NEXT GENERATION SEQUENCING OPENS NEW VIEWS ON VIRUS EVOLUTION AND EPIDEMIOLOGY. 16th International WAVLD symposium, 10th OIE Seminar

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1 NEXT GENERATION SEQUENCING OPENS NEW VIEWS ON VIRUS EVOLUTION AND EPIDEMIOLOGY S. Van Borm, I. Monne, D. King and T. Rosseel 16th International WAVLD symposium, 10th OIE Seminar

2 Viral livestock threaths: sequencing applications FMDV RNA 8 kbp ASFV DNA 170 kbp AIV RNA 14 kbp 11/06/2013 2

3 Fast virus evolution o o o Large population sizes (RNA & DNA viruses) Rapid replication kinetics, short generation time Fast generation of genetic diversity: DNA replication: enzymes with proofreading activity Error rates <1/10 7-1/10 9 > RNA replication: RNA polymerase lacks proofreading activity error rates <1/10 3-1/10 6 > e.g. for 10 Kbp genome, 1/10 5 means 1 error per replication cycle RNA virus quasispecies: population of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. 11/06/2013 3

4 RNA virus as quasispecies Consensus: :Consensus Large population passage Selection, bottleneck e.g. infect next cell, individual, farm, e.g. cell culture passage e.g. new host, treatment, immune response,.. e.g. plaque purification 11/06/2013 4

Sequencing technologies «first generation»: Sanger Sequencing Chain

nucleotide (A T G C) Capillary electrophoresis with resolution of 1

Illumina, Roche/454, Solid, Iontorrent, Clonal amplification of

sequencing of clonal DNA s 100 000 s of reads from clonal DNA s

5 Sequencing technologies «first generation»: Sanger Sequencing Chain termination sequencing reaction Fluorescently labeled terminal nucleotide (A T G C) Capillary electrophoresis with resolution of 1 nt 1 Consensus sequence of a DNA sample «Second generation» Illumina, Roche/454, Solid, Iontorrent, Clonal amplification of single target molecules from DNA library Massive parallel sequencing of clonal DNA s s of reads from clonal DNA s representing the genetic variation within a sample Real picture of quasispecies 11/06/2013 5

6 Toolbox: 2nd Generation Sequencing technologies (NGS) o Sample -> DNA library -> fragmentation/size selection -> including adapters o o o o Clonal amplification of single target molecules from DNA library empcr (Roche, Iontorrent) Bridge PCR (Illumina) Massive parallel sequencing of physically separated clonal DNA s Pyrosequencing (Roche) Reversible dye terminator sequencing (Illumina) s of reads from clonal DNA s DATA ANALYSIS! 6

7 applications of NGS in veterinary virology: examples Data selected from Epi-SEQ «Molecular epidemiology of epizootic diseases using next generation sequencing technology (Emida Era- Net project) o Avian influenza evolution in outbreak (I. Monne) o In vivo FMDV evolution (D. King) o In vitro AIV evolution under antiviral drug pressure (S. Van Borm) 11/06/2013 7

8 The AI H7N1 epidemic in Italy I. Monne et al., IZSVe, Padova, IT 11/06/2013 8

9 The AI H7N1 epidemic in Italy 11/06/2013 Consensus sequencing 148 viruses 9

10 11/06/

11 HPAI: amino acid signatures 11/06/

12 Application of NGS: new questions can be tackled: o Confirm LPAI is progenitor of HPAI? o Mixed populations? o Study emergence of HPAI from LPAI in the field with quasispecies resolution! o Representative LPAI & HPAI samples 11/06/

NGS of AIV o Targeted RT-PCR amplification of genome

and 3 ) directly from clinical samples (strong

Illumina sequencing libraries o Sequencing on Illumina

13 NGS of AIV o Targeted RT-PCR amplification of genome segments (primers <-> conserved regions noncoding 5 and 3 ) directly from clinical samples (strong positives pre-selected by RT-qPCR) o Preparation of Illumina sequencing libraries o Sequencing on Illumina MiSeq o Data analysis using consensus reference genomes 11/06/

14 HPAI signatures at low frequency in LPAI precursor viruses. Directly detected in clinical samples (analysis ongoing) 11/06/

15 LPAI signatures at low frequency in HPAI precursor viruses. Directly detected in clinical samples (analysis ongoing) 11/06/

16 NGS on clinical AIV samples during epidemic: conclusion: o The NGS study confirmed the low and highly pathogenic epidemics occurred as the result of a single viral introduction o Amino acid signatures characteristic for the HP virus cluster were already present with low frequency within several host viral populations from the first months of the H7N1 epidemic although the linkage between these amino acid sites could not be ascertained o The precise role of all mutations identified in the H7 HPAI viruses is still unclear. However, having identified minority variants with HP key mutations in the LP samples that became prevalent in the HP viral population, confirms the importance of implementing prompt and effective eradication measures in case of an LP H7 outbreak to prevent the appearance of viruses with selective advantages and unpredictable pathogenic properties 11/06/

17 In vivo evolution of Foot-and-Mouth disease virus 11/06/

In vivo evolution : within host methods needle inoculation O 1 -BFS (cell culture adapted) within host pathways NGS offer step-change increase in the amount of sequence data that can be generated

18 In vivo evolution : within host methods needle inoculation O 1 -BFS (cell culture adapted) within host pathways NGS offer step-change increase in the amount of sequence data that can be generated from a sample Study used reversible terminator chemistry (Illuminia) Foot lesions Back right foot Front left foot Sequence diversity generated within a single animal at two sites and compared to the inoculum 11/06/

19 Genome coverage 3 samples, 2 technical replicates of PCR amplification and sequencing (A and B) 11/06/

20 Variability in samples polymorphisms (selection) (cell culture) Equal to consensus genome inoculum Mutation compared to consensus inoculum fixed (100% present) 11/06/

21 Sequence changes that arise during transmission in cattle C1 C2 C3 C4 Direct contact Direct contact Direct contact x9 samples x7 samples x2 samples 11/06/

22 Sub-consensus changes: C1 C2 C3 C4 Transmission direction Lost Drifting Fixed Wright et al., 2011 J. Virol. ; Morelli et al., /06/

23 FMDV in vivo evolution: conclusion o NGS: step-change in resolution of sequence data With quasispecies resolution Both low & high frequency mutations Within-host evolution (selection of certain mutations relative to cell-culture adapted inoculum) Transmission dynamics (loss, drift, fixation) 11/06/

24 In vitro evolution of a LPAI H5N1 in presence of oseltamivir (neuraminidase inhibitor) Known resistance mutations H274Y, R292K, N294S (E119V facilitates H 2 O binding) 11/06/

25 LPAI H5N1 + oseltamivir: experiment Wild type P1selection 0,005 mm oseltamivir 10³ EID50 P2 0,01mM oseltamivir 10² EID50 P3 0,01 mm oseltamivir 10 1 EID50 P1 amplification P2 amplification P3 amplification clone L Plaque purified NGS (Roche Gsjunior) & phenotype (NA activity, NA inhibition, ) 11/06/

LPAI H5N1 + oseltamivir: the phenotype Infectious dose (TCID50) Oseltamivir concentration Virus (RT-qPCR) NA activity (substrate) IC50 oseltamivir tolerance of remaining NA activity P1 P2 P3 o o

26 LPAI H5N1 + oseltamivir: the phenotype Infectious dose (TCID50) Oseltamivir concentration Virus (RT-qPCR) NA activity (substrate) IC50 oseltamivir tolerance of remaining NA activity P1 P2 P3 o o Adapted virus has a defective neuraminidase The remaining NA activity resists oseltamivir better. Neuraminidase genotype? Compensatory mutations for lack of NA activity decrease by mutation as well as inhibition? Makes virus less dependent on NA activity? (HA?) 11/06/

386,08 555,35 398,72 554,82 335,63 785,85 P1 14470 294,7 247,36 318,9 697,5 302,02 439,63 255,81 866,06 P2 18374 328,6

27 LPAI H5N1 +oseltamivir: the genotype o Data: full genome coverage Ref assembly sanger genome clone average coverage virus reads PB2 PB1 PA HA NP NA M NS wildtype ,5 243,35 224,43 515,24 316,21 981, ,26 clone ,87 435,38 386,08 555,35 398,72 554,82 335,63 785,85 P ,7 247,36 318,9 697,5 302,02 439,63 255,81 866,06 P ,6 412,82 352,61 655,38 388,75 699,58 480,1 1171,23 P ,59 481,14 345,95 504,86 592,29 550, , ,08 11/06/

28 Usual mutations oseltamivir resistance are not involved Known mutation Wildtype Clone P1 P2 P3 E 199 V E E E /G (0,1%) E E H 275 Y H H H H H R 293 K R / G (0,1%) / K (0,06%) R / G (0,5%) / K (0,1%) R R R N 295 S N / S (0,06%) N N / S (0,06%) N N 11/06/

29 base frequency base frequency base frequency Sustainably introduced nonsynonymous mutations in cds (low frequency mutations not shown) 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 PB1 128 T S 1 1 0,0476 0,1627 0,0091 wt clone 1 pass 2 pass 3 pass C T G A 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 0,011 0 HA 205 K E 0, wt clone 1 pass 2 pass 3 pass C T G A 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0, ,0064 NA 434 N S 0, wt clone 1 pass 2 pass 3 pass C T G A 11/06/

Compensatory mutation for decreased NA: + charged to - charged AA near

30 HA/NA activity balance o NA Asn434Ser HA Lys205Glu Not in active site. How does this affect NA activity? Compensatory mutation for decreased NA: + charged to - charged AA near receptor binding site: receptor-binding (Kaverin, Matrosovich et al. Virus Res (2) 123) 11/06/

31 Conclusions o NGS: step change in resolution of available sequencing data: pictures the quasispecies population o Application in Molecular epidemiology: HPAI markers present in LPAI precursors Infectiology: Within-host evolution Host-to-host transmission In vitro research: Evolution of viral escape strategies against antiviral compounds, immunological selection pressure, 11/06/

Thanks! www.epi-seq.eu Padova: Isabella Monne, V. Valastro, A. Schivo, A.

Freimanis, N.Knowles Glasgow: Dan Haydon, M. Morelli, R.

32 Thanks! Padova: Isabella Monne, V. Valastro, A. Schivo, A. Milani Glasgow: P. Murcia, J. Hughes Pirbright: Don King, C. Wright, G. Freimanis, N.Knowles Glasgow: Dan Haydon, M. Morelli, R. Orton Brussels: Toon Rosseel, M. Steensels, G. Nguyen, O. Ozhelvaci, B. Lambrecht 11/06/

ANIHWA ERA-Net Animal Health and Welfare

ANIHWA ERA-Net Animal Health and Welfare Final Dissemination Conference Epi-SEQ Steven Van Borm steven.vanborm@coda-cerva.be www.episeq.eu This project has received funding from the European Union s Seventh