 376
Maruta A, Tanabe J, Hashimoto C, Kato K, Kanamori H, Fukawa H, et al. Long-term
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[28]
Ernst D, Greer M, Akmatova R, Pischke S, Wedemeyer H, Heiken H, et al. Impact of
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GB virus C viraemia on clinical outcome in HIV-1-infected patients: a 20-year follow-
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up study. HIV Med 2014; 15(4): 245-50. 17
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Table 1: Clinical characteristics of HPgV (+) and HPgV (-) patients ACCEPTED MANUSCRIPT
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HPgV(+) 39 26 (10-40) 50 (18-69) n % 19 49 20 51 n % 23 59 8 21 4 10 0 0 2 5 1 3 1 3 0 0 n % 27 69 11 28 1 3 n % 4 10 19 49 12 31 3 8 1 3 n % 23 59 16 41 n % 13 33 17 44 3 8 6 15 n % 34 87 5 13 n % 10 26 29 74 n % 22 56 17 44 n % 8 21 3 8 5 13 23 59 n % 17 44 19 49 3 8
P .2879 .5911 .0036
.0001
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HPgV(-) 78 22 (5-39) 52 (19-70) n % 59 76 19 24 n % 26 33 25 32 4 5 10 13 2 3 7 9 3 4 1 1 n % 41 53 33 42 4 5 n % 4 5 45 58 20 26 1 1 8 10 n % 33 42 45 58 n % 30 38 32 41 11 14 5 6 n % 66 85 12 15 n % 35 45 43 55 n % 56 72 22 28 n % 25 32 6 8 12 15 35 45 n % 38 49 28 36 12 15
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All patients 117 22 (5-40) 51 (18-70) n % 78 67 39 33 n % 49 42 33 28 8 7 10 9 4 3 8 7 4 3 1 1 n % 68 58 44 38 5 4 n % 8 7 64 55 32 27 4 3 9 8 n % 56 48 61 52 n % 43 37 49 42 14 12 11 9 n % 100 85 17 15 n % 45 38 72 62 n % 78 67 39 33 n % 33 28 9 8 17 15 58 50 n % 59 50 43 37 15 13
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Patients, n Median Follow-up, months (range) Median Age, years (range) Sex Male Female Primary disease Acute myeloid leukaemia MDS/MPS/MDPS Acute lymphoid leukaemia Lymphoma/chronic lymphoid leukaemia Chronic myeloid leukaemia Multiple myeloma Severe aplastic anaemia Othera Disease status at transplant Complete remission Partial Remission/Progression/Relapse NA Disease Risk Index Low Intermediate High Very High NA Conditioning MAC RIC Donor Type Matched Sibling Matched Unrelated Donor Mismatched Relative Mismatched Unrelated Donor Cell source PBSC BM In vivo TCD No ATG ATG Ex vivo TCD No Yes CMV status D-/RD-/R+ D+/RD+/R+ Immunosuppression CNI, MMF CNI, MTX Cy,Tacrolimus, MMF
.0673
.198
.0162
.1142
.6836
.005
.0184
.2303
.300
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19
Statistical analyses were doneACCEPTED using X2 or Fischer exact tests for categorical variables and MANUSCRIPT
384
Mann-Whitney test for continuous variables. Myeloid malignancies include acute myeloid
385
leukemia, MDS/MPS/MDPS and chronic myeloid leukemia. Lymphoid malignancies include
386
acute lymphoid leukemia, lymphoma/chronic lymphoid leukaemia and myeloma. acategory
387
other includes haemoglobinopathies. HPgV: human pegivirus; HPgV (+): HPgV-infected;
388
HPgV (-): HPgV non-infected; PBSC: peripheral blood stem cell; BM: bone marrow; TCD:
389
T-cell depletion; ATG: antithymoglobulin; D: donor; R: recipient; CNI: calcineurin inhibitor;
390
MMF: mycophenolate mofetil; MTX: methotrexate; CY: cyclosporine A.
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Figure legends
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Figure 1. Next-generation sequencing analysis. (A) Circular abundance plot of virus
395
families identified by next-generation sequencing in 40 allo-HSCT patients.
396
indicate the number of patients in whom each of the virus families have been identified. (B)
397
Grid plot of viruses identified in 40 allo-HSCT patients. Each line represents one virus and
398
each column one patient’s blood sample. The numbers under the grid indicate the number of
399
distinct viruses identified in each sample. The numbers on the right indicate the number of
400
patients in whom genome sequences of the indicated virus have been identified. The colors
401
represent the approximate number of reads (indicated in the legend) matching the indicated
402
virus’ genome detected in each sample.
403
Figure 2. HPgV real-time RT-PCR screening. Each boxplot represents one time point of
404
screening. N represents the number of HPgV-positive patients, and total the total number of
405
patients screened, at the indicated time point. The percentage is the proportion of positive
406
patients for each time point. BT: before transplantation; d: day
407
Figure 3. Estimates of time to engraftment, survival, aGVHD and relapse in HPgV (+)
408
and HPgV (-) patients. (A) Engraftment. (B) Survival: a. Overall survival (OS); b.
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Progression-free survival (PFS); c. Graft-Versus-Host Disease-Free, Relapse-Free Survival ACCEPTED MANUSCRIPT
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(GRFS). (C) Acute graft-versus-host disease (aGvHD). (D) Relapse.
411
HPgV(+) patients had a median of 17 days (range 12-37 days) and HPgV(-) patients had a
412
median of 16 days (range 12-34 days) before engraftment. OS was calculated from the date of
413
allo-HSCT to death or last follow-up. PFS was calculated from the date of allo-HSCT until
414
disease relapse/progression or last follow-up. GRFS was calculated from the date of allo-
415
HSCT until a GRFS-defining event or last follow up. Events determining GRFS included
416
grade 3-4 aGvHD, systemic therapy-requiring chronic GvHD, relapse, or death. P values
417
compare HPgV (+) versus HPgV (-) patients. Percentages indicate the outcome at 12 months
418
post-transplantation with 95% CI.
419
Figure 4. Immune reconstitution in HPgV (+) and HPgV (-) patients from 0 to 12 months
420
after transplantation. (A) Total CD4+, CD8+ and NK cell count. (B) Proportion of distinct
421
subsets within CD4+, CD8+ and NK cells in HPgV(-) or HPgV(+) patients.
422
At one month, HPgV(+) patients had a median of CD4 count of 0 cell/µl (range 0-199) and of
423
CD8 count of 0 cell/µl (range 0-478) while HPgV(-) patients had a median of CD4 count of
424
35.31 cell/µl (range 0-503, P=.0326) and a median of CD8 count of 81 cells/µl (range 0-449,
425
P=.0445). At 12 months, HPgV(+) patients had higher NK cell counts (median 185 cells/µl,
426
range 50-1090, versus 125 cells/µl, range 10-460, P=.0472). HPgV(+) patients had a lower
427
proportion of CD8 TEMRA T cells (median 26%, range 5%-77%; HPgV(-) patients: median
428
45%, range 12%-93%; P=.0142). EM: effective memory; CM: central memory; NK: natural
429
killer T cells. * for comparison in the rate of TEMRA cells between HPgV(+) and HPgV(-)
430
patients.
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431 432 433
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B
A
a.
100 Pegivirus Pegivirus +
P=.404 100
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Engraftment
64% (95%CI 46%-78%)
80
60
53% (95%CI 39%-65%)
40
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OS (%)
50
Pegivirus Pegivirus +
0 0
10
20
30
20
40
Time since HSCT (days)
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0
C
0
100 Pegivirus Pegivirus + 80
40
46% (95%CI 30%-61%)
PFS (%)
D
60
20
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0 6
12
18
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Pegivirus Pegivirus +
46% (95%CI 34%-58%)
0 0
c.
6
12
18
24
Time since HSCT (months)
P=.297 80
P=.185
60
30% (95%CI 20%-41%)
40
66% (95%CI 49%-79%)
100
Pegivirus Pegivirus +
80
P=.108
40
24
GRFS (%)
Relapse cumulative incidence (%)
100
24
60
20
Time since HSCT (months)
D
18
Time since HSCT (months)
80
59% (95%CI 47%-70%)
0
12
100
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aGvHD 2-4 grade cumulative incidence (%)
b.
P=.196
6
45% (95%CI 29%-60%)
60
40 Pegivirus Pegivirus +
20
36% (95%CI 24%-47%)
20 0
18% (95%CI 8%-32%)
0 0
6
12
18
Time since HSCT (months)
0 24
6
12
18
Time since HSCT (months)
24
B
A
80
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100
Pegivirus Pegivirus +
Pegivirus-
%
60
P=.0326
40
20
0
CM Naive EM CM Naive
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Pegivirus+
EM
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P=.0142*
100
%
80
P=.0445
TEMRA EM Pegivirus-
CM
60
Naive
40
TEMRA
20
Pegivirus+
EM CM Naive
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0
100
P=.0472
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80
Pegivirus-
CD56dim CD56bright
Pegivirus+
CD56dim CD56bright
60
40
20
0
S1198-743X(18)30410-5
DOI:
10.1016/j.cmi.2018.05.004
Reference:
CMI 1316
To appear in:
Clinical Microbiology and Infection
Received Date: 9 January 2018 Revised Date:
11 April 2018
Accepted Date: 1 May 2018
Please cite this article as: Vu D-L, Cordey S, Simonetta F, Brito F, Docquier M, Turin L, van Delden C, Boely E, Dantin C, Pradier A, Roosnek E, Chalandon Y, Zdobnov EM, Masouridi-Levrat S, Kaiser L, Human pegivirus persistence in the human blood virome after allogeneic haematopoietic stem cell transplantation, Clinical Microbiology and Infection (2018), doi: 10.1016/j.cmi.2018.05.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED Human pegivirus persistence in the MANUSCRIPT human blood virome after allogeneic
2
haematopoietic stem cell transplantation
3
Diem-Lan Vu1,6*, Samuel Cordey2,3*, Federico Simonetta4*, Francisco Brito3,5, Mylène
4
Docquier3, Lara Turin2,3, Christian van Delden1,3,6, Elsa Boely6, Carole Dantin4, Amandine
5
Pradier4, Eddy Roosnek3, Yves Chalandon3,4, Evgeny M. Zdobnov3,5, Stavroula Masouridi-
6
Levrat4, Laurent Kaiser1,2,3
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7 1
Division of Infectious diseases, University of Geneva Hospitals, Geneva, Switzerland
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2
Laboratory of virology, Division of Laboratory Medicine, University of Geneva Hospitals,
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Geneva, Switzerland
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3
Faculty of medicine, Geneva, Switzerland
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4
Division of Haematology, University of Geneva Hospitals, Geneva, Switzerland
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5
Swiss Institute of Bioinformatics, Faculty of medicine, Geneva, Switzerland
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6
Swiss transplant cohort study
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*equal contribution.
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Category: original article
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Abstract words count: 237; manuscript words count: 2496
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Running title: Pegivirus in allo-HSCT blood virome
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Corresponding author:
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Diem-Lan Vu
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Laboratory of enteric viruses, Department of Genetics, Microbiology and Statistics
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Faculty of Biology, University of Barcelona
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Av. Diagonal 643, 08028 Barcelona, Spain 1
ACCEPTED MANUSCRIPT
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Tel: +34603361321
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Fax: +412203729830
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Email: [email protected]
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Abstract
32
Objectives: As commensal viruses are defined by the immunological tolerance afforded to
33
them, any immunomodulation, such as is received during haematopoietic stem cell
34
transplantation, may shift the demarcation between innocuous viral resident and disease-
35
causing pathogen.
36
Methods: We analysed by deep-sequencing the plasma virome of 40 allogeneic
37
haematopoietic stem cell transplant (allo-HSCT) patients 1 month post-transplantation. As
38
human pegivirus (HPgV) was highly prevalent, we performed a one-year screening of 122
39
plasma samples by specific rRT-PCR assay. We used log rank test and the Gray test to assess
40
association with outcomes and Mann-Whitney test and multivariable linear regression model
41
to assess association with T-cell reconstitution.
42
Results: Polyomaviruses (PyV) (20/40 patients), anelloviruses (16/40), pegiviruses (14/40)
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and herpesviruses (14/40) were most frequently identified, including 10 cytomegalovirus, 3
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Epstein-Barr, 2 herpes simplex-1, 1 human herpesvirus (HHV)-6b and 1 HHV-7; 18 Merkel
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cell-PyV, 2 BK-PyV, 3 PyV-6 and 1 JC-PyV. Papillomavirus and adenovirus were identified
46
in 11 and 2 patients, respectively. The HPgV-specific rRT-PCR screening identified 51/122
47
positive samples, high viral loads and persistent infections up to one year post-transplantation.
48
Comparison between patients with or without HPgV infection at time of transplantation didn’t
49
reveal significant difference in infections, engraftment, survival, graft-versus-host disease,
50
relapse or immune reconstitution.
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Conclusion: The blood virome after allo-HSCT includes several DNA viruses notably
52
herpesviruses and polyomaviruses. Among RNA viruses, HPgV is highly prevalent, persisting
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for several months and thus may deserve special attention in further research on immune ACCEPTED MANUSCRIPT
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reconstitution.
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3
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Introduction
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Throughout life, humans encounter countless viruses. While the majority do not cause overt
57
disease, each leaves an indelible mark on the immune system and some may persist as
58
commensal residents, tolerated under a tight immunological restraint [1]. Defining pathology
59
as the binary presence/absence of a pathogen is then a reductive analysis of a more complex
60
interaction, where immunological crosstalk from commensal tolerance is able to influence the
61
immune response to pathogens, determine their pathology or have an indirect effect on host
62
health [1]. Next-generation sequencing (NGS) can elucidate the true diversity of the human
63
virome, providing a more holistic definition of viral pathogenicity in context of the greater
64
microbiome. The human virome plays a diverse and important role in host biology, either by
65
interacting with its immune system or with other components of its microbiome [2]. As
66
virome is essentially comprised of a collection of antigens and pathogen-associated molecular
67
patterns, it can influence the host response to other infections [3], or enhance susceptibility to
68
inflammatory diseases [4].
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Allogeneic haematopoietic stem cell transplant (allo-HSCT) recipients’ virome is of particular
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interest, due to their high state of immunosuppression: the consequences of shifts in
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immunocompetence towards commensal viruses are still unknown. Investigating HSCT
73
virome could change the shape of routine viral screening in transplant donors/recipients: there
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is already evidence that complications experienced by HSCT recipients such as graft-versus-
75
host disease (GvHD) may be modulated by the presence of commensal viruses [5].
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Using NGS, we analysed the human blood virome of HSCT patients at the peak of
78
immunosuppression and after multiple transfusions. According to the high prevalence of
79
human pegivirus (HPgV) identified and its potential for immune modulation [6], we then
4
more deeply investigated itsACCEPTED potential influence on immune reconstitution and patient MANUSCRIPT
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outcome.
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Methods
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Study design (Figure S1)
84
The primary cohort, comprising 40 plasma specimens of allo-HSCT recipients (Table S1),
85
was retrospectively analysed by NGS. Plasma specimens were collected at a median of 33
86
days post-transplantation (range 26-40 days).
87
A specific HPgV investigation included the first cohort plus 82 additional allo-HSCT patients:
88
we performed an HPgV-specific real-time RT-PCR (rRT-PCR) assay on stored plasma at
89
days 0 and 30 post-transplantation for all 122 samples, and at four additional time points
90
(days before, +100, +180, +365 from transplantation), when specimens were available. For
91
the immune reconstitution and outcome analyses, we selected patients transplanted for the
92
first time (excluding 5/122 patients) and considered the time point d0 post-transplantation to
93
categorize patients as infected (HPgV (+)) or non-infected (HPgV (-)).The study was
94
approved by the cantonal ethic committee.
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NGS and sequence analysis
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Unbiased NGS (DNA and RNA-seq library preparation, paired-end sequencing by using the
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100-base-pair protocol with indexing on a HiSeq 2500 sequencer (Illumina, San Diego, CA,
99
USA) was performed on the plasma samples. We analysed results using the ezVIR pipeline
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HPgV rRT-PCR assay
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190 ul of plasma were spiked with 10 µl of standardized canine distemper virus of known
104
concentration and extracted with the NucliSENS easyMAG (bioMérieux, Geneva,
105
Switzerland) nucleic acid kit in a 25 ul elution volume, according to the manufacturer’s
106
instruction. Extracted RNA was used for HPgV-specific rRT-PCR screening analysis, using a
107
previously published assay [8]. PCR assay reaction was performed using the QuantiTect 6
Probe RT-PCR Kit (Qiagen, ACCEPTED Valencia, CA. MANUSCRIPT USA) on a StepOnePlus instrument (Applied
109
Biosystems, Rotkreuz, Switzerland) under the following cycling conditions: 50°C for 30 min;
110
95°C for 15 min; 45 cycles of 15 s at 94°C; and 1 min at 55°C. Data were analysed with the
111
StepOne software V.2 (Applied Biosystems). Analytical sensitivity was assessed with a
112
plasmid-derived transcribed RNA including the target region (kindly provided by Professor
113
JT Stapleton) and showed a 100% detection limit corresponding to 5 RNA copies/reaction
114
(approximately 132 RNA copies/ml of plasma).
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Flow cytometry
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Anti-coagulated blood (0.5-1 ml) obtained at 1, 3, 6, 9 and 12 months post-transplantation,
118
was lysed with ammonium chloride and stained with fluorochrome-conjugated antibodies
119
directed against CD3, CD4, CD8, CD56, CCR7 and CD45RA. Samples were incubated 15
120
minutes in the dark and FACS analysis was performed using a Navios flow cytometer
121
(Beckman Coulter, Nyon, Switzerland). FACS data were analyzed using FlowJo software.
122
NK cells were identified as CD3- CD56+ lymphocytes and further divided in CD56bright and
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CD56dim NK cells. CD3+ CD4+ and CD3+ CD8+ T cell subpopulations were further
124
identified based on CCR7 and CD45RA expression.
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Statistical analyses
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X2 or Fischer exact tests were used for categorical variables. Mann-Whitney test or Wilcoxon
128
matched pairs signed rank test were used for continuous variables. Log-rank test was used for
129
survival analyses. We used multivariable regression models to adjust for confounding factors
130
(Table 1). Cumulative incidence estimates of acute GvHD (aGvHD), relapse and infections
131
were calculated using the Gray test with death from other causes as a competitive event. A P
132
value < .05 was considered statistically significant. Statistics were performed using GraphPad
133
Prism version 7, R version 3.2.0 with the EZR graphical user interface and Stata/IC 13.1.
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Results
135
NGS allo-HSCT blood virome study
136
Among the 40 allo-HSCT patients screened by NGS, three (7.5%) were negative for any viral
137
sequences. Thirty-two (80%) and 19 (47.5 %) had at least one detectable DNA and RNA viral
138
sequence, respectively. Among the 37 positive patients, the number of concomitantly detected
139
viruses ranged from one to 5 viruses in a single patient. Figure 1A provides the virus families
140
identified and the number of patients concerned. Among DNA viral sequences, the most
141
frequent were polyomaviruses (PyV) (20/40 patients, 50%), followed by anelloviruses (40%),
142
herpesviruses (35%), papillomaviruses (HPV) (27.5%) and adenoviruses (5%). For RNA viral
143
sequences, the most frequent were HPgV (35%), followed by hepatitis C virus (HCV) (5%),
144
and Rubella virus (2.5%).
145
Figure 1B shows the specific viral sequences identified for each patient according to their
146
respective number of reads. The anelloviruses were mostly Torque Teno (TT) virus (15/21);
147
the other cases were TT Midi Virus and TT Mini Virus. Five patients had more than one
148
species detected. Among herpesviruses, cytomegalovirus (CMV) was the most frequent
149
(10/17), followed by Epstein-Barr virus (EBV), herpes simplex-1 (HSV-1), human
150
herpesvirus (HHV)-6b and HHV-7. Two different types of herpesviruses were simultaneously
151
detected in three patients. The polyomaviruses included Merkel cell (MCPyV) (18/24), BK
152
virus, PyV-6 and JC virus. Four patients were positive for two different polyomaviruses. HPV
153
matching to the 107 genotype was predominant among papillomaviruses (6/11), followed by
154
genotypes 17, 4, 15, and FA75. No multiple infections were identified for HPV.
155
There was no differences in the blood virome composition between patients receiving non-
156
and myeloablative conditioning (Mann-Whitney test, data not shown).
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Confirmatory testing by r(RT-)PCR
8
Our r(RT-)PCR specific assays for HPgV, HCV, adenovirus, BK, JC polyomavirus, CMV, ACCEPTED MANUSCRIPT
160
EBV and HHV-6 confirmed all cases except two EBV, one BK polyomavirus and one
161
adenovirus. Oppositely, three CMV and one EBV, with relatively low viral loads (< 3.2x10E3
162
copies/ml) were detected by rPCR but missed by NGS. One BK virus was filtered by NGS as
163
a contaminant although then confirmed by rPCR. The HSV-1 and the Rubella cases were not
164
confirmed by r(RT-)PCR. We didn’t screen the HHV-7, the anelloviruses nor the
165
papillomaviruses by rPCR. Figure S2 provides the HPgV-positive samples phylogenetic tree.
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166
HPgV investigation
168
Given the high frequency of HPgV detection, the lack of knowledge on its infection kinetics
169
and its potential immunomodulatory role, we performed an HPgV-specific rRT-PCR
170
screening on 122 consecutive allo-HSCT patients with a mean follow-up of 339 days post-
171
transplantation (Figure 2). Overall, 51 patients (41.8%) were positive for HPgV at least once
172
during the follow-up period. 32% of patients were positive before transplantation, of whom all
173
except one patient remained positive until the last time point (d180-365). In 7/51 positive
174
patients, the infection appeared after transplantation; we screened the donor serum from 6 of
175
these patients: one donor was positive for HPgV, whose recipient became viraemic at the time
176
of transplantation. The median viral load of positive cases ranged between 1.7x104 and 5.2
177
x105 copies/ml of plasma (Figure 2).
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HPgV and transplantation outcomes
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We next assessed the association between HPgV infection and transplantation outcomes.
181
Table 1 presents the patients’ characteristics. HPgV(+) group included higher proportions of
182
patients with myeloid malignancies (85% vs 68%; p=0.0072) and lower proportions of
183
patients with lymphoid malignancies (13% vs 27%; p=0.0208). Accordingly, higher
184
proportions of HPgV(+) patients received a myeloablative conditioning. No difference was 9
185
observed in the time to neutrophil engraftment (Figure 3A), in overall survival, progressionACCEPTED MANUSCRIPT
186
free survival or GRFS (Figure 3B a-c), nor in cumulative incidences of grades II–IV aGvHD
187
(Figure 3C), disease relapse (Figure 3D) or infections (Figure S3A-D and Table S2 for
188
microbial agents identified) between HPgV(+) and HPgV(-) patients.
189
HPgV association with cellular and humoral immunity
191
We compared cellular immune reconstitution in HPgV(+) and HPgV(-) patients over one year
192
post-transplantation. At 1 and 12 months post-transplantation, we detected significantly lower
193
numbers of CD4 and CD8, and higher NK cell counts in HPgV(+) patients, respectively
194
(Figure 4A). However, multivariable linear regression analysis, taking into account clinical
195
variables differing between both groups at the studied time points (i.e. sex and disease status
196
at 1 month and sex, donor type and T-cell depletion at 12 months), did not support these
197
differences. Similar analyses on cell subsets distributions among T and NK cells identified
198
lower proportions of CD8 TEMRA T cells in HPgV(+) patients at 12 months, although this
199
difference was not confirmed after adjustment (Figure 4B).
200
Our analyses also failed to detect any significant impact of IVIG administration on HPgV
201
titres (Figure S4).
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Discussion
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We reported all RNA and DNA viruses detectable by unbiased NGS following allo-HSCT.
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Our results reveal that so-called commensal viruses are a major component of the allo-HSCT
206
blood virome, where HPgV was identified in more than 30% of patients.
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Overall, polyomavirus, anellovirus and pegivirus were most frequently identified. The median
209
number of concomitant viral sequences was 2 per patient, with a maximum of 5 distinct
210
sequences identified in four patients. According to a previous study, the DNA blood virome
211
of solid organ transplant recipients (SOT) is composed of 70% of Anelloviridae, 13% of
212
Herpesviridae and 5% of Polyomaviridae and 0% of Papillomaviridae [9]. The equivalent
213
findings in our cohort is 28%, 22%, 32% and 14% respectively. This divergence could be
214
partially explained by critical differences in the protocols used for genetic material extraction
215
and bio-informatics pipelines. If comparing with the gut virome of allo-HSCT patients [5], the
216
same persistent viruses than ours were identified (Anelloviridae, Polyomaviridae,
217
Herpesviridae, and Papillomaviridae found in 70%, 34%, 27% and 18% of patients,
218
respectively); differences between both studies mainly rely on the viruses’ tropism
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(Flaviviridae found exclusively in blood and exogenous enteric viruses in the gut).
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Anelloviruses are known to cause prolonged viremia and multiple co-infections. Two thirds of
222
our study population were positive for at least one anellovirus, corresponding to previously
223
reported prevalence [10]. While it has not been linked with overt organ disease, TTV has been
224
proposed as a proxy of immune suppression in both SOT [11], and allo-HSCT recipients [12].
225
MCPyV and PyV 6 are found on the skin of healthy individuals. While MCPyV has been
226
associated with the Merkel cell carcinoma, PyV 6 is currently not associated with any disease.
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Peripheral blood mononuclear cells (PBMC) are a reservoir for many Polyomaviridae [13]; as
228
we studied cell-free plasma samples, this could explain the relatively low number of reads in
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the PyV(+) samples. Similarly, papillomaviruses can persist in PBMCs, and HPV DNA is ACCEPTED MANUSCRIPT
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generally found in 8-15% of healthy blood donors [14], suggesting that HPV can circulate in
231
blood and are not necessarily skin contaminants.
232
The r(RT-)PCR assays couldn’t confirm the HSV-1 and the Rubella results. Clinical history
234
didn’t indicate any HSV infection and the Rubella-positive patient was serologically positive
235
before transplantation, as for measles and mumps, suggesting previous vaccination. Although
236
the low number of reads detected in these cases suggests non-specific sequences, HSV
237
viremia can occur in up to 26% of critically ill patients [15], including immunocompromised
238
patients without organ disease [16]. Moreover, the cumulative burden of dsDNA viruses
239
detected in plasma has been associated with an increased mortality in HSCT recipients [17],
240
although they rarely caused end-organ disease and were infrequently the direct cause of death.
241
Similarly, in children with primary immunodeficiencies, cases of chronic skin granuloma
242
induced by persistent rubella vaccine strains exist [18]. Thus, commensal viruses may either
243
have an indirect role in pathology or cause unexpected clinical syndrome.
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HPgV viremia is found in 1-5% of healthy blood donors in developed countries [6]. Viral load
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can reach 107 copies/ml and transmission through blood transfusion is well-described [19].
247
HPgV primarily infects haematopoietic stem cells [20], but can also be found in T, B, NK
248
cells and monocytes [6]. HPgV genome can persist in serum micro vesicles and be further
249
transmitted to PBMCs in vitro, leading to active replication and production of infectious viral
250
particles [8]. Our HPgV prevalence is similar to the one of other studies [21-26] (Table S3),
251
but our investigation could suggest an HPgV transmission by the donor in one patient,
252
although a pre-transplant undetectable but positive low viral load is not excluded and that firm
253
evidence are lacking in absence of sequencing. Previous studies have demonstrated that HPgV
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can persist for up to 9 years [26]. Besides, weMANUSCRIPT provided detailed quantitative viremia kinetics, ACCEPTED
255
definitely proving that HPgV is a persistent infection.
256
Although
257
immunomodulatory effect in HIV patients [6]. We assessed the potential impact of HPgV on
258
T cell reconstitution. We identified a difference in the total CD4 and CD8 cell count at 1
259
month and the rate of CD8 TEMRA at 12 months between HPgV positive and negative cases,
260
which could be comparable to what is seen in HIV patients [27]. The effect did not persist
261
after multivariable analyses, and our results can thus not identify any effect of HPgV on cell
262
reconstitution; nevertheless, many confounders can attenuate the potential effect of HPgV, as
263
this was the case in HPgV co-infected HIV patients who experienced immune reconstitution
264
after HAART [28]. Similarly, our results did not uncover an association between HPgV
265
infection and allo-HSCT outcomes, but we found a significant difference in the primary
266
disease between HPgV(+) and HPgV(-) patients, potentially suggesting an association
267
between HPgV and some haematological malignancies, similarly to what we recently reported
268
for TT virus [12].
no
known
HPgV-associated
organ
disease,
HPgV
has
an
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there
Our virome NGS study focused on a single time point post-transplantation without
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comparison to the virome before transplantation and we focused our investigation on HPgV
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due to its immunomodulatory properties, of interest in the context of allo-HSCT. Yet, Legoff
273
et al have recently demonstrated an increase in the proportion of the eukaryotic viruses in the
274
gut virome after allo-HSCT [5]; in addition, picobirnavirus infection was specifically
275
predictive of aGvHD, reinforcing the need to investigate the role of so-called orphan disease
276
viruses in non-infectious complications.
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277 278
In conclusion, our study highlights that most of allo-HSCT recipients have at least one virus
279
circulating in their blood after transplantation. Our observations that HPgV is predominant 13
280
and persistent in allo-HSCT blood virome, should promote further research into the potential ACCEPTED MANUSCRIPT
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impact that commensal viruses can altogether have on immunosuppression.
282
Fundings
284
This work was supported by grants of the Ernst and Lucie Schmidheiny foundation to L.K.;
285
the Henri-Dubois-Ferrière Dinu Lipatti Foundation to D-L.V.; and the Faculty of medicine of
286
Geneva.
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Acknowledgments
289
We thank Pr Jaques Schrenzel, Dr Vladimir Lazarevic and Dr Nadia Gaia for their
290
contribution to the bio-informatics analyses and Mary-Anne Hartley for editorial assistance.
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Potential conflict of interest. All authors: No reported conflicts of interest.
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References
294
[1]
142-50.
Barton ES, White DW, Cathelyn JS, Brett-McClellan KA, Engle M, Diamond MS, et
AC C
[3]
al. Herpesvirus latency confers symbiotic protection from bacterial infection. Nature
299
2007; 447(7142): 326-9.
300 301
Virgin HW. The virome in mammalian physiology and disease. Cell 2014; 157(1):
EP
[2]
297 298
TE D
borders in clinical virology. Clin Microbiol Infect 2017; 23(10): 688-90.
295 296
Vu D-L, Kaiser L. The concept of commensal viruses almost 20 years later: redefining
[4]
Cadwell K, Patel KK, Maloney NS, Liu TC, Ng AC, Storer CE, et al. Virus-plus-
302
susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes
303
in intestine. Cell 2010; 141(7): 1135-45.
14
304
[5]
Legoff J, Resche-RigonACCEPTED M, Bouquet J,MANUSCRIPT Robin M, Naccache SN, Mercier-Delarue S, et
305
al. The eukaryotic gut virome in hematopoietic stem cell transplantation: new clues in
306
enteric graft-versus-host disease. Nat Med 2017; 23(9): 1080-85.
307
[6]
Chivero ET, Stapleton JT. Tropism of human pegivirus (formerly known as GB virus C/hepatitis G virus) and host immunomodulation: insights into a highly successful
309
viral infection. J Gen Virol 2015; 96(Pt 7): 1521-32.
310
[7]
RI PT
308
Petty TJ, Cordey S, Padioleau I, Docquier M, Turin L, Preynat-Seauve O, et al. Comprehensive human virus screening using high-throughput sequencing with a user-
312
friendly representation of bioinformatics analysis: a pilot study. J Clin Microbiol
313
2014; 52(9): 3351-61. [8]
Chivero ET, Bhattarai N, Rydze RT, Winters MA, Holodniy M, Stapleton JT. Human
M AN U
314
SC
311
315
pegivirus RNA is found in multiple blood mononuclear cells in vivo and serum-
316
derived viral RNA-containing particles are infectious in vitro. J Gen Virol 2014; 95(Pt
317
6): 1307-19. [9]
De Vlaminck I, Khush KK, Strehl C, Kohli B, Luikart H, Neff NF, et al. Temporal
TE D
318 319
response of the human virome to immunosuppression and antiviral therapy. Cell 2013;
320
155(5):1178-87.
Med Virol 2010; 20(6): 392-407.
322 323
[11]
Focosi D, Antonelli G, Pistello M, Maggi F. Torquetenovirus: the human virome from
bench to bedside. Clin Microbiol Infect 2016; 22(7): 589-93.
324 325
Maggi F, Bendinelli M. Human anelloviruses and the central nervous system. Rev
EP
[10]
AC C
321
[12]
Masouridi-Levrat S, Pradier A, Simonetta F, Kaiser L, Chalandon Y, Roosnek E.
326
Torque teno virus in patients undergoing allogeneic hematopoietic stem cell
327
transplantation for hematological malignancies. Bone Marrow Transplant 2016; 51(3):
328
440-2.
15
329
[13]
Toracchio S, Foyle A, ACCEPTED Sroller V, ReedMANUSCRIPT JA, Wu J, Kozinetz CA, et al. Lymphotropism
330
of Merkel cell polyomavirus infection, Nova Scotia, Canada. Emerg Infect Dis 2010;
331
16(11): 1702-9.
332
[14]
Chen AC, Keleher A, Kedda MA, Spurdle AB, McMillan NA, Antonsson A. Human papillomavirus DNA detected in peripheral blood samples from healthy Australian
334
male blood donors. J Med Virol 2009; 81(10): 1792-6.
335
[15]
RI PT
333
Ong DS, Bonten MJ, Spitoni C, Lunel FM, Frencken JF, Horn J, et al. Epidemiology of multiple herpes viremia in previously immunocompetent patients with septic shock.
337
Clin Infect Dis 2017; 64(9):1204-10.
338
[16]
SC
336
Lepiller Q, Sueur C, Solis M, Barth H, Glady L, Lefebvre F, et al. Clinical relevance of herpes simplex virus viremia in Intensive Care Unit patients. J Infect 2015; 71(1):
340
93-100.
341
[17]
M AN U
339
Hill JA, Mayer BT, Xie H, Leisenring WM, Huang ML, Stevens-Ayers T, et al. The cumulative burden of double-stranded DNA virus detection after allogeneic HCT is
343
associated with increased mortality. Blood 2017; 129(16): 2316-25
344
[18]
TE D
342
Neven B, Perot P, Bruneau J, Pasquet M, Ramirez M, Diana JS, et al. Cutaneous and Visceral Chronic Granulomatous Disease Triggered by a Rubella Virus Vaccine Strain
346
in Children With Primary Immunodeficiencies. Clin Infect Dis 2017; 64(1): 83-6. [19]
of transfusion-associated hepatitis G virus infection and its relation to liver disease. N
348
Engl J Med 1997; 336(11): 747-54.
349 350
Alter HJ, Nakatsuji Y, Melpolder J, Wages J, Wesley R, Shih JW, et al. The incidence
AC C
347
EP
345
[20]
Bailey AL, Lauck M, Mohns M, Peterson EJ, Beheler K, Brunner KG, et al. Durable
351
sequence stability and bone marrow tropism in a macaque model of human pegivirus
352
infection. Sci Transl Med 2015; 7(305): 305ra144.
16
353
[21]
Skidmore SJ, Collingham KE, Harrison P, Neilson JR, Pillay D, Milligan DW. High ACCEPTED MANUSCRIPT
354
prevalence of hepatitis G virus in bone marrow transplant recipients and patients
355
treated for acute leukemia. Blood 1997; 89(10): 3853-6.
356
[22]
Corbi C, Traineau R, Esperou H, Ravera N, Portelette E, Benbunan M, et al. Prevalence and clinical features of hepatitis G virus infection in bone marrow allograft
358
recipients. Bone Marrow Transplant 1997; 20(11): 965-8.
359
[23]
RI PT
357
Rodriguez-Inigo E, Tomas JF, Gomez-Garcia de Soria V, Bartolome J, Pinilla I, Amaro MJ, et al. Hepatitis C and G virus infection and liver dysfunction after
361
allogeneic bone marrow transplantation: results from a prospective study. Blood 1997;
362
90(3): 1326-31. [24]
Akiyama H, Nakamura N, Tanikawa S, Sakamaki H, Onozawa Y, Shibayama T, et al.
M AN U
363
SC
360
364
Incidence and influence of GB virus C and hepatitis C virus infection in patients
365
undergoing bone marrow transplantation. Bone Marrow Transplant 1998; 21(11):
366
1131-5. [25]
Ljungman P, Halasz R, Hagglund H, Sonnerborg A, Ringden O. Detection of hepatitis
TE D
367 368
G virus/GB virus C after allogeneic bone marrow transplantation. Bone Marrow
369
Transplant 1998; 22(5): 499-501.
liver function of recipients with hepatitis G virus infection after bone marrow
371
transplantation. Bone Marrow Transplant 1999; 24(4): 359-63.
372 373
[27]
Rydze RT, Bhattarai N, Stapleton JT. GB virus C infection is associated with a
reduced rate of reactivation of latent HIV and protection against activation-induced T-
374
cell death. Antivir Ther 2012; 17(7): 1271-9.
375 376
Maruta A, Tanabe J, Hashimoto C, Kato K, Kanamori H, Fukawa H, et al. Long-term
EP
[26]
AC C
370
[28]
Ernst D, Greer M, Akmatova R, Pischke S, Wedemeyer H, Heiken H, et al. Impact of
377
GB virus C viraemia on clinical outcome in HIV-1-infected patients: a 20-year follow-
378
up study. HIV Med 2014; 15(4): 245-50. 17
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Table 1: Clinical characteristics of HPgV (+) and HPgV (-) patients ACCEPTED MANUSCRIPT
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HPgV(+) 39 26 (10-40) 50 (18-69) n % 19 49 20 51 n % 23 59 8 21 4 10 0 0 2 5 1 3 1 3 0 0 n % 27 69 11 28 1 3 n % 4 10 19 49 12 31 3 8 1 3 n % 23 59 16 41 n % 13 33 17 44 3 8 6 15 n % 34 87 5 13 n % 10 26 29 74 n % 22 56 17 44 n % 8 21 3 8 5 13 23 59 n % 17 44 19 49 3 8
P .2879 .5911 .0036
.0001
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HPgV(-) 78 22 (5-39) 52 (19-70) n % 59 76 19 24 n % 26 33 25 32 4 5 10 13 2 3 7 9 3 4 1 1 n % 41 53 33 42 4 5 n % 4 5 45 58 20 26 1 1 8 10 n % 33 42 45 58 n % 30 38 32 41 11 14 5 6 n % 66 85 12 15 n % 35 45 43 55 n % 56 72 22 28 n % 25 32 6 8 12 15 35 45 n % 38 49 28 36 12 15
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All patients 117 22 (5-40) 51 (18-70) n % 78 67 39 33 n % 49 42 33 28 8 7 10 9 4 3 8 7 4 3 1 1 n % 68 58 44 38 5 4 n % 8 7 64 55 32 27 4 3 9 8 n % 56 48 61 52 n % 43 37 49 42 14 12 11 9 n % 100 85 17 15 n % 45 38 72 62 n % 78 67 39 33 n % 33 28 9 8 17 15 58 50 n % 59 50 43 37 15 13
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Patients, n Median Follow-up, months (range) Median Age, years (range) Sex Male Female Primary disease Acute myeloid leukaemia MDS/MPS/MDPS Acute lymphoid leukaemia Lymphoma/chronic lymphoid leukaemia Chronic myeloid leukaemia Multiple myeloma Severe aplastic anaemia Othera Disease status at transplant Complete remission Partial Remission/Progression/Relapse NA Disease Risk Index Low Intermediate High Very High NA Conditioning MAC RIC Donor Type Matched Sibling Matched Unrelated Donor Mismatched Relative Mismatched Unrelated Donor Cell source PBSC BM In vivo TCD No ATG ATG Ex vivo TCD No Yes CMV status D-/RD-/R+ D+/RD+/R+ Immunosuppression CNI, MMF CNI, MTX Cy,Tacrolimus, MMF
.0673
.198
.0162
.1142
.6836
.005
.0184
.2303
.300
382
19
Statistical analyses were doneACCEPTED using X2 or Fischer exact tests for categorical variables and MANUSCRIPT
384
Mann-Whitney test for continuous variables. Myeloid malignancies include acute myeloid
385
leukemia, MDS/MPS/MDPS and chronic myeloid leukemia. Lymphoid malignancies include
386
acute lymphoid leukemia, lymphoma/chronic lymphoid leukaemia and myeloma. acategory
387
other includes haemoglobinopathies. HPgV: human pegivirus; HPgV (+): HPgV-infected;
388
HPgV (-): HPgV non-infected; PBSC: peripheral blood stem cell; BM: bone marrow; TCD:
389
T-cell depletion; ATG: antithymoglobulin; D: donor; R: recipient; CNI: calcineurin inhibitor;
390
MMF: mycophenolate mofetil; MTX: methotrexate; CY: cyclosporine A.
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Figure legends
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Figure 1. Next-generation sequencing analysis. (A) Circular abundance plot of virus
395
families identified by next-generation sequencing in 40 allo-HSCT patients.
396
indicate the number of patients in whom each of the virus families have been identified. (B)
397
Grid plot of viruses identified in 40 allo-HSCT patients. Each line represents one virus and
398
each column one patient’s blood sample. The numbers under the grid indicate the number of
399
distinct viruses identified in each sample. The numbers on the right indicate the number of
400
patients in whom genome sequences of the indicated virus have been identified. The colors
401
represent the approximate number of reads (indicated in the legend) matching the indicated
402
virus’ genome detected in each sample.
403
Figure 2. HPgV real-time RT-PCR screening. Each boxplot represents one time point of
404
screening. N represents the number of HPgV-positive patients, and total the total number of
405
patients screened, at the indicated time point. The percentage is the proportion of positive
406
patients for each time point. BT: before transplantation; d: day
407
Figure 3. Estimates of time to engraftment, survival, aGVHD and relapse in HPgV (+)
408
and HPgV (-) patients. (A) Engraftment. (B) Survival: a. Overall survival (OS); b.
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Progression-free survival (PFS); c. Graft-Versus-Host Disease-Free, Relapse-Free Survival ACCEPTED MANUSCRIPT
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(GRFS). (C) Acute graft-versus-host disease (aGvHD). (D) Relapse.
411
HPgV(+) patients had a median of 17 days (range 12-37 days) and HPgV(-) patients had a
412
median of 16 days (range 12-34 days) before engraftment. OS was calculated from the date of
413
allo-HSCT to death or last follow-up. PFS was calculated from the date of allo-HSCT until
414
disease relapse/progression or last follow-up. GRFS was calculated from the date of allo-
415
HSCT until a GRFS-defining event or last follow up. Events determining GRFS included
416
grade 3-4 aGvHD, systemic therapy-requiring chronic GvHD, relapse, or death. P values
417
compare HPgV (+) versus HPgV (-) patients. Percentages indicate the outcome at 12 months
418
post-transplantation with 95% CI.
419
Figure 4. Immune reconstitution in HPgV (+) and HPgV (-) patients from 0 to 12 months
420
after transplantation. (A) Total CD4+, CD8+ and NK cell count. (B) Proportion of distinct
421
subsets within CD4+, CD8+ and NK cells in HPgV(-) or HPgV(+) patients.
422
At one month, HPgV(+) patients had a median of CD4 count of 0 cell/µl (range 0-199) and of
423
CD8 count of 0 cell/µl (range 0-478) while HPgV(-) patients had a median of CD4 count of
424
35.31 cell/µl (range 0-503, P=.0326) and a median of CD8 count of 81 cells/µl (range 0-449,
425
P=.0445). At 12 months, HPgV(+) patients had higher NK cell counts (median 185 cells/µl,
426
range 50-1090, versus 125 cells/µl, range 10-460, P=.0472). HPgV(+) patients had a lower
427
proportion of CD8 TEMRA T cells (median 26%, range 5%-77%; HPgV(-) patients: median
428
45%, range 12%-93%; P=.0142). EM: effective memory; CM: central memory; NK: natural
429
killer T cells. * for comparison in the rate of TEMRA cells between HPgV(+) and HPgV(-)
430
patients.
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431 432 433
21
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B
A
a.
100 Pegivirus Pegivirus +
P=.404 100
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Engraftment
64% (95%CI 46%-78%)
80
60
53% (95%CI 39%-65%)
40
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OS (%)
50
Pegivirus Pegivirus +
0 0
10
20
30
20
40
Time since HSCT (days)
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0
C
0
100 Pegivirus Pegivirus + 80
40
46% (95%CI 30%-61%)
PFS (%)
D
60
20
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0 6
12
18
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Pegivirus Pegivirus +
46% (95%CI 34%-58%)
0 0
c.
6
12
18
24
Time since HSCT (months)
P=.297 80
P=.185
60
30% (95%CI 20%-41%)
40
66% (95%CI 49%-79%)
100
Pegivirus Pegivirus +
80
P=.108
40
24
GRFS (%)
Relapse cumulative incidence (%)
100
24
60
20
Time since HSCT (months)
D
18
Time since HSCT (months)
80
59% (95%CI 47%-70%)
0
12
100
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aGvHD 2-4 grade cumulative incidence (%)
b.
P=.196
6
45% (95%CI 29%-60%)
60
40 Pegivirus Pegivirus +
20
36% (95%CI 24%-47%)
20 0
18% (95%CI 8%-32%)
0 0
6
12
18
Time since HSCT (months)
0 24
6
12
18
Time since HSCT (months)
24
B
A
80
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100
Pegivirus Pegivirus +
Pegivirus-
%
60
P=.0326
40
20
0
CM Naive EM CM Naive
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Pegivirus+
EM
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P=.0142*
100
%
80
P=.0445
TEMRA EM Pegivirus-
CM
60
Naive
40
TEMRA
20
Pegivirus+
EM CM Naive
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D
0
100
P=.0472
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%
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80
Pegivirus-
CD56dim CD56bright
Pegivirus+
CD56dim CD56bright
60
40
20
0