Viruses in Transplantation - Not Always Enemies

Viruses in transplantation - not always enemies

Virome and transplantation ECCMID 2018 - Madrid

Prof. Laurent Kaiser Head Division of Infectious Diseases Laboratory of Virology Geneva Center for Emerging Viral Diseases University Hospital of Geneva ESCMID eLibrary © by author Conflict of interest

None

ESCMID eLibrary © by author The human virome: definition?

Repertoire of viruses found on the surface of/inside any body fluid/tissue • Eukaryotic DNA and RNA viruses • Prokaryotic DNA and RNA viruses (phages) 25 • The “main” viral community (up to 10 bacteriophages in humans) Haynes M. 2011, Metagenomic of the human body • Endogenous viral elements integrated into host chromosomes (8% of the human genome) • NGS is shaping the definition Rascovan N et al. Annu Rev Microbiol 2016;70:125-41 Popgeorgiev N et al. Intervirology 2013;56:395-412 Norman JM et al. Cell 2015;160:447-60 ESCMID eLibraryFoxman EF et al. Nat Rev Microbiol 2011;9:254-64 © by author Viruses routinely known to cause diseases (non exhaustive) Upper resp./oropharyngeal HSV 1 Influenza CNS Mumps virus Rhinovirus JC virus RSV Eye Herpes viruses Parainfluenza HSV Measles Coronavirus Adenovirus LCM virus Cytomegalovirus Flaviviruses Rabies HHV6 Poliovirus Heart Lower respiratory HTLV-1 Coxsackie B virus Rhinoviruses Parainfluenza virus HIV Coronaviruses Respiratory syncytial virus Parainfluenza virus Adenovirus Respiratory syncytial virus Coronaviruses Gastro-intestinal Influenza virus type A and B Human Bocavirus 1 Adenovirus Hepatitis virus type A, B, C, D, E Those that cause asymptomatic reactivations/infectionsRotavirus Yellow fever Norovirus Astrovirus Coxsackie B virus Skin /mucosa are often considered as part of the virome VZV, CMV, HHV8 Human papillomavirus Genital tract Human polyomavirus HSV 2 (Merkel) Systemic / blood Human papillomavirus Parvovirus B19 Mumps virus Rubella HHV6 HIV, HBV Rubella Adenovirus Measles HTLV-1 HSV Measles Epstein-Barr virus Enteroviruses HIV Enteroviruses Cytomegalovirus Flavivirus Yellow fever VZV, CMV, HHV8 Human polyomavirus ESCMIDParvovirus B19 Hepatitis eLibraryvirus type A, B, C, D, E © by author Virome: the definition has evolved with the technology (NGS)

Vertebrates: 1’500 viral species ≈ 180 viruses known to infect or detected at least once in humans P Lemercier ViralZone

ESCMID eLibrary © by author NGS & the black box: you need to trust…

The sample preparation The sequencing procedure The quality of the database The bioinformatics pipeline You need a tool to communicate with the average microbiologist

ESCMID eLibrary © by author NGS and the basic I learned at school

A diagnostic test should have excellent: Sensitivity and specificity Positive and negative predictive values PCR-based assays: Everything is done to avoid contamination

ESCMID eLibrary © by author NGS at the analytical level

Sensitivity: can be high but also very low

Limit of detection/quantification: copies/mL vs. number of reads

Specificity: could be perfect or "catastrophic“ Viral sequences in reagents… Banana bunchy top virus Endless list of “false-positive” results ESCMID eLibrary © by author CSF negative control report (RNA and DNA protocols) Canine distemper virus (positive control)

Merkel cell p. Polyomavirus HSV 1 Papillomavirus

ESCMID eLibrary Known as a reagent contaminant (vector ) © by author NGS and the eukaryotic human virome

Viral sequences ≠ replication competent viruses We do not see the virus or any viral antigens Serological assays: not available for many viruses

Association with disease: confirmed, probable, possible, none

Contamination and uncertainty are part of the game

ESCMID eLibrary DL Vu et al. CMI 2017 © by author Eukaryotic viruses in blood of « asymptomatic » humans (non exhaustive)

Pubmed search with algorithms of keywords and MeSH > 300 papers investigating the virome in humans

ESCMIDDiem Lan Vu Cantero Samuel CordeyeLibraryMarie-Céline Zanella © by author Blood Virome Association with diseases (confirmed, probable, possible or none)

Herpesviridae HSV1-2, VZV, EBV, CMV, HHV6, HHV8 HHV7 Polyomaviridae BK virus, JC virus Merkel, TdS PyV HPyV 6-7-9 Adenoviridae adenovirus A-F Parvoviridae parvovirus B19 bocavirus, parvo 4 Papillomaviridae Different serotypes Anelloviridae TTV, TTMV, TTMDV Cycloviridae cyclovirus Vietnam Circoviridae human circovirus Picornaviridae enterovirus cardiovirus, salivirus Flaviviridae human pegivirus 1-2 Picobirnaviruses ESCMID eLibrary © by author Blood DNA virome

8’000 individuals Healthy or with comorbidities/chronic diseases “by-product” of a large human genome study Viral sequences mapped 94 viruses Prevalence: age dependent ESCMID eLibraryMoustafa A et al. 2017 PLoS Pathog © by author HHV7 Primary infection during childhood : roseola Seroprevalence > 90% in adults

Tropism : CD4 T lymphocytes Lusso P PNAS 1994

SOT: primary infection or reactivation in up to 46% of patients HSCT: primary infection or reactivation in 44% (adults)

ESCMID eLibraryAgut H et al. Human Herpesviruses 6A, 6B, and 7. Microbiol Spectr . 2016 Jun;4(3) © by author HHV7: possible associations with diseases

Fever and acute myelitis Fever, rash, hepatitis, pneumonitis in IC hosts Bronchiolitis obliterans syndrome after lung transplantation Allograft rejection and dysfunction in case of co-infection with HHV-6 Association with CMV infection and disease

ESCMID eLibraryAgut H et al. Human Herpesviruses 6A, 6B, and 7. Microbiol Spectr . 2016 Jun;4(3) © by author NGS/virome in transplant recipients

ESCMID eLibrary © by author DNA virome in Heart & Lung transplant recipients

Cell free DNA in plasma (656 samples) Anelloviridae 68% Heart recipients: pediatric (24) and Herpesvirales 13% adults (41) Caudovirales 5% Lung recipients: adults (31) Polyomaviridae 5% Adenoviridae 2% Poxviridae 1% Retroviridae 1% Other 5%

ESCMID eLibraryI De Vlaminck. Cell. 2013 Nov 21;155(5):1178 -87. © by author Response to immunosuppression & antiviral therapy (valaciclovir / tacrolimus)

Temporal association ? Effect on adenoviruses?

ESCMID eLibraryI De Vlaminck. Cell. 2013 Nov 21;155(5):1178 -87. © by author Polyomaviridae Bialasiewicz Am J Transplant 2016, Bialasiewicz J Clin Virol 2009, Moens Patholog Res Int 2011, Rockette PlosOne 2013, Dialanis Virology 2013 Seroprevalence DNA detection in clinical samples Children Adult blood urine GI skin resp CNS

HPyV1 BK polyomavirus 73% 82-87.6%   ? ?   HPyV2 JC polyomavirus 21% 35-58%    ?   HPyV3 Karolinska Insitute polyomavirus 56% 55-91.3 ? ?  ?  ? HPyV4 Washington University polyomavirus 54% 69-97.4%   HPyV5 Merkel cell polyomavirus 23-34% 25-70.4%    HPyV6 unkown 69-73.7%   HPyV7 unkown 35%   Trichodysplasia spinulosa associated HPyV8 polyomavirus unkown 80.9%    HPyV9 20% 17.6-47%   HPyV10 Malawi polyomavirus unkown 99.1%   HPyV11 Saint Louis polyomavirus 34-61% 90-93.3%  HPyV12 34-80% 90-97.3%  ESCMIDHPyV13 New Jersey polyomavirus 7.5 -33%eLibrary 31.4-57.5%  © by author Post-transplant Merkel cell carcinoma

Neuroendocrine skin cancer Very rare but 66 to 182 fold increased risk in transplant recipients

V Koljonen Acta Dermato Venerologica 2016 ESCMID eLibraryFeng Science 2008 © by author Trichodysplasia Spinulosa Polyomavirus

Rare skin disease (> 35 cases reported) Acute primary infection (not reactivation/chronic infection) with high viral load (skin, blood and CSF) may be the cause of complications

ESCMID eLibraryE van der Meijden J Infect Dis. 2016 © by author New Jersey polyomavirus

Pancreas transplantation Acute myopathy, retinal blindness and skin lesions High viral load in skin lesions and blood Histopathology suggest endothelial tropism

ESCMID eLibraryN Mishra J Infect Dis. 2014 © by author Disease association: confirmed, probable, possible, none Seroprevalence DNA detection in clinical samples Children Adult blood urine GI skin resp CNS

CMV D+/R+, EBV D+/R+ December: acute grade 3 intestinal GVHD -> high dose steroids

BK virus: urine 3.59 copies/mL, 2.84 in plasma CMV: 2.42 copies/mL in plasma, 2.72 copies/mL in BAL EBV in plasma: 4.24 copies/mL Adenovirus : 6.52 copies/mL in plasma (and prolonged stool carriage) Norovirus: prolonged stool carriage ESCMID eLibrary © by author dsDNA viruses in the plasma of HSCT recipients

Weekly samples during 100 days post HSCT PCR for EBV, CMV, HHV-6, ADV, BK virus Positive detection of multiple viruses: 90% had ≥1 62% had ≥2 28% had ≥3, and 5% had 4 or 5 viruses Co-detection 2 viruses : 37% of patients 3-4 viruses : 12% of patients ESCMIDHill JA et al. Blood. 2017 eLibraryApr 20;129(16):2316-2325, CID 2018, YT Huang Biol Blood Marrow Transplant 2017 . © by author Impact of viral infections and reactivations after HSCT

Cumulative viral load (EBV, CMV, HHV-6, adenovirus, BK virus) is a predictor of early and late mortality [aHR 1.36, [1.25, 1.49], and 1.04 [1.0, 1.08] detection and kinetics have a temporal relationship with outcomes “dose-dependent” association with increased mortality.

ESCMIDHill JA et eLibraryal. Blood. 2017, Hill JA CID 2018, YT Huang Biol Blood Marrow Transplant 2017 © by author Pre- and post- transfusion A Kapoor mBio 2015

Stem cells Blood products

« ESCMIDpost-transfusion » TTV or pegivirus eLibraryinfections (not only herpes viruses ) © by author Blood DNA and RNA virome in HSCT recipients

To define the landscape of the blood virome at day 30 post transplantation using NGS Has the intrinsic and « transfused » virome any impact on immune reconstitution or other outcomes?

• 40 consecutive HSCT recipients, plasma at day 30: no case selection • Unbiased NGS (DNA and RNA-seq library, HiSeq 2500) • 40-50 millions of read/sample

• Updated EzVir pipeline JCM 2014 ESCMID eLibraryDiem Lan Vu et al. CMI in revision 2018 © by author

Results Viruses

Patients & number of “virus/cases” ESCMID eLibrary © by author The most frequent viral sequences NGS « Snapshot »

Multiple reactivation/acquisition of viruses Up to 5 different « viruses », only one without any viral sequence Recovered in >20% of cases: • Merkel cell PyV: 18/40 • Anelloviruses: 15/40 Different species/strains • Pegivirus: 14/40 • Papillomavirus: 11/40 • CMV: 10/40 SeeESCMIDalso abstract O0555 A Bal eteLibrary al. © by author False positive results vs. real findings?

Adenovirus – XRMV: contaminants

Merkel cell: « similar » strain across all cases but different patterns Polyomavirus: Different serotypes but some may be false-positive Papillomavirus: Different serotypes Skin contamination…?

Blood collected though implanted catheter! ESCMID eLibrary © by author Pegivirus: expanded cohort (n=122)

• >30% HPgV-positive before transplantation and remained positive • 51 (41.8%) HPgV positive at least once • 7/51 patients (13.7%) were negative before Tx and became positive • High viral loads

ESCMID eLibrary © by author Human Pegivirus

Flavivirus: Related to hepatitis C Previously GBV-C Several genotypes

New type recently identified in blood MG Berg PlosPathogens 2015

ESCMID eLibrary Chivero 2015 © by author Human Pegivirus previously GBV-C

25% of persistent or protracted infection: up to 10E7 copies/ml in blood Viral clearance occurs within ~ 2 years: Anti E2 antibodies Viremic blood donors: North: 1-5%, South: up to 20%

2018: not associated to a clinical disease New pegivirus (HpgV-2) associated with hepatitis C/HIV co-infection Berg PlosPathogen 2015 Wang CID 2018 Reported as a possible cause of encephalitis during a primary infection in Denmark H Fridholm 2016

ESCMID eLibrary Chivero 2015 © by author Pegivirus

Chivero 2015

Reduces HIV-associated mortality Stapleton et al., PLoS One 2012, Tillmann et al., NEJM 2001 Improved Ebola survival in young co-infected patients Lauck J Virol 2014

Receptors and primary permissive cell unknown Probable infection of HSC precursors and naïve T cells Lymphotropic: CD4 T cells, CD8 and B cells Rate of PBMC infection similar to HIV and CD4+ T cells Reduced T cell activation and proliferation Inhibition of HIV cell entry: Decrease CCR5 & CXCR4 and production of ligands targeting HIV cell receptors ESCMID eLibrary © by author Kaplan-Meier likelihood of survival, acute GVHD and relapse (pegivirus + vs. -)

Pegivirus positive patients may have a better outcome (?) No significant association with clinical outcomes or immune-reconstitution ESCMID eLibraryDiem Lan Vu in revision 2018 © by author DNA virus RNA virus Association with diseases (diarrhea, acute flaccid paralysis, others…) Confirmed, probable, possible, none

Adenoviridae Caliciviridae DNA Prokaryotic viruses adenovirus norovirus RNA prokaryotic viruses sapovirus Astroviridae Polyomaviridae Picornaviridae Anelloviridae novel human TTV, TTMV, TTMDV enterovirus Anelloviridae astrovirus Reoviridae human gyrovirus Picobirnaviridae rotavirus Parvoviridae picobirnavirus Astroviridae human bocavirus classical Astrovirus Picornaviridae bufavirus cardiovirus tusavirus cosavirus husavirus Circoviridae kobuvirus cyclovirus Gut virome rosavirus circovirus salivirus Genomoviridae ESCMIDhuman torovirus eLibrarygemicircularvirus © by author Circoviridae

Small, non enveloped, ss circular DNA: circovirus and cyclovirus

Circovirus (animal): Swan, Gull, Porcine… Beak & feather disease Cyclovirus (animal and humans)

High detection rate (>40%) in pigs & chicken (oro-fecal) Li J Virol 2010 Todd VetESCMIDMiobiol 2004, Gillespie J Vet Intern Med 2009, Sauvage eLibraryJournal of Virology 2011 © by author Cyclovirus in humans: possible associations

Human blood: 10% of Dengue negative tropical febrile illnesses NL Yozwiak PlosNTD 2012 CNS infections in Vietnamese children: 4% of 642 cases, none of 122 CSF of non infectious etiology Le Van Tan mBio 2013

7-17% of non-polio acute flaccid paralysis Li J Virol 2010

10%-15% of CSF/serum of unexplained paraplegia in Malawi SL Smits EID 2013

Stools with or without gastroenteritis Vietnam and Africa, HIV patients Garigliani Science Report 2014

Nasopharyngeal swabs of Chilean children with respiratory infections TG Phan J. of General Virology 2014 Italy: 21% blood detection rate in HIV+ patients but low in transplant recipients (2%), 0% in healthy individuals l Macera JCV 2016 ESCMID eLibrary © by author HSCT recipients and the gut virome

2017

Longitudinal study in 44 HSCT recipients ESCMID eLibrary © by author Gut virome in HSCT recipients J Legoff Nature Medicine 2017

• Increase in rates and number of DNA virus sequences after transplantation • Persistent viruses in 50% of samples (anello-, herpes, papilloma-, polyoma-). • Decreased richness of verterbate viruses during first weeks but increased rate of herpesviruses in GVHD cases (not predictive) • Reduced phage richness • Association with expansion of Lactobacillales and loss of Clostridiales • Picobirnavirus detected in 41% ESCMID eLibrary © by author Picobirnaviruses

Small, non-enveloped, bi-segmented dsRNA, capsid, discovered High diversity not completely characterized Recovered in animal feces and in sewage worldwide Human strains related to porcine species Can be shed abundantly Viral persistence or prolonged shedding, relapsing infections

ESCMIDAlfieri eLibrary AF 1989, Ganesh B Virus Diseases 2014, Ganesh Rev Med Virol 2012 © by author Picobirnavirus: pathogenicity (?)

Discovered in the 1980s in children with acute gastroenteritis Pereira Lancet 1988

Sole pathogen or co-infection in HIV patients with diarrhea Giordano JAIDS 1998

Acute diarhhea in children China G Sun Archives of Virology 2015 Recovered in human with or without diarrhea More frequent in HIV/IC hosts? Pathogenicity: not established No isolation yet (?) No serological assays ESCMID eLibraryGanesh B Rev Med Virol 2012 and Virus Diseases 2014 © by author HSCT recipients: the gut virome

Picobirnavirus detected in 41% of all cases, more frequently at baseline Multivariate analysis: associated with severe GVHD (HR 2.66, 1.46-4.86)

Non enteric GVH Enteric GVH

ESCMID eLibraryJ. Legoff. Nature Medicine . 2017 Sept 23; (9): 1080-1085 © by author NGS and unexpected viral infections or viral discovery

Kidney transplant living donor/recipient pairs (n=30)

Phylogenetic analysis confirmed transmission of JCPyV from donors to recipients in 6 out of 7 cases Role of JCPyV infection after renal transplantation? ESCMID eLibrary © by author Use of NGS for respiratory viral infections

Lung Transplant Recipients D Lewandowska PlosOne 2017 60 episodes of respiratory symptoms in LTRs Identified 4 cases of human rhinovirus compared to routine panel The majority of samples were TTV and HHV7 positive

NGS could detect divergent strains but the added value of NGS compared to multiplex panels seems limited

Viruses recently «discovered» in transplant patients (non exhaustive)

Astroviruses: VA1/MLB2 SN Naccache, Clin Inf Dis 2015, JR Brown Clin Infect Dis 2015

New poxvirus NS Lakis Clin Infect Dis 2015 Dandenong virus (arenavirus) G. Palacios N. Engl. J. 2008

New Jersey polyomavirus N. Mishra J Infect Dis 2014 Mastoadenovirus (adenoviridae) in a HSTC recipients Hage JGV 2017 Usutu Cavrini Eurosurveillance 2009

Tick Born Encephalitis D. Lipowski J Infect Dis 2017 ESCMID eLibrary © by author Novel astroviruses and CNS complications Diem Lan Vu JCV 2016, Viruses 2017

Mamastrovirus 1 HAstV-PS Classical HAstV 4 - Encephalitis in a boy with an X-linked agammaglobulinemia - Fever and multiple organ failure in an infant after HSCT Quan et al, EID 2010 Wunderli et al, PlosOne 2011

HAstV-VA1/HMO-C-UK1 - Encephalitis in an infant after HSCT Brown et al, CID 2015

- Encephalitis in an adult after HSCT Naccache et al, CID 2015 Mamastrovirus 6 HAstV-VA1/HMO-C-PA -Encephalitis in a boy with an X-linked agammaglobulinemia Fremond et al, J Pediatric Infect Dis Soc 2015 HAstV MLB-2 - Child with febrile neutropenia, rash and URT symptoms HAstV-VA1/HMO-C Holtz et al, EID 2011, Wylie et al., Plosone 2012 -Upper respiratory tract infection in an immunocompetent child Brain biopsy Cordey et al, in preparation - Meningitis in an immunocompetent young adult and a HSCT recipient Cerebrospinal fluid Cordey et al, EID 2016

Plasma/serum Mamastrovirus 9 - Asymptomatic immunocompromised child Stools Cordey et al, EID 2016

Nasopharyngeal swab Urine Other : vesicleESCMIDswab, bone marrow, heart, lung, spleen, kidney. eLibrary © by author Novel poxvirus NS Lakis CID 2015

Reoviridae (rotavirus): dsRNA isolated in mammalian hosts Case reports in humans: respiratory disease, meningitis, encephalitis and gastroenteritis Steyer JCM 2013, Chua PNAS 2007, Johansson SJID 1996, Tyler JID 2004, Ouatarra JID 2011

Persistent mammalian orthoreovirus, seroconverted Lewandoska BMC Inf Dis 2018

Bornavirus: negative RNA virus with a large animal reservoir Fatal disease in horses near Leipzig in 1885 Cluster of fatal encephalitis in Germany in 2011-13 in squirrels breeders Hoffmann B NEJM 2015 Variegated squirrel bornavirus 1 (VSBV-1)

March 2018: 3 solid organ recipients from a single donor, 2 of them died from encephalitis ECDC March 26, 2018 Bornavirus disease virus-1 (natural host bicoloured white-toothed shrew) distinct from the VSBV-1 ESCMID eLibrary © by author Conclusions

The landscape is quite well defined

The technology may be misleading We need tools that overcome NGS weaknesses Need to differentiate acute primary infection from reactivation ESCMID eLibrary © by author Viruses in transplantation - not always enemies

Obviously many viruses are enemies Some are opportunists Role for «large spectrum antivirals»? Some may just be innocent bystanders (TTV) or friends (pegivirus?) Invisible for the immune system? danger signals? Immune evasion? New players: picobirnaviruses? Could be prokaryotic phages Krishnamurthy Virology 2018 « new » polyomaviruses / others NGS and virus discovery : «new viruses = rare events» ESCMID eLibrary © by author Commensal viruses: complex interactions

Interactions with other components of the microbiome, mainly in the GI tract The microbiota, it composition and impact on clinical outcomes (e.g. specific bacteria like Blautia and GVH)

HW Virgin Cell 2015, Cadwell Immunity 2015, DL Vu CMI 2017 RR Jeng BBMT 2015, A Staffas Bloiod 2016, Shono Science ESCMID Transl Med 2016, Fuji Frontiers Oncology 2014 eLibrary © by author Acknowledgment

Division of Infectious Diseases Division of Haematology Diem Lan Vu Federico Simonetta Marie Céline Zanella Stavroula Masouridi-Levrat Christian van Delden Yves Chalandon Laboratory of Virology Swiss Institute of bioinformatics and Samuel Cordey Faculty of medicine, Geneva Lara Turin Francisco Brito Gaël Vieille Evgeny Zdobnov Florian Laubscher Mylène Docquier Isabelle Arm-Vernez ESCMID eLibrary © by author