Genotyping near full-length hepatitis C viruses Snoeck Joke Rega - - PowerPoint PPT Presentation

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Genotyping near full-length hepatitis C viruses Snoeck Joke Rega - - PowerPoint PPT Presentation

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Genotyping near full-length hepatitis C viruses Snoeck Joke Rega Institute for Medical Research, KU Leuven, Leuven, Belgium AREVIR Meeting 2010 J. Snoeck April 22th Introduction AREVIR Meeting 2010 J. Snoeck April 22th Hepatitis C

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SLIDE 1

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Genotyping near full-length hepatitis C viruses

Snoeck Joke Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

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SLIDE 2

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Introduction

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SLIDE 3

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Hepatitis C Virus

Anzola et al., Expert Reviews in Molecular Medicine, 2003

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SLIDE 4

AREVIR Meeting 2010

  • J. Snoeck – April 22th

HCV replication cycle

  • (a) binding and internalisation
  • (b) cytoplasmic release and

uncoating

  • (c) translation and polyprotein

processing

  • (d) RNA replication
  • (e) assembly
  • (f) maturation and release

Moradpour et al., Nature Reviews 2007

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SLIDE 5

AREVIR Meeting 2010

  • J. Snoeck – April 22th

HCV drugs

  • (a) entry inhibitor
  • (b)
  • (c) protease inhibitor
  • (d) polymerase inhibitor
  • cyclophilin inhibitor
  • (e) glucosidase inhibitor
  • (f)
  • New interferons, p7 inhibitors,

host factor inhibitors, immunomodulators, …..

Moradpour et al., Nature Reviews 2007

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SLIDE 6

AREVIR Meeting 2010

  • J. Snoeck – April 22th

HCV diversity

  • 6 genotypes, more than 70

subtypes

  • 30-35% between genotypes on
  • nuc. level
  • 20-25% between subtypes on
  • nuc. level
  • In the patient : quasispecies

Irshad, Reviews in Medical Virology, 2008

Selective pressure

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SLIDE 7

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Why perform genotyping ?

>NS5B CTAaACCTCaaAGAAaAACCAAACGTAATACCAACCGCCGCCCACAGGACGTTAAGTTCCCGGGCGGCGGCCAGATCGT TGGTGGAGTTTACTTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAGGAAGACTTCCGAGCGGTCGC AACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGGCCCGAGGGCAGGACCTGGGCTCAGCCCGGGTACCCT TGGCCCCTCTATGGCAACGAGGGCATGGGGTGGGCAGGATGGCTCCTGTCACCCCGCGGCTCCCGGCCTAATTGGGG CCCCACAGACCCCCGGCGTAGGTCGCGTAACTTGGGTAAGGTCATCGATACCCTCACATGCGGCTTCGCCGATCTCAT GGGGTACGTTCCGCTCGTCGGTGCCCCCCTAGGGGGCGTTGCCAGGGCCCTGGCGCATGGCGTCCGGACTCTGGAG GACGGCGTGAACTATGCAACAGGGAACTTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTGGCTTTGCTGTCCTGTTTGAC CATCCCAGCTTCCGCTTATGAAGTGCGCAACGTGTCCGGGGTGTACCATGTCACGAACGACTGCTCCAACTCAAGCATT GTGTATGAGGCAGCGGACATGATCATGCACACCCCCGGGTGCGTGCCCTGCGTTCGGGAAGCCAACTCCTCCCGCTG CTGGGTAGCGCTCACCCCTACGCTTGCGGCCAGGAACGCTAGTGTCCCCACCGTGACAATACGACGCCATGTCGATTT

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SLIDE 8

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Method

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SLIDE 9

AREVIR Meeting 2010

  • J. Snoeck – April 22th

General method (1)

RNA extraction (Qiagen viral RNA extraction) cDNA synthesis (Expand RT from Roche) PCR amplification (Expand High Fidelity from Roche) Capillary sequencing

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SLIDE 10

AREVIR Meeting 2010

  • J. Snoeck – April 22th

General method (2)

5’UTR-NS2 inner PCR E2-NS5A inner PCR NS4B-NS5B inner PCR 5’UTR-NS2 outer PCR E2-NS5A outer PCR NS4B-NS5B outer PCR cDNA synthesis from 3’UTR cDNA synthesis from NS5A cDNA synthesis from NS2

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SLIDE 11

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Results

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SLIDE 12

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Validation of the assay (1)

Partial PCR HCV1a HCV1b 1st (5'UTR-NS2) 935 (170-1700) 1700 (170-1700) 2nd (E2-NS5A) 1700 (170-17000) 1700 (1700-17000) 3rd (NS4B-NS5B) 1700 (170-170000) 17000 (1700-170000)

Mean detection limit based on a dilution series of 10 samples Reproducibility based 50 (1a) or 35 (1b) samples tested in triplicate

# postitive PCRs3 1st (5'UTR-NS2) 2nd (E2-NS5A) 3rd (NS4B-NS5B) 1st (5'UTR-NS2) 2nd (E2-NS5A) 3rd (NS4B-NS5B) 0 positive

0 (0%) 0 (0%) 2 (6%) 0 (0%) 2 (4%) 2 (4%)

1 positive

0 (0%) 1 (3%) 2 (6%) 2 (4%) 5 (10%) 7 (14%)

2 positive

1 (3%) 1 (3%) 3 (8%) 17 (34%) 23 (46%) 17 (34%)

3 positive

34 (97%) 33 (94%) 28 (80%) 31 (62%) 20 (40%) 24 (48%)

HCV1a1 HCV1b2

Performance of the sequencing primers :

  • HCV1a succes rate between 70-100%
  • HCV1b succes rate between 80-100%
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SLIDE 13

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Validation of the assay (2)

G e n

  • t

y p e 1 c Genotype 4 G e n

  • t

y p e 5 G e n

  • t

y p e 6 G e n

  • t

y p e 3 G e n

  • t

y p e 2 0.05

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SLIDE 14

Data interpretation

AREVIR Meeting 2010

  • J. Snoeck – April 22th
  • Genotyping data allows us to

– Study appearance of drug resistance-related variants under drug selective pressure – Study evolution of the virus under drug/immune/… selective pressure – Study epidemiology of the virus – Study the mode of action of new anti-HCV drugs – …

  • Full genome information allows us to :

– Study the mode of action of new anti-HCV drugs – Study mutations in different genes at the same time – …

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SLIDE 15

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Discussion

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SLIDE 16

Is full genome data needed for drug resistance screening ?

AREVIR Meeting 2010

  • J. Snoeck – April 22th
  • Is there a clear relationship between the protein targetted

by the administrated drug and the appearance of mutations in the gene coding for the targetted protein ?

– On first sight : YES – Upon closer examination : not necessarily

Protease Active site: binding site for inhibitor and natural substrate Inhibitor cannot bind  resistant protease Substrate can still bind  reduced function

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SLIDE 17

Interferon - ribavirin

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Fukuhara et al., Journal of Hepatology 2010 Yi et al., Virus Research 2009 Taylor et al., Science 1999

Core and NS5A NS4B E2

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SLIDE 18

Protease inhibitors

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Moradpour et al., Nature Reviews 2007 Sarazin and Zeuzem, Gastroenterology 2010

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SLIDE 19

Polymerase inhibitors

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Sarazin and Zeuzem, Gastroenterology 2010 Moriishi et al., Reviews in Medical Virology 2007

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SLIDE 20

Cyclophilin inhibitors

AREVIR Meeting 2010

  • J. Snoeck – April 22th

In vitro resistance-related mutations

Puyang et al., Antimicrobial Agents and chemotherapy 2010

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SLIDE 21

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Is full genome feasible in a clinical setting ?

  • The assay as it is, is quite expensive

– about 100 euro material to amplify the 3 parts of the genome – on average (depending on the genotype) 40 sequencing primers, 5 euro per reaction = 200 euro for sequencing (by a commercial company) 300 euro per patient sample without the personnel cost

  • Personnel cost

– Amplifying the genome does not take that much time, but the analysis of the sequencing results is quite labour-intensive !

 Best seems to stick to what we know at the moment

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SLIDE 22

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Conclusions and future perspectives

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SLIDE 23

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Conclusion (1)

  • We describe a near-full length genotypic assay for HCV genotypes

1a and 1b, based on PCR amplification and capillary sequencing.

  • The sensitivity for both the assays is good for the 5’UTR-NS2 and

NS2-NS5A region, 1700 viral copies/ml or lower. The detection limit for the NS4B-3’UTR region for both assays was less. Such a sensitivity is sufficient to investigate the in vivo mode of action of new anti- HCV drugs, and the method can be used to monitor HCV therapy response.

  • The sets of sequencing primers that we designed are able to

sequence the near-full length genome of HCV1a and 1b strains in both directions. This enables us to reliably detect quasispecies and use the assay for determining genotypic drug resistance in patient strains.

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SLIDE 24

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Conclusion (2)

  • Routine full genome sequencing does not seem feasible at

the moment, it is a good idea though for research questions  determine the gene(s) that needs to be sequenced to study resistance towards a particular drug

  • For the moment, stick to what is known : clear relationships

between the appearance of mutations and the susceptibility of the virus to the antiviral, for example

– Polymerase inhibitors  NS5B – Protease inhibitors  NS3

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SLIDE 25

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Future perspectives

  • Expand the assay to other genotypes, using the same

strategy

  • Upscale the assay to a more high throughput assay
  • Reduce the time of the assay (faster enzymes, methods,

…)

  • Explore the use of next-generation sequencing

– Go deeper than 25% detection of quasispecies – Easier analysis of sequencing data if there is adequate software available – Faster : pool samples using barcoding

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SLIDE 26

AREVIR Meeting 2010

  • J. Snoeck – April 22th

Acknowledgments

  • Prof. Anne-Mieke Vandamme and Lien Kerremans from

the Rega Institute for Medical Research, KULeuven, Belgium

Rega Institute for Medical Research