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Transcript
A ROAD MAP FOR HCV GENOTYPING:
HOW DO WE GET THERE –
AND WHAT WILL WE FIND?
Scott Bowden
Molecular Microbiology
VIDRL
Adjunct Professor
Dept of Microbiology
Monash University
HCV Genotyping
1. How we get there – evolution of genotypes
2. What do we find – genotyping technology
First Methods of Viral Characterization
were Based on Serotyping & Phenotyping
• Virus characterization has undergone
continual refinement as a consequence of the
development of new technologies
• Initial methods based on phenotyping growth in eggs, cell culture, CPE etc.
• Polyclonal and monoclonal antibodies could
serotype isolates which grew in culture
Serotyping
• 1970s - some classes of viruses could be
identified using specific antibodies capable of
neutralizing their infectivity.
• Serotyping, raised to type-specific antigens, has
been an important tool for classifying viruses
– poliovirus (types 1, 2 and 3)
– hepatitis B virus (adw, adr, ayw and ayr)
– dengue virus (types 1, 2, 3 and 4)
These methods not suitable for all viruses
• The dawn of the molecular biology era allowed
viruses to be classified genotypically
• Initially using cloning & nucleic acid hybridization
technology and more recently with the aid of
amplification technology, such as PCR followed by
DNA sequence analysis
• Next Gen Sequencing not yet widely used
• Requires some form of sequence interrogation
Definition of Viral Genotype
• Genetic sequences within species are subdivided
into one or more groups. These classifications are
commonly termed genotypes or clades
• There can be further subdivisions - subgenotypes
• The degree of variation seen between these
classifications is species specific and no
standardisation in nomenclature exists between
viral species
• An important job for the ICTV!!
Viral Genotypes
• HIV groups subdivided into clades which share around
30% nucleotide identity within the envelope region and
viruses within a clade vary by around 15%.
• HCV genetic diversity is classified on three levels.
– level 1 defines genotypes which differ by >35 % in nucleotide
sequence over the whole 9.5kb genome (Genotype 1-7)
– level 2 divides genotypes into subtypes whose nucleotide
sequence differs by >15% (subtypes 1a, b, c, etc)
– level 3 defines isolates, within a subtype, which differ by <13%.
Hepatitis C Virus Genotypes:
World View
1a,1b,2b,3a
1a,3a
1b,1a,2b,3a
4,1a,1b
4 4
4
1,2
4
1b,1a,3a
5a,1b,1a,2
3a
2a
1b
1b,2a,1a
1b,6a
6,6c-6l,1b,2
3a,6a-6b
3k,6g
3a,1a,1b,2
1a,1b,3a
HCV Divergence
• Adaptive change – selection pressure
– Immunoselection
– Antiviral therapy
> Change of phenotype
• Neutral sequence drift
– Changes which have little or no effect on fitness
– Accumulate and become fixed in the viral population
– In geographically distinct regions, become
genetically diverse
– > GENOTYPES
> Little or no change of phenotype
Simmonds (2004) J Gen Virol 85:3173
Interferon Therapy for HCV
HCV Can Modify the Innate Immune System
Evolution of HCV Genotypes
• Hypothesis:
Immune selection has been a driving force in
divergence of HCV genotypes
Viral factors inhibiting the immune response
have played a role in the genotype-specific
response to interferon
Genotype 2 older than genotype 3 > 4 > 1
Pang et al 2009 PLoS ONE 4: e6579
Will Genotype be Important in the
Era of Direct-Acting Antivirals?
Shinazi et al Liver Int 2014
Positive recommendation
- ledipasvir/sofosbuvir
- sofosbuvir
- daclatasvir
- genotype 1-6
- all patients (no comment about F stage)
- general section rather than S100
- peginterferon containing treatment is not cost-effective
Resistance Associated Variants to
DAAs
1. HCV exists as a swarm of related variants
2. HCV with amino acid substitutions known to
confer resistance pre-exist even in the
absence of drug exposure
3. Naturally occurring polymorphisms may
encode resistance
4. Minor variants can also be selected and
amplified by antiviral therapy
Selection of a minor quasispecies
Amplification by selection pressure
Mode of Action of New HCV DAAs
1. NS3/4 Protease inhibitors
2. NS5A inhibitors
3. NS5B non-nucleoside inhibitors
4. NS5B nucleoside inhibitors
1. NS3/4A Protease
– Essential for Initiating Replication
Cleavage
P7
NS2
NS3/4A
Protease NS4B
E2
NS5A
E1
C
Kwong A, et al. Beyond interferon and ribavirin: Antiviral therapies for hepatitis C virus.
Drug Discovery Today: Therapeutic Strategies. 2006;3:211-220.
NS5B
Protease Inhibitors Prevent Cleavage – Bind to
the Catalytic Site and Prevent Replication
Protease inhibitor
binds to protease
Downstream
cleavage
is halted
Protease
Kwong A, et al. Beyond interferon and ribavirin: Antiviral therapies for hepatitis C virus.
Drug Discovery Today: Therapeutic Strategies. 2006;3:211-220.
Protease Inhibitor Resistance
• Binding site includes R155 (arginine at amino acid
position 155 of the protease), A156 & D168
• Thus changes at these positions influence efficacy
• Some genotype differences – R155 with HCV 1a
(R155K = nt AGG to AAG - for 1b = nt CGG to AAG);
A156 with HCV 1b
• Simeprevir efficacy reduced by Q80K, a naturally
occurring polymorphism (HCV genotype 1a)
– In Australia, HCV 1a makes up approx 35%
– Q80K frequency is 5-10%
HCV Genome and Targets for DAAs
Shinazi et al Liver Int 2014
2. HCV NS5A Inhibitors
Daclatisvir (BMS); Ledipasvir (Gilead); Ombitasvir (AbbVie)
• NS5A is a phosphoprotein involved in replication
and host-cell interactions
• Mechanism of inhibition unclear (SVR 1b> 1a)
• Resistance associated variants map to domain I
(of three domains) of the protein – L31 and Y93
• These changes cause cross-resistance among
other NS5A inhibitors
• Naturally occurring polymorphisms associated
with resistance occur in HCV genotype 1b
Genotype/Subtype will Remain
Important in the Era of Current DAAs
Progress of an Online Genotypic Drug
Resistance Testing and Monitoring Program
for Chronic Hepatitis C
Lilly Yuen 1, Francesco Moriconi 2, Michael Beard 3, Scott Bowden 1 and Stephen Locarnini 1
1
2
3
VIDRL, North Melbourne, VIC 3051;
Hepatology Unit, University Hospital of Pisa, Italy;
IMVS & The University of Adelaide, Adelaide, SA 5000.
Aim
To develop a platform (SeqHepC) for detecting and measuring antiviral
drug resistance to DAA compounds in patients
Main Goals
• Aid in individualized patient management
• Identification of risk factors associated with drug resistance
• Identification of novel clinically significant drug associated resistance
amino acid substitutions that may emerge in chronic patients on DAA
therapies
Sequence Analysis
Unrooted tree of 228 Full length, Unique, Confirmed and Naïve HCV genomes
4a
5a
1b
6
1c
100
100
100
100
Similar results were obtained from
unrooted trees of the HCV regions
coding for the DAAs targets:
100
100
1a
100
6a
100
100
100
•NS3
1866nt
•NS5A
1530nt
•NS5B
1782nt
100
3
100
100
3a
100
100
7a
2a
Final Reference Set (n = 32)
Input Sequence Name
NC004102_H77 (1a)
EF407429_7043 (1a)
EF407417_4040 (1a)
AJ238799_Con1 (1b)
D50483_HCVK1S (1b)
EU781828_TN28 (1b)
AY051292 (1c)
AF177036_pJ6CF (2a)
AB047639_JFH1 (2a)
AY232742_MD2b71 (2b)
AB559564 (2b)
D50409_BEBE1 (2c)
DQ155561_D54 (2i)
AB031663_VAT96 (2k)
D28917_K3A (3a)
GQ275355 (3a)
D49374_HCVTr (3b)
D63821_JK049 (3k)
DQ516084_25 (4a)
DQ988075_Eg4 (4a)
AF064490_SA1 (5a)
DQ480522_6a72 (6a)
EU246930_D9 (6a)
D84262 (6b)
D84263 (6d)
c0046_HCV (6f)
HK6554_HCV (6g)
D84265 (6h)
KM41_HCV (6k)
EU408330_DH012 (6u)
EU798761_KM046 (6v)
EF108306_QC69 (7a)
Length
9646
9086
9383
9605
9410
9359
9441
9711
9678
9406
9511
9513
9370
9488
9454
9442
9444
9450
9309
9057
9386
9358
9376
9628
9615
9454
9462
9621
9430
9451
9429
9357
HCV full length sequences
Report Screen
HCV Genotyping
1. How we get there – evolution of genotypes
2. What do we find – genotyping technology
Methods to Determine Genotype
•
•
•
•
•
Direct sequencing post PCR - 5’ UTR, core, NS5
RFLP post-PCR
Type-specific PCR primers
Genotype-specific antibodies (NS4)
Real-time PCR and melting curve analysis
• AUSTRALIA (TGA)
• Hybridization assays (LiPA – 5’ UTR, Core)
• Abbott RealTime HCV Genotype II
Line Probe Assay (LiPA) – Innogenetics
> Bayer > Siemens Versant LiPA 2.0
Requires initial
amplification by PCR amplicon tested by LiPA
Drawbacks - LiPA 1.0
• Relied on 5’ UTR for determination of genotype
• Some banding patterns not compatible with the
Interpretation Chart
• Could not discriminate many HCV genotype 6 from 1
• Inability to clearly subtype some HCV genotype 1
Unusual Band Interpretation Chart
www.VIDRL.org.au > Laboratories > Hepatitis Reference
Courtesy JM Pawlotsky
Advantages - LiPA 1 versus LiPA 2
• LiPA 2 distinguishes HCV genotype 6 c-l from genotype 1b
• Also discriminates HCV 1a from HCV 1a/1b (and HCV 1)
Abbott RealTime HCV Genotype II
• 5’ UTR for Genotyping – NS5B for HCV 1 Subtyping
Drawbacks
• Some reports of cross-reactivity. True?
• Despite NS5B primers, some subtyping of HCV
genotype 1is unsuccessful
• Difficulties distinguishing HCV genotype 6
Prevalence and Genotype Distribution in Asia Pacific – Select
Countries
Korea
3
China
2
1b
2a
1b
Japan
2b 3a 1a
2a
Pakistan
4 1
1b
Taiwan
2
2
3
2010 Prevalence
Rate
< 1%
1b
India
3b 4 1a 1b
2
3a
2b
Thailand
1a
3b 6
1b
1 - 1.9%
2 - 2.9%
> 3%
Vietnam
Australia
4 6
6
1
3a
2a
2c
3
1
2a
2
Not Studied
Source: Kromite – Center for Disease Analysis, International Conquer C Coalition (I-C3)
39
Abbott RealTime HCV Genotype II
versus
VERSANT LiPA 2.0
Method
Abbott RealTime HVC GT
II
Versant HCV Genotyping
2.0 assay (LiPA)
Genotyping technology
Real-time PCR
Line Probe Assay: End Point
PCR followed by reverse
hybridization
Total turnaround time
- 5 hours for 24 samples
- 10 hours for 20 samples
Result interpretation
Genotyping result generated Subjective interpretation of
automatically by the software
results
Sample identification
Samples barcoded for
traceability
Manual labelling of reactions
and strips
Internal control in sample to Internal controls of samples Internal controls only for PCR
monitor extraction efficiency from extraction to PCR, and steps; Control bands on all
and possible inhibition
external controls to monitor strips, on external controls.
the run.
HCV subtypes
1a, 1b
Most subtypes
LL of D
500 IU/ml
∼1,000 IU/ml
Liu et al JCM 2015 53: 1754
Abbott HCV Genotype Plus (RUO)
• Designed for detection of HCV 1a,1b & 6 only
• Can be used as a reflex assay after obtaining
results from HCV Genotype II assay (same RNA)
• Or used directly for detection of HCV 1a,1b & 6
HCV Genotype Assay Comparison – 1
Routine Genotype Samples
SAMPLE ID
VERSANT-LiPA 2.0
Abbott RealTime II
15549869
1a
1a
15513934
1a
1a
15549832
1a
1a
15545272
1a
1a
15548755
1a & 3a
1a Reactivity with 3
15554931
1b
1b
15549558
2b
2
15548113
3a
3
15524732
3b
3
15544270
3a
3
15546297
3a
3
15552064
4
4
High concordance between assays
HCV GT Plus RUO
1a; 1b
HCV Genotype Assay Comparison – 2
LiPA Problematic Samples
SAMPLE ID
VERSANT-LiPA 2.0
CORE SEQUENCING
Abbott RealTime II
HCV GT Plus RUO
15513206
346N
6f
1
6
15546665
34N
no product
1a
15546667
band 24
no product
1a
15547928
345A
1a
1a
15548329
band 25
1a
1a
15548332
345N
1a
1a
15508997
346A
1a
1a
15517954
345A
1a
1a
15550297
345A
1a
1a
15550456
band 24
unsuccessful
1a
15553443
3 4 (5) 6 N
to be done
1a
15501602
no band
1a
1b
15555305
no band
to be done
1b
15531224
band 9
2a
2
15544256
no band
no product
3
15545261
band 24
3a
3
The Abbott HCV Genotype II can resolve LiPA problematic genotyping
HCV Genotype Assay Comparison – 3
HCV Genotype 6 c-l Samples
LAB NO
14541062
14547094
14547624
14548133
14551922
14552770
14553472
14556716
14561666
14571514
14580039
14579962
14582079
14586009
14588162
14588373
14596632
14605594
15502180
15524079
15524727
LIPA
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
6C-L
HCV GT Plus RUO
6
6
6
1a; 6
6
ND
6
6
6
1a;6
6
6
6
6
6
6
6
6
6
6
6
HCV GT Plus can successfully distinguish HCV genotypes 6c-l
Conclusions
• Our data and previous published comparisons
show both LiPA 2.0 and Abbott Gen II have a high
concordance rate
• Assays complement each other for problematic
samples
• Shortcomings of the Abbott Gen II assay are
resolved with the HCV GT Plus (RUO)
• Both LiPA and the Abbott assays offer accurate
genotyping – choice will be based on whichever
method suits workflow of individual laboratories
Acknowledgements
• Lilly Tracy
• Prof Alexander Thompson (pinched a few slides)
• Staff of Molecular Microbiology
• Adam & Jennifer - Abbott