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TRANSCRIPT
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Vall dHebron Institut de Recerca (VHIR)Hospital Universitari Vall dHebron (HUVH)
Institut dInvestigaci Sanitria del Instituto de Salud Carlos III (ISCIII)
Lab. Malalties Hptiques 006. VHIR-HUVH
Ciber Enfermedades Hepticas y Digestivas (Ciberehd) del Instituto de Salud Carlos III
Universitat Autnoma de Barcelona
Josep Quer PhD
josep .q uer@v hir.org
Uso de la Secuenciacin masiva para el estudio del Virus
de la Hepatitis C
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Viruses that infect the Liver (Hepatotropic)
-Epstein Barr virus (Herpesviridae) dsDNA 185kb-Citomegalovirus (Herpesviridae) dsDNA 229kb-Yellow fever virus (Flaviviridae) ssRNA 11kb-Varicela Zoster (Herpesviridae) dsDNA 125kb-Herpes Simplex (Herpesviridae) dsDNA 152kb-Coxsackie virus (Picornaviridae) ssRNA+ 7.5kb-Lassa fever (Arenaviridae) ssRNA bisegm (7.2kb and 3.5kb).
-Marburg (Filovirus) ssRNA- 19.1kb-Ebola (Filovirus) ssRNA- 18.9kb-Rift valley fever (Bunyaviridae) ssRNA- trisegmented-Measles (Paramyxovirus) ssRNA- 16kb
-HEPATITIS A (HAV)-HEPATITIS B (HBV)-HEPATITIS C (HCV)-HEPATITIS delta D (HDV)-HEPATITIS E (HEV)
Viruses that primarily infect the liver causinga clinically relevant and widely extended
lesion:
VIRAL HEPATITIS
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H V
HBV
HCV
HDV
37bp
5 3
27bp68bp
1 7194ORF3(328)
ORF1(5079)ORF2(1980)
CAPSIDAPEPTIDO SEALMT Proteasa Helicasa Replicasa
NO ESTRUCTURAL ESTRUCTURAL
ESTRUCTURAL
p73NCR5NCR
ESTRUCTURAL NO ESTRUCTURAL
C E1 E2 2 3 4B 5A 5B4Ap7
NO ESTRUCTURALVP2 VP3VP45NCR
VpgVP1 2A 2B 2C 3Cpro 3Dpol3A 3B
3NCR
AAAA
HEV
GENOMES. High level of variation in natural isolates.
Cellular RNA polimerase II
Polymerase with Reverse
Transcriptase activity
HCV RNA DependentRNA Polymerase
HIV Reverse Transcriptase
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6Simmonds P & Smith D. Chapter 43: Evolution of hepatitis viruses pp575-586 in Viral Hepatitis. 4th ed.
Ed.Thomas, Lok, Locarnini & Zuckerman. John Wiley & sons. Oxford 2014.
Simmonds et al. Hepatology 2005
HIV
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High level of replication: HBV 1011// HCV 1012// HIV 1.03x1010particles/day
LACK OF PROOFREADING MECHANISMS.
Rate of accumulation (fixation) of mutations (rate of evolution) = substitutions/base/year
HAV: 1x10-3 -10-4
HBV: 1.4-7.9 105
HCV: 1.5x10-3
HDV: 3.05.0x10-3
HEV: 1.401.72x10-3
Mutation rate =substitution/base/replication cycle
HIV 3.4 x 10-5
~106-fold higher
than for cellu lar
DNA
DNA VIRUS
10-7to 10-9
Human Herpesvirus (HHV): 1,58x10-8
Mean: 0,003 mutations per genome (Dr ake&Hwang Genetics2005:170:969-970)
2.-VARIABILITY
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CONSENSUS
MUTANTESPECTRA
**
* *
* **
* **
*
*
* **
* *
**
*
*
*
*
QUASIESPECIES
Holland JJ et al. Science 1982; 215(4540):1577-1585Domingo E & Holland JJ. Evolutionary biology of viruses. 1994Martell et al. J.Virol. 1992; 66(5):3225-3229Vignuzzi Nature 2006; 439:344-348Vignuzzi M, et al. Nature 2005.
QUASIESPECIESSTRUCTURE
Any RNA viral population is composed of a
complex mixture of different but closelyrelated genomes, known as QUASISPECIES.
Quasispecies composition is subjected tocontinuous change following Principles ofDarwinian evolution
Reproduction with genetic variationCompetitionSelectionInteraction and Cooperation
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HCV prevalence (%) in General Population
< 0.5%
0.5-1%
1.1-1.5%
1.6-2.5%
2.6-3.5%
3.6-5%
>5%
14,9%
Chronically infected patients
2,3% general population
160 millions
HCV
Quer J& Esteban JI. In ViralHepatitis 4th edition. 2013.
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2.5%
1.3%
2%
3%
5%
1%
1%
2%
2%2%
1.5%
1%1%
2%
3%
0.2%
0.6%
2%3%
0.8%
0.2%
0.2%
0.5%
0.2%
3%
1%
0.1%
1%
1%
1.3%
1.5%
2.5%
2.5%
0.2%
< 0.5%
0.5-1%
1.1-1.5%
1.6-2.5%
2.6-3.5%
> 3.6%
Ucrania 4%
3%
JI.Esteban, S.Sauleda, J.Quer J.Hepatol.2008;48:148-162
PREVALENCE OF HCV CHRONIC INFECTION IN EUROPE
HVC GENOTYPES AND SUBTYPES
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Smith D, Bukh J, et al. Expanded classification of HCV 7genotypes and 67 subtypes, updated criteria and assignment web
source.Hepatology 2013 Oct 1. doi: 10.1002/hep.26744.
HVC GENOTYPES AND SUBTYPES
7a
NS5B *Complete genome
*Adaptation H.T. Hraber et al.Virology Journal 2006;3:103-
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World DISTRIBUTION of HCV Genotypes
http://hcv.lanl.gov/components/sequence/HCV/geo/geo.comp
5
6
4
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Hraber HT et al Virology Journal 2006; 3:103Zein NN ClinMicrobRev 2000;13:223-235 (review)Simmonds et al. Hepatology. 2005
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HCV
NATURAL HISTORY
Acute
infection
Chronic
infection
Compensated
cirrosis
Decompensated
cirrosis
Hepatocarcinoma
Hemorrhagic
varices
Hepatic
encephalopathy
AscitisCuration
Antiviral treatment Death
Liver
Transplant
6 weeks 1-2 years to 10-20 years 20-30 years
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Follow-up patients / year assocated to Liver damage % Patients(estimation)
Moderate Liver Damage (F0-F3) 308# 70
Compensated Cirrhosis 1.831* 15
Decompensated Cirrhosis 21.688* 10
Hepatocelular Carcinoma 34.595* 3
Liver Transplant (Mean value) 104.542# 2
Postransplant Follow-up 29.911**adaptation in Razavi et al Hepatol. 2013; 57(6):2164-2170. doi: 10.1002/hep.26218
# data from Diari Oficial Generalitat (DOG 6326 - 1.3.2013) CVE-DOGC-A-13051031-2013. RESOL.
SLT/353/2013
SOCIAL & ECONOMICAL COST. NOT TO TREAT.
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SVR=Sustained Virologic Response
HCV-RNA neg. 24weeks after stopping Treatment.
THERAPY Non-cirrhotic Patients
SVR G1 G2,3,5,6 G4
pegIFN+RBV (24weeks) 38% 75-80% 38%
pegIFN+RBV (48weeks)42% 70%
TREATMENT of CHRONIC HCV
STANDARD-OF-CARE pegIFN alfa 2a/2b + RIBAVIRINA
ANTI HCV TREATMENT What next?
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SOC
pIFN + RBV
(~40% SVR in G1)
SOC 1
pIFN + RBV + DAA
(30% increase SVR in G1)
2011 2011-2013
ANTI-HCV TREATMENT. What next?
2011-2012. Federal Drug Administration (FDA) & European Medicines Agency (EMA) approved two NS3
protease inhibitors (to be used in Triple Therapy with pIFN+RBV):
Telaprevir(VX-950). (INCIVO. Tibotec. Janssen. J&J /// INCIVEK. VertexN.America-). EMA Sep 20, 2011
Boceprevir(SCH503034). (VICTRELIS. Merck Sharp & Dohme). 2012.
TRIPLE THERAPY G1 PATIENTS (Naive v Treatment Experienced patients (TE)
SVR NAIVE Relapsers Partial Responders Null-Responders
pegIFN+RBV 38% 29% 7% 5%
pegIFN+RBV+BOC 63-66% 75% 52% 40%
pegIFN+RBV+TVR 72-75% 83-88% 54-59% 29-33%
pegIF+RBV+BOC:Bacon BR. et al. N Engl J Med 2011; 364:1207-1217. // Poordad F et al. N Engl J Med 2011: 364: 1195-1206 // Bronowicki JP. etal. J Hepatol 2012: 56: S6
pegIFN+RBV+TVR:Sherman KE et al. N Engl J Med 2011; 365: 1014-1024. // Jacobson IM et al. N Engl J Med 2011; 364 : 2405-16. // Zeuzem S. etal. N Engl J Med 2011;364:241728
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CUPIC TRIALPATIENTS Liver fibrosis stage F3/F4.SVR to pegIFN+Rbv: 17-26%
56%
Summary from EASL 2013 for Hepatitis C - New HCV DAAs on their way soon: what do the phase III studies tell us?
ITT = intent to treat
ANTI-HCV TREATMENT What next?
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SOC
pIFN + RBV
(~40% SVR in G1)
SOC 1
pIFN + RBV + DAA
(Goal: 65%-80% SVR in G1)
Future SOC 2
Improved pIFN(?) + RBV(?) + DAA(Goal: 80% SVR in G1)
DAA(s) + RBV only
(Spare pIFN)
DAA Combination
Spare IFN and RBV
pIFN + RBV + DAAs
(Quad therapy)
2011 2014 & Next2011-2013
pIFN + DAAs
(Spare RBV)
ANTI HCV TREATMENT. What next?
COMBINATION of DAAsIFN-free therapies
NEW oral DAAs approval to be used in triple therapywith pegIFN/Rbv:
Simeprevir- OLYSIOby Janssen(NS3 2nd gen. DAAmacrocyclic) to treat Genotype 1.
Approved by FDA Nov. 23rd 2013Approved by EMA 2014
Sofosbuvir-Sovalsiby Gilead. (NS5B inhibitor Ni) pan-genotyping activity to treat Genotypes 1, 2, 3 and 4Approved by FDA Dec. 6th 2013Approved by EMA January 25th 2014
AASLD. The Liver Meeting 2013. Nov. Washington USA.
SOCIAL COST. TREATMENT.
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20Jacobson I, et al. AASLD 2010, Abstract 211
% of patients with
Telaprevir 12 wks +
pegIFN/RBV
N=363
Telaprevir 8 wks +
pegIFN/RBV
N=364
PegIFN/RBV
(control)
N=361
Any adverse event* 99 99 98
Fatigue 57 56 57
Pruritis 50 45 36
Headache 41 43 39
Nausea 43 40 31
Rash 37 35 24
Anemia 37 39 19
Insomnia 32 32 31
Diarrhoea 28 32 22
Influenza-like illness 28 29 28
Pyrexia 26 30 24
ADVERSE EVENTS
ECONOMICAL COST. TREATMENTS.
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Treatment COST. CATSalut.
DRUGTELAPREVIR (T) BOCEPREVIR (B)Unit price (Lab sales price) 52,60 9Dosage 750 mg/8h 800 mg/8hCost per day 315,6(6 tablets/day) 108(12 tablets/day)
Cost complet treatment
12weeks T + PR24 weeks 31145 (26510 (T) + 4635 (PR24)) ...
12w T + PR48 w 35781(26510(T) + 9271(PR48)) ...24w B + PR28 w ... 23551(18144 (B) + 5407(PR28))32w B + PR48 w ... 33463(24192 (B) + 9271(PR48))44w B + PR48 w (cirrhosis) ... 42535(33264 (B) + 9271(PR48))
PR=pegIFN+Ribavirin
Estimated COST.
Therapy SOF 12w (G1 & G2) 60.000
Therapy SOF 24w (G3) 120.000
Triple therapy pegIFN+RBV+SMV 60.000
COST TREATMENT + FOLLOW-
UP / PATIENT / YEAR
pegIFN+RBV 9.371,88
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Response factorsIFN + RBV + DAA
orDAAs-IFNfree
Treatment regimen
PegIFN exposureRBV exposureDAA exposure
Host factors
Age, gender, race,obesity, ETOH,
Genetic factors (IL28B)
Disease featuresFibrosis, steatosis,
coinfection (HBV, HIV)
Viral factorsGenotype
HCV RNA level
Quasispecies(baseline resistance)
PREDICTORS OF ANTI-HCV RESPONSE
Efectiveness of anti-HCV therapy depens on:
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1.- Quasispecies diversity. Viral load.
py p
ACUTEPHASE
CHRONICPHASE
STANDARD TREATMENTpegIFN alfa + RBV
80-90%
G145% SVR
56% Non response
G2 & 3)
80% SVR
20% Non response
SVR=Sustained virologic response
HCV-RNA neg. 24 weeks after stopping treatment.
40-50%
HCV
Efectiveness of anti-HCV therapy depens on:
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1.- Quasispecies diversity. Viral load.
Efectiveness of anti HCV therapy depens on:
2. Resistance mutations to DAAs
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HCV GENOME.
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1 190
191-383 384-746
747-809
810-1026
1027-1657
1658-1711
1712-1972
1973-24202421-3011
Core
protein
Envelope
glycoprotein 1
Envelope
glycoprotein 2
Putative ionchannel
Autoprotease
Helicase and
Serine protease
NS3 co-
factor
RNA-dependent RNA
polymeraseFormation of
replication
complex
Phosphoprotein.
Regulation
replication
-PREVIR-ASVIR
-BUVIR
RESISTANCE MUTATIONS to NS3.
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NS3 (67kDa): 631 aa 1893 nts
PROTEINASE 189aa (567nts) / HELICASE 442aa (1326nts)
Faldaprevir=FDVSimeprevir=SMV
TVR
BOC
RESISTANCE MUTATIONS to NS3.
Sarrazin C, Hzode C, Zeuzem S & Pawlotsky JM J.Hepatol 2012; S88-S100
RESISTANCE MUTATIONS to NS5A.
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N5A: 1344 nts // 448 aa
1 (6248) to 1344 (7601)NT1 (1973) to 448 (2420)
aa
GS-5885
G1a
G1b
RESISTANCE MUTATIONS to NS5A.
M28 R30 L31M Q54HP58 E62 Y93H
E62D
Q30R-E62D Galmozzi 2012 Hepatol.
Q54H-Y93HGalmozzi 2012 Hepatol.
RESISTANCE MUTATIONS to NS5B.
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1 (7602) to 1776 (9377)NT 1 (2421) to 591 (3011)aa
NUCLEOS(T)IDIC INHIBITORS
NON NUCLEOS(T)IDIC INHIBITORS
Sofosbuvir GS-7977
BMS791325
Deleobuvir
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Efectiveness of anti-HCV therapy depens on:
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1.- Quasispecies diversity. Viral load.
2.- Presence of resistance mutations to DAAs.3.- HCV genotypeis the most important viral factor
predicting IFN treatment responsiveness.
G1 = SVR 38-41%
G2 = SVR 93%
G3 = SVR 79%
G4 = Difficult-to-treat virus. SVR rates similar than G1.
G5 = Easy-to-treat virus. SVR rates similar than G2 & 3 (67% SVR).
G6 = Intermediate level of response between 1 and 2.
Higher SVR Rates With BOC in Pts With HCV
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Genotype 1b vs 1a
BOC RGT
5950
Genotype 1aGenotype 1b
Treatment Naive[1]BOC/PR48 BOC/PR48BOC RGT
Treatment Experienced[2]
1. Poordad F, et al. N Engl J Med. 2011;364:1195-1206.2. Bacon BR, et al. N Engl J Med. 2011;364:1207-1217.
66 63
70
6165
73
0
20
40
60
80
100
SV
R(%)
RGT= response guided-therapy
4. Subtype-dependent efficacy of DAAs
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FDV= FaldaprevirSMV= Simeprevir=TMC435SOF=SofosbuvirDCV=Daclatasvir
PI = Protease inhibitor
Sarrazin et al., Gastroenterology 2010; Sullivan, et al. EASL 2011;Jacobson et al., NEJM 2011; Poordad et al., NEJM 2011; Manns et al.,EASL 2013; Jacobson et al., EASL 2013; Ferenci et al., EASL 2013
pegIFN+RBV+PI (NS3)NS5A NS3 +
NS5B(Ni)NS5B
SVR rateNAIVE patients
TVR BOC SMV FDV DCV Faldaprevir+Deleobuvir
SOF+RBV
G1a 71% 59% 75% 69% 58% 11%84%(naive)
10% (noresponse prio
therapy)G1b 79% 66% 85% 84% 87% 57%
EFFICACYG1b - G1a 8% 7% 10% 15% 29% 46%
G1
TURQUOISE-II study by ABBVIE.Compensated cirrhosis
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Compensated cirrhosis
RITONAVIR [ABT-450](NS3)+
OMBITASVIR [ABT-267](NS5A)
+DASABUVIR [ABT-333](NS5B)+
Rbv
DASABUVIR [ABT-333](NS5B) is not active on Genotype 4 (PEARL-I study Hezode et al)
Phase II ION-1and ION-2 study by GILEAD:
LEDIPASVIR [GS 5885] (NS3)
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LEDIPASVIR [GS-5885] (NS3)+
SOFOSBUVIR [GS7977] (NS5B) pan-genotyping
SVR LDV+SOF:12w= 99%24w= 99%
SVR LDV+SOF+Rbv:No further benefit
Very mild AE.
SVR LDV+SOF:12w= 94
24w= 98%
SVR LDV+SOF+Rbv:No further benefit
COST SOFOSBUVIR 12w= $84000 ($1000/tablet) Addition of LDV will increase the cost.
Egypt 1Mpeople to be treated Cost SOF 12w= 900.
ION-1 n=440 ION-2 n=865
G1a & G1b
PHASE III STUDIES FISSION, POSITRON & VALENCE by GILEAD:
SOFOSBUVIR [GS7977] (NS5B) pan genotyping
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Relapse with RAVmutation to NS5B =S282T is G2b patient
SOFOSBUVIR [GS7977] (NS5B) pan-genotyping+
RIBAVIRIN (Rbv)Approved FDA / EMA
G2 & G3
1 Quasispecies diversity Viral load
Efectiveness of anti-HCV therapy depens on:
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1.- Quasispecies diversity. Viral load.
2.- Presence of resistance mutations to DAAs.
3.- HCV genotype is the most important viral factor
predicting IFN treatment responsiveness.
4.- HCV subtypeis as a key determinant of the efficacy of
direct antiviral therapy.
Efectiveness of anti-HCV therapy depens on:
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1.- Quasispecies diversity. Viral load.
2.- Presence resistance mutations to DAAs.
3.- HCV genotypeis the most important viral factor
predicting IFN treatment responsiveness.
4.- HCV subtypeis as a key determinant of the efficacy ofdirect antiviral therapy.
5.- Infection with more than one subtypeat the same time.
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CLASSIC (VIRUS):
CULTURE:In most cases it is not possible (HCV, HBV, ...). Problems of falsenegatives.
SEROLOGY:Antibodies Detection:
- Cross reactions
- Limited specificity to identify genotypes/types/subtypes.
Antigen detection: In most cases it is inaccessible or has low sensitivity.
MOLECULAR BIOLOGY:
DNA and RNA detection and sequencing, using PCR, is the most sensitive andefficient method.
DIAGNOSTIC TECHNIQUES
AVAILABLE TECHNOLOGY in ROUTINE DIAGNOSTICSLABORATORY.
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Reverse hybridising assay (Versant HCV Genotype 2.0 System (LiPA) (Siemens). Lmit de detecci >5%. Usesimmobilised biotinylated oligonucleotides specific to different genotypes and subtypes. Indentify genotypes 1-
6 and differentiate 1a and 1b subtypes but not others. Study of 5-core.
Direct sequence analysis (Trugene HCV 5NC genotyping kit) (Siemens). Determines the HCV genotype andsubtype by direct analysis of the nucleotide sequence. The accuracy of subtyping is poor because of theexclusive analyses of the 5UTR. 20% erroneous classification (17%1a as 1b and 3% 1b as 1a.Chevaliez PlosONE 2009; 4:e82009).
Real-time PCR technology (RealTime HCV Genotype II assay) (Abbott) . Genotype 1a and 1b were wellclassified, but single genotype 2, 3, 4, and 6 isolates were misclassified at the genotype level. Study of NS5B.
None of these technologies allows:high-throughput and confident subtypingdetection of mixed infections
detection of resistance mutations
or:
indication of recombinant genomes
presence of compensatory / multiple mutations in the same
genome.
Cloningand Sanger Sequencing. Difficult/impossible to be implemented for routine diagnostics lab.
Vall dHebronInstitut de Recerca (VHIR)Hospital Universitari Vall dHebron(HUVH)Inst i tut dInvestigaci Sanitria d el Ins titut o d e Salud Carlos III (ISCIII)
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Inst i tut d InvestigaciSanitria d el Ins titut o d e Salud Carlos III (ISCIII)
454 / GS-Junior
-100.000 reads- 800nts 454 / GS-FLX+
-1.000.000 reads- 800-900nts
High resolution HCV subtyping MANUAL
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High resolution HCV subtyping . MANUAL
BLOOD EXTRACTION (5-10mL). SERUM
High resolution HCV subtyping MANUAL
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SERUM SAMPLE
High resolution HCV subtyping . MANUAL
High resolution HCV subtyping
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High resolution HCV subtyping
RNA EXTRACTION
HCV STUDIES. UDPS
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RT-PCR-NESTED
HCV 3NCR5NCRSTRUCTURAL NO STRUCTURAL
C E1 E2 7 NS2 NS3 NS5A NS5BNS4B4A
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genome 3 NCR5 NCRNS5B (HCV RNA Dependent
RNA Polymerase)
8254 8707
RT-PCR (35 cycles)
Hemi-Nested M13 (30 cycles)
ReNested MID (15 cycles)
M13f M13r
M13f M13r
3 Specific primer + 5Universal M13 forward
3 Specific primer + 5Universal M13 reverse
Primer 1
Primer 1
Primer 3
Primer 2
MID MID
C E1 E2 p7 NS2 NS3 NS5A NS5BNS4B4A
M13fOligo A+ TCAG MID Primer 1 M13f TCAG+Oligo BMIDPrimer 3
Sanger sequencing High-resolution HCVsubtyping (454/GS-Junior)
454nts
428nts
499nts
Oligo A+ TCAG TCAG+Oligo B
339nts
339nts
High resolution HCV subtyping . MANUAL
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AGAROSE GEL PURIFICATION
High resolution HCV subtyping . MANUAL
HIGH-RESOLUTION HCV SUBTYPING 454/GS-Junior
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Original file (GS-Junior software)
RAW DATA includes 150.000 reads (sequences)
HIGH RESOLUTION HCV SUBTYPING
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Approved January 2014: 7 Genotypes and 67 subtypes.
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Smith D, Bukh J, et al. Expanded classification of HCV 7genotypes and 67 subtypes, updated criteria and assignment web
source.Hepatology 2013 Oct 1. doi: 10.1002/hep.26744.
pp y yp yp
References are continuously updated.
If classification changes, sequences can be easly re-subtyped.
G1 (7): a,b,c,e,g,h,lG2 (11): a,b,c,d,e,i,j,k,m,q,rG3 (6): a,b,g,h,i,kG4 (17): a,b,c,d,f,g,k,l,m,n,o,p,q,r,t,v,wG5 (1): aG6 (24): a,b,c,d,e,f,g,h,i,j,k,l,m,n,o,p,q,r,s,t,u,v,w,xaG7 (1): a
HCV genotypes and subtypes. Region Discriminating power.
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While the NS5B region is highlyvariable and discriminates betterbetween subtypes, the 5 core ismore stable and better suited forsampling from patients.
The table besides gives the nearestsubtype to each subtype, both innumber of differences and in % identity.
Pos: 5core 168:469,NS5B 8283:8618
HIGH RESOLUTION HCV SUBTYPING
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HIGH RESOLUTION HCV SUBTYPING
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53G1b
Infection with more than one subtype at the same time.
HIGH RESOLUTION HCV SUBTYPING
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PATIENT CASE:
Infection with more than one subtypeat the same time.
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Infection with more than one subtypeat the same time
HIGH RESOLUTION HCV SUBTYPING
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yp
G1a
Infection with more than one subtypeat the same time.
HIGH RESOLUTION HCV SUBTYPING
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PATIENT CASE:
yp
Infection with more than one subtypeat the same time.
HIGH RESOLUTION HCV SUBTYPING
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58G4d
Infection with more than one subtypeat the same time = Mixed infection.
5HIGH RESOLUTION HCV SUBTYPING
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PATIENT CASE:
Patient 37 is infected by:
G1a (1506 reads -60.53%-)+ G4d (982 reads -39.47%)
COMPARATIVE study
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Sangersequencing
NS5BHigh-resolution HCVsubtyping GS-Junior
NS5B
Versant HCVGenotype 2.0LiPA Siemens
5-core
Real Time HCVGenotype II. Abbott
5-coreNS5B (1a/1b)
Patient code
Direct NS5B
sequencing
Versant HCV Genotype
2.0
Abbott Real time HCV
genotype II.
UDPS HCV Subtyping
454/GS-Junior*P1-P17 1a 1a 1a 1aP18-61 1b 1b 1b 1b
P62 1a 1 1 1aP63 1a 1a 1 1a
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G1 samples(n=82)
P63 1a 1a 1 1aP64 1a 1 1a 1aP65 1a 1a 1 1aP66 1a 1 1 1aP67 1a 1 1 1a
P68 1a 1 1 1aP69 1a 1a 1 1aP70 1b 1 1b 1bP71 1b 1 1 1bP72 1b 1 1b 1bP73 1b 1 1b 1bP74 1b 1 1b 1bP75 1b 1 1 1bP76 1b 1b 1 1b
P77 1b 1b 1b 1b (43%)+3a(35%)+1a (22%)P78 1b 1b 1 1b
P79 1b 1/1a 1b 1b
P80 1b 1 1b 1bP81 1b 1b 1 1bP82 1b 1b 1 1b
Table summary Untyped G1 samplesNumber of patients Versant/Siemens Rt-PCR/Abbott
G1a 25 5 (20%) 7 (28%)G1b 57 8 (14%) 6 (10%)
Total 82 13 (16%) 13 (16%)
16 %of Genotype 1 samples cannot be subtyped by the commercial techniques used in routine labs.
Non-G1 samples
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Patient CodeDirect PCR Sanger
sequencing
Versant HCV Genotype 2.0
LiPA. Siemens
Real time HCV genotype II.
Abbott
High-resolution HCV
subtyping 454/GS-Junior
P-IND-1 2c 2a/c 2 2c
P-IND-2 2c 2a/c 2 2c
P-IND-3 3a 3 3 3a
P-IND-4 3a 3a ND 3a
P-IND-5 3a 3a 3 3a
P-IND-6 3a 3a 3 3a
P-IND-7 4a 4 4 4a
P-IND-8 4a 4a/c/d 4 4a
P IND 9 4d 4 4 4d
Non-G1 samples
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P-IND-9 4d 4 4 4d
P-IND-10 4d 4 4 4d
P-IND-11 4d 4a/c/d 4 4d
P-IND-12 4d 4a/c/d 4 4d
P-IND-13 4f 4f 4 + 5* 4f
P-IND-14 4f 4f 4 + 5* 4f
P-IND-15 4f Ind 4 + 5* 4f
P-IND-16 4f Ind 4 + 5* 4f
P-IND-17 4f Ind 4 + 5* 4f
P-IND-18 4f Ind 4 + 5* 4f
P-IND-19 4f Ind 4 + 5* 4f
P-IND-20 4f Ind 4 + 5* 4f P-IND-21 4f Ind 4 + 5* 4f
P-IND-22 4f Ind 5 4f
P-IND-23 4f Ind 5 4f
P-IND-24 4f Ind 5 4f
P-IND-25 4f Ind 5 4f
P-IND-26 4r Ind 1 + 4 4r
P-IND-27 5a 5a 5 5a
P-IND-28 6c neg 5 6c
* 4 or 5
Mixed InfectionsMixed infections
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P-IND-29 4d Ind 4 4d (72%)+1a (28%)
P-IND-30 2j 2 ND 2j (95%)+4f (5%)
P-IND-31 4p Ind 3 + 4 4p (53%)+3a (47%)P-IND-32 4d Ind 1a + 4 4d (87%) +1a (13%)
2,52 %mixed infections in patients
visited in the Hospitaloutpatient clinics
HCV SUBTYPE PREVALENCE HUVH BARCELONA 2012-13.
24%
56%
9%
7%1% 3%
1a
1b
3a
4d
4f
Other
109 patients analyzed by 4th March 2013
SPECIAL CASE:
HIGH RESOLUTION HCV SUBTYPING
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PATIENT INFECTED with 1b + 3a + 2c + 4d + 1a.
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Patient treated two years ago with TVR: Alignment of the 48 different amplified nucleotide sequencesHere we show 150 from total 443 nts.
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Patient treated two years ago w ith TVR: Al ignment of 48 amino acid sequences
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68V36A = 0.2% Q80R = 0.27%
NS3 (67kDa): 631 aa 1893 nts
PROTEINASE 189aa (567nts) / HELICASE 442aa (1326nts)
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Faldaprevir=FDV
Simeprevir=SMV
TVR
BOC
Sarrazin C, Hzode C, Zeuzem S & Pawlotsky JM J.Hepatol 2012; S88-S100
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Kwong AD, Najera I, et al. Gastroenterology 2011Mar;140(3):755-60. doi:10.1053/j.gastro.2011.01.029
SUMMARY
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SUMMARY
1.- Massive sequencing allows high-resolution studies on viral infections. Variabilitystudies. Detection of minority mutants some of them can be clinically relevant.
2.- HCV subtype is a key determinant of the efficacy of direct antiviral therapy. High-
resolution HCV subtyping based in massive sequencing and phylogeny represents a
maximum precision methodology for HCV subtyping and for quantification of mixedinfections.
3.- High resolution HCV sequencing allows detection of resistance mutations.
Antonio MadejnJavier Garca-Samaniego F i R d F
Ricardo MorenoPaloma Sanz-Cameno
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Javier Garca-Samaniego Francisco Rodrguez-FrasMara Buti
David Garca-Cehic
Rosario CasillasMara BlasiMara Homs
David TaberneroLeo Nieto
Mara CuberoSilvia Sauleda
Marta BesAndrea CaballeroRafael EstebanJaume Guardia
Juan Ignacio Esteban
Sofa Prez del Pulgar
Xavier Forns
Esteban DomingoCelia PeralesJulie Sheldon
Carlos Briones
Manolo Romero-GmezJose Antonio del Campo
Josep GregoriMiguel Alvrez-TejadoJos Manuel MuozJordi Gmez
Javier SalmernPaloma Muoz de Rueda
Rosa Quiles-Prezngela Extremera
Rosa Lpez-Rodrguez
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THANK YOU !
Why genotypes have different sensitivity to pegIFN?
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IL28B (=IFN3),IL28A (= IFN2)i IL29 (=IFN1)
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IL28B(=IFN3)
rs12979860(chr19:39738787)
IL28A(=IFN2)
IL29(=IFN1)
CC
SVR topegIFNbased
treatments
Favorable
rs368234815(chr19:39739154)
-G
IL28B(=IFN3)
rs12979860(chr19:39738787)
IL28A(=IFN2)
IL29(=IFN1)
SVR topegIFNbased
treatments
Unfavorable CT / TT
rs368234815(chr19:39739154)
TT
IFN4
Carlos Fernndez-Carrillo, P.Gonzlez, G.Crespo, M.Coto-Llerena, M.Navasa, X.Forns, Sofia Prez del PulgarXXXIX Congreso AEEH Madrid, 19-21 Febrero 2014
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NS3 V36A/M/L Q41 F43C/S T54A/S V55A Q80R/K R155K/T/Q A156T A156S/V/I V158I D168A/V V170A/T
Telaprevir
Boceprevir
Vaniprevir
ITMN-191
BILN-2061
TMC-435
BI-201335
Low level resistance 100 x
Not done
NS3 resistance mutation (protease-helicase)
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Identification of non-primate hepaciviruses (NPHV)
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A novel hepacivirus identified by high-throughput
sequencing of respiratory samples from dogs
Animals from respiratory disease outbreaks in two sheltersfound positive
The novel virus is the closest known homologue to HCV
Kapoor PNAS 2011
High prevalence of NPHV in horses
Serology identified high prevalence (35%) of NPHV specific
antibodies in horse sera
8 (22%) of immuno-reactive samples were positive for NPHV
by RT-PCR
One equine isolate was nearly identical to the canineviruses!!!
Epidemiology, disease association, transmission?
New hepacivirus isolates in rodents and bats!
Burbelo JVI 2012 Kapoor mBio 2013 Quan PNAS 2013
Import 2012 % pes relatiu/total
Taula 102. Import dels medicaments dispensats a pacients en rgim ambulatori als hospitals de Catalunya
concertats i de lICS, 2012
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Import 2012 % pes relatiu/total
Antiretrovirals 153.286.409 23,6
Citosttics 150.838.716 23,3
Immunosupressors selectius 99.378.048 15,3Altres medicaments d's hospitalari 83.793.753 12,9
Esclerosi mltiple 41.061.364 6,3
Eritropoetina 26.327.776 4,1
Hormona de creixement 18.723.454 2,9
Factors antihemoflics 18.724.676 2,9
Nutrici enteral (NE) per sonda a domicili ms material NE 18.659.082 2,9
Tractaments hepatitis C 15.095.863 2,3Immunoglobines humanes inespecfiques 11.891.718 1,8
Factors d'estimulants de colnies 6.731.840 1
Medicaci estrangera 2.973.335 0,5
Medicaci i solucions per a la nutrici parenteral (NP) intravenosa 656.824 0,1
Medicaments per al tractament de la fibrosi qustica 223.953 0
Total 648.366.813 100
Font: Gerncia dAtenci Farmacutica i Prestacions Complementries.
Pagina 210. Memoria 2012 CatSalut.
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