Non-targeted diagnosis of respiratory viral infections by means of shotgun

metagenomic using deep sequencing

Christophe Rodriguez, Slim Fourati, Guillaume Gricourt, Vanessa Demontant, Stéphane Chevaliez, Jean-Michel Pawlotsky

Microbiology dpt University Hospital Henri Mondor

INSERM U955 Team 18, NGS platform IMRB,

Créteil, France,

Introduction

MetagenomicsDefinition : the direct genetic analysis of genomes contained within a sample-> instead of targeting specific genomic regions of predetermined targets, short RNA/DNA sequences (‘reads’) are generated from the full range of genomic material present in the sample

Great potential method to characterize host transcriptome, identify viruses, bacteria, and fungi from a range of biological samples in clinical diagnostic

Objectives

• Develop an unbiased metagenomic screening tool to diagnose acute respiratory infections:– Pan-pathogens

– On nasopharyngeal swabs and broncho-alveolar lavage

• Use metagenomic for viral characterization and host-viral interaction

– Genotyping

– Intra-individual viral diversity (minority variants)/resistance

– Semi-quantification (number of reads compared to viral loads)

– Host-pathogens interactions by transcriptome analysis

Methods➢ Thirty three clinical routine samples (BAL, NP swabs) from patients infected

with one respiratory virus (8 viruses tested, Ct<30)

12 HDV

5 HAV, 1 Rhinovirus, 1 Enterovirus

8 HIV

3 HEV

41 HCV, 1 Dengue, 1 Zika

2 Coronavirus 229E

1 Inf A, 1 Inf B

13 HBV

1 HSV1, 1 HSV2, 1 VZVZ, 1 EBV, 8 HCMV, 1 HHV6

1 Adenovirus

1 BKV

24 HRSV, 1 HMPV, 1 HPIV

1 Chikungunya

Cover a wide range of different viral properties

BAL or NPAcontaining viruses identified by qPCR

Library preparation

DNA/RNA adjusted protocols for

metagenomic

DNA/RNA extraction

DNA Lib prep (adapted from Nextera XT) +

RNA Lib prep (adapted from TruSeq RNA)

NextSeq500 Sequencing 2*150bp(Illumina)

>20 M reads/sample

MetaMIC® (experimental and software are patented)

TissueCellular fluids

Acellular fluids

MetaMIC® Analysis (quality, filtering, identification, genome reconstruction and comparison)

Sequencing

The method includes eliminating host rRNA, (≈80% of total cellular RNA)

MethodsTechnical procedures

Dpt of Clinical Microbiology

Clinicians

NGS platformMg prescription

Pre-PCR extraction Library prep Sequencing Analysis

Methods

Results

➢1) Screening : All 33 positive samples were correctly identified by metagenomics :

• Adenovirus• Rhinovirus, Enterovirus, Coronavirus• Influenza A and B• hRSV, hMPV, hPIV

➢2) Coverage of full-length viral genome (>98%) obtained in all cases (Ct <30)

ResultsCoverage plots of the virus panel samples

Influenza A (8 segments)

Human Coronavirus 229E

genome coverage by nuc positions (x-axes) sequencing depth (y-axes)

dep

thd

ep

th

Human Rhinovirus

Human Respiratory Syncytial Virus

ResultsCoverage plots of the virus panel samples

de

pth

de

pth

ResultsWithin-host viral diversity

G

hRSV minority variants

Double populations in some regions of the G protein reflecting immune pressure

F L

Resistance analysis

RASs to nuc analogs (Lumicitabine)

628 789 795 796

Fears R Antiviral res 2016; Perron M. Antimicrob Agents Chemother 2016

ResultsWithin-host viral diversity

Minority populations of escape variants 140 141 142 143 144

RASs to fusion inhibitors (presatovir)

NP Transcriptome immunocompetent P

NP Transcriptome immunocompromised PGRIN 2B

KCNQ2

HLA-C

HLA-DRB1

HSP70-1

HLA-A

HLA-C

CXCL10

HLA-DRA

BPIFA1

HLA-C

HLA-A

HLA-DRB1

cadherin 4

RHOT2

ZNRF1

HLA-DRA

HSP70-2

HLA-DRA1

HLA-DQB

ResultsHost Transcriptome

Comparison of whole transcripts levels obtained from : -NP from RSV infected Immunocompetent adults at TOD-NP from RSV infected Immunocompromised adults at TOD

Conclusion

• Screening/identification – Unbiased Metagenomic approach with full concordance with qPCR

– in clinical diagnosis from various sample matrix when conventional methods are not conclusive

– Theoretically, no limit to detect any virus (provided VL is not too low, ≈ Ct<35)

• Molecular characterization– Full length virus genome analysis

– Viral diversity/ resistance genotyping with minority variants

– Transcriptome analysis to identify host-pathogens interaction pathways

INSERM U955 Equipe 18 CréteilPr. Pawlotsky Jean-MichelDr Ahmed-Belkacem AbdelhakimAhnou NazimBaudesson CamilleBerry FrançoisBrillet RozennDr Brouillet ArthurDr Bruscella PatricePr. Chevaliez StéphaneDemontant VanessaPr. Dhumeaux DanielDi Donato JuliaGricourt GuillaumeHamadat SabahDr Lafdil FouadMercier-Darty MélanieN’Debi MelissaPoiteau LilaDr Rodriguez ChristopheDr Rousseau BenoîtDr Ruiz IsaacSoftic laurentSoulier AlexandreDr Texeira-Clerc Fatima

Acknowledgments