1
Vall d’Hebron Institut de Recerca (VHIR)
Hospital Universitari Vall d’Hebron (HUVH) Institut d’Investigació Sanitària del Instituto de Salud Carlos III (ISCIII)
Lab. Malalties Hèpàtiques 006. VHIR-HUVH
Ciber Enfermedades Hepáticas y Digestivas (Ciberehd) del Instituto de Salud Carlos III
Universitat Autònoma de Barcelona
Josep Quer PhD
Uso de la Secuenciación masiva para el estudio del Virus
de la Hepatitis C
2
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 causing
a clinically relevant and widely extended
lesion:
VIRAL HEPATITIS
3
Group IV (ssRNA+) VII (dsDNA-RT) IV (ssRNA+) V (ssRNA-) IV (ssRNA+) VI (ssRNA-RT)
Order Picornavirales Unassigned Unassigned Unassigned
Non assigned
Unassigned
Family Picornaviridae Hepadnaviridae Flaviviridae Unassigned
Non assigned
Retroviridae
Genus Hepatovirus Orthohepadna
virus Hepacivirus Deltavirus Hepevirus Lentivirus
Names
HEPATITIS A VIRUS (HAV)
HEPATITIS B VIRUS (HBV)
HEPATITIS C VIRUS (HCV)
HEPATITIS DELTA (HDV)
HEPATITIS E VIRUS (HEV)
HUMAN IMMUNO DEFICIENCY
1 /2 (HIV1/HIV2)
Genome ssRNA+ lineal dsDNA parcial
circular ssRNA+ lineal
ssRNA- circular ssRNA+ lineal
ssRNA+ lineal
Size 7.5kb 3.0-3.3kb 9.5kb 1.7kb 7.17kb 9.8kb
Virion Non enveloped Enveloped
Enveloped
HBV Enveloped Non
enveloped Enveloped
HEPATITIS VIRUS HIV
4
5
HAV
HBV
HCV
HDV
37bp
5’ 3’
27bp 68bp
1 7194 ORF3(328)
ORF1(5079) ORF2(1980) CAPSIDA
PEPTIDO SEÑAL MT Proteasa Helicasa Replicasa
NO ESTRUCTURAL ESTRUCTURAL
ESTRUCTURAL
p7 3‘NCR 5‘NCR
ESTRUCTURAL NO ESTRUCTURAL
C E1 E2 2 3 4B 5A 5B 4 A
p7
NO ESTRUCTURAL VP2 VP3 VP4 5‘NCR
Vpg VP1 2A 2B 2C 3Cpro 3Dpol 3A 3B
3‘NCR
AAAA
HEV
GENOMES. High level of variation in natural isolates.
Cellular RNA polimerase II
Polymerase with Reverse
Transcriptase activity
HCV RNA Dependent
RNA Polymerase
HIV Reverse Transcriptase
6 Simmonds 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
7
High level of replication: HBV 1011 // HCV 1012 // HIV 1.03x1010 particles/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 10−5
HCV: 1.5x10-3
HDV: 3.0–5.0x10-3
HEV: 1.40–1.72x10-3
Mutation rate =substitution/base/replication cycle
HIV 3.4 x 10-5
~106-fold higher than for cellular
DNA
DNA VIRUS
10-7 to 10-9
Human Herpesvirus (HHV): 1,58x10-8
Mean: 0,003 mutations per genome (Drake&Hwang Genetics2005:170:969-970)
2.-VARIABILITY
8
CONSENSUS
MUTANT
ESPECTRA
* *
* *
* * *
* * *
*
*
* * *
* *
* *
*
*
*
*
QUASIESPECIES
Holland JJ et al. Science 1982; 215(4540):1577-1585
Domingo E & Holland JJ. Evolutionary biology of viruses. 1994
Martell et al. J.Virol. 1992; 66(5):3225-3229
Vignuzzi Nature 2006; 439:344-348
Vignuzzi M, et al. Nature 2005.
QUASIESPECIES STRUCTURE
Any RNA viral population is composed of a complex mixture of different but closely related genomes, known as QUASISPECIES.
Quasispecies composition is subjected to continuous change following Principles of Darwinian evolution
Reproduction with genetic variation Competition Selection Interaction and Cooperation
9
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 Viral
Hepatitis 4th edition. 2013.
10
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
11
Smith D, Bukh J, et al. “Expanded classification of HCV 7
genotypes 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-
12
World DISTRIBUTION of HCV Genotypes
http://hcv.lanl.gov/components/sequence/HCV/geo/geo.comp
5
6
4
13
Hraber HT et al Virology Journal 2006; 3:103
Zein NN ClinMicrobRev 2000;13:223-235 (review)
Simmonds et al. Hepatology. 2005
14
HCV
NATURAL HISTORY
Acute infection
Chronic infection
Compensated cirrosis
Decompensated cirrosis
Hepatocarcinoma
Hemorrhagic varices
Hepatic encephalopathy
Ascitis Curation
Antiviral treatment Death
Liver Transplant
6 weeks 1-2 years to 10-20 years 20-30 years
15
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.
16
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
17
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. Vertex –N.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. et al. 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. et al. N Engl J Med 2011;364:2417–28
18
CUPIC TRIAL
PATIENTS 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
19
SOC
pIFN + RBV
(~40% SVR in G1)
SOC 1
pIFN + RBV + DAA
(Goal: 65%-80% SVR in G1)
Future SOC 2
Improved pIFN(λ?) + RBV(?) + DAAs
(Goal: ≥80% SVR in G1)
DAA(s) + RBV only
(Spare pIFN)
DAA Combination
• Spare IFN and RBV
pIFN + RBV + DAAs
(Quad therapy)
2011 2014 & Next 2011-2013
pIFN + DAAs
(Spare RBV)
ANTI-HCV TREATMENT. What next?
COMBINATION of DAAs
IFN-free therapies
NEW oral DAAs approval to be used in triple therapy
with pegIFN/Rbv:
Simeprevir- OLYSIO by Janssen(NS3 2nd gen. DAA
macrocyclic) to treat Genotype 1. Approved by FDA Nov. 23rd 2013
Approved by EMA 2014
Sofosbuvir- Sovalsi by Gilead. (NS5B inhibitor Ni) pan-
genotyping activity to treat Genotypes 1, 2, 3 and 4
Approved by FDA Dec. 6th 2013
Approved by EMA January 25th 2014
AASLD. The Liver Meeting 2013. Nov. Washington USA.
20 •Jacobson 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
SOCIAL COST. TREATMENT.
21
Treatment COST. CATSalut.
DRUG
TELAPREVIR (T) BOCEPREVIR (B) Unit price (Lab sales price) 52,60 € 9 €
Dosage 750 mg/8h 800 mg/8h
Cost 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€
ECONOMICAL COST. TREATMENTS.
COST TREATMENT + FOLLOW-
UP / PATIENT / YEAR
pegIFN+RBV 9.371,88 €
pegIFN+RBV+BOC26.192,04 €
CIRRHOTIC 44.012,04 €
pegIFN+RBV+TPV 31.932,18 €
22
Response factors IFN + RBV + DAA
or DAAs-IFNfree
Treatment regimen PegIFN exposure
RBV exposure DAA exposure
Host factors Age, gender, race, obesity, ETOH,…
Genetic factors (IL28B)
Disease features Fibrosis, steatosis,
coinfection (HBV, HIV)
Viral factors Genotype
HCV RNA level Quasispecies
(baseline resistance)
PREDICTORS OF ANTI-HCV RESPONSE
23
1.- Quasispecies diversity. Viral load.
Efectiveness of anti-HCV therapy depens on:
ACUTE
PHASE
CHRONIC
PHASE
STANDARD TREATMENT
pegIFN alfa + RBV 80-90%
G1
45% 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
24
1.- Quasispecies diversity. Viral load.
Efectiveness of anti-HCV therapy depens on:
25
2. Resistance mutations to DAAs
26
1 190
191-383 384-746
747-809
810-1026
1027-1657
1658-1711
1712-1972
1973-2420 2421-3011
Core
protein
Envelope
glycoprotein 1
Envelope
glycoprotein 2
Putative ion
channel
Autoprotease
Helicase and
Serine protease
NS3 co-
factor
RNA-dependent RNA
polymerase Formation of
replication
complex
Phosphoprotein.
Regulation
replication
HCV GENOME.
-PREVIR -ASVIR
-BUVIR
27
NS3 (67kDa): 631 aa 1893 nts PROTEINASE 189aa (567nts) / HELICASE 442aa (1326nts)
Faldaprevir=FDV Simeprevir=SMV
TVR
BOC
RESISTANCE MUTATIONS to NS3.
Sarrazin C, Hézode C, Zeuzem S & Pawlotsky JM J.Hepatol 2012; S88-S100
28
N5A: 1344 nts // 448 aa
1 (6248) to 1344 (7601) NT
1 (1973) to 448 (2420) aa
GS-5885
G1a
G1b
RESISTANCE MUTATIONS to NS5A.
M28 R30 L31M Q54H P58 E62 Y93H
E62D
Q30R-E62D Galmozzi 2012 Hepatol.
Q54H-Y93H Galmozzi 2012 Hepatol.
29
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
RESISTANCE MUTATIONS to NS5B.
30
1.- Quasispecies diversity. Viral load.
2.- Presence of resistance mutations to DAAs (Direct-acting antivirals).
Resistance to inhibitors:
Treatment-experienced patients (quasispecies memory) (Ruiz-Jarabo et al J.Virol 2000;74:3543; Briones J.Mol.Bio. 2003;331:213.). Ex. HBV,
HIV… (Le T et al. PLos One 2009;4(6)e6079:1-8.)
Untreated patients (basal resistance).
Ex. HCV,… (Cubero et al. Virology 2008;370:237-245)
Efectiveness of anti-HCV therapy depens on:
31
1.- Quasispecies diversity. Viral load.
2.- Presence of resistance mutations to DAAs.
3.- HCV genotype is the most important viral factor predicting IFNα 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.
Efectiveness of anti-HCV therapy depens on:
32
Higher SVR Rates With BOC in Pts With HCV Genotype 1b vs 1a
BOC RGT
59
50
Genotype 1a
Genotype 1b
Treatment Naive[1]
BOC/PR48 BOC/PR48 BOC 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
61 65
73
0
20
40
60
80
100
SV
R (
%)
RGT= response guided-therapy
33
FDV= Faldaprevir
SMV= Simeprevir=TMC435
SOF=Sofosbuvir
DCV=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 rate NAIVE patients
TVR BOC SMV FDV DCV Faldaprevir+
Deleobuvir SOF+RBV
G1a 71% 59% 75% 69% 58% 11%
84% (naive)
10% (no
response prior
therapy) G1b 79% 66% 85% 84% 87% 57%
EFFICACY
G1b - G1a 8% 7% 10% 15% 29% 46%
G1
4. Subtype-dependent efficacy of DAAs
34
TURQUOISE-II study by ABBVIE.
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)
35
Phase II ION-1and ION-2 study by GILEAD:
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
36
Relapse with RAV
mutation to NS5B =
S282T is G2b patient
PHASE III STUDIES FISSION, POSITRON & VALENCE by GILEAD:
SOFOSBUVIR [GS7977] (NS5B) pan-genotyping
+
RIBAVIRIN (Rbv)
Approved FDA / EMA
G2 & G3
37
1.- Quasispecies diversity. Viral load.
2.- Presence of resistance mutations to DAAs.
3.- HCV genotype is the most important viral factor predicting IFNα treatment responsiveness.
4.- HCV subtype is as a key determinant of the efficacy of direct antiviral therapy.
Efectiveness of anti-HCV therapy depens on:
38
1.- Quasispecies diversity. Viral load.
2.- Presence resistance mutations to DAAs.
3.- HCV genotype is the most important viral factor predicting IFNα treatment responsiveness.
4.- HCV subtype is as a key determinant of the efficacy of direct antiviral therapy.
5.- Infection with more than one subtype at the same time.
Efectiveness of anti-HCV therapy depens on:
39
CLASSIC (VIRUS):
CULTURE: In most cases it is not possible (HCV, HBV, ...). Problems of false negatives.
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 and efficient method.
DIAGNOSTIC TECHNIQUES
40
AVAILABLE TECHNOLOGY in ROUTINE DIAGNOSTIC’S LABORATORY.
Reverse hybridising assay (Versant® HCV Genotype 2.0 System (LiPA) (Siemens). Límit de detecció >5%. Uses immobilised 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 5’NC genotyping kit) (Siemens). Determines the HCV genotype and subtype by direct analysis of the nucleotide sequence. The accuracy of subtyping is poor because of the exclusive analyses of the 5’UTR. 20% erroneous classification (17%1a as 1b and 3% 1b as 1a. Chevaliez Plos ONE 2009; 4:e82009).
Real-time PCR technology (RealTime™ HCV Genotype II assay) (Abbott) . Genotype 1a and 1b were well classified, 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 subtyping • detection of mixed infections • detection of resistance mutations • or:
• indication of recombinant genomes • presence of compensatory / multiple mutations in the same
genome.
Cloning and Sanger Sequencing. Difficult/impossible to be implemented for routine diagnostic’s lab.
41
Vall d’Hebron Institut de Recerca (VHIR)
Hospital Universitari Vall d’Hebron (HUVH) Institut d’Investigació Sanitària del Instituto de Salud Carlos III (ISCIII)
454 / GS-Junior
-100.000 reads
- 800nts 454 / GS-FLX+
-1.000.000 reads
- 800-900nts
42
High resolution HCV subtyping . MANUAL
BLOOD EXTRACTION (5-10mL). SERUM
43
SERUM SAMPLE
High resolution HCV subtyping . MANUAL
44
High resolution HCV subtyping
RNA EXTRACTION
45
RT-PCR-NESTED
HCV STUDIES. UDPS
HCV
genome 3‘NCR 5‘NCR
STRUCTURAL NO STRUCTURAL
NS5B (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 + 5’
Universal M13 forward 3’ Specific primer + 5’
Universal M13 reverse
Primer 1
Primer 1
Primer 3
Primer 2
MID MID
C E1 E2 p7 NS2 NS3 NS5A NS5B NS4B 4A
M13f Oligo A+ TCAG MID Primer 1 M13f TCAG+Oligo B MID Primer 3
Sanger sequencing High-resolution HCV
subtyping (454/GS-Junior)
454nts
428nts
499nts
Oligo A+ TCAG TCAG+Oligo B
339nts
339nts
47
AGAROSE GEL PURIFICATION
High resolution HCV subtyping . MANUAL
48
Original file (GS-Junior software)
RAW DATA includes 150.000 reads (sequences)
HIGH-RESOLUTION HCV SUBTYPING 454/GS-Junior
49
HIGH RESOLUTION HCV SUBTYPING
50
Smith D, Bukh J, et al. “Expanded classification of HCV 7
genotypes and 67 subtypes, updated criteria and assignment web
source.” Hepatology 2013 Oct 1. doi: 10.1002/hep.26744.
Approved January 2014: 7 Genotypes and 67 subtypes.
References are continuously updated.
If classification changes, sequences can be easly re-subtyped.
G1 (7): a,b,c,e,g,h,l
G2 (11): a,b,c,d,e,i,j,k,m,q,r
G3 (6): a,b,g,h,i,k
G4 (17): a,b,c,d,f,g,k,l,m,n,o,p,q,r,t,v,w
G5 (1): a
G6 (24): a,b,c,d,e,f,g,h,i,j,k,l,m,n,o,p,q,r,s,t,u,v,w,xa
G7 (1): a
51
While the NS5B region is highly
variable and discriminates better
between subtypes, the 5’ core is
more stable and better suited for
sampling from patients.
The table besides gives the nearest
subtype to each subtype, both in
number of differences and in % identity.
Pos: 5’ core 168:469,
NS5B 8283:8618
HCV genotypes and subtypes. Region Discriminating power.
52
HIGH RESOLUTION HCV SUBTYPING
53 G1b
HIGH RESOLUTION HCV SUBTYPING
54
PATIENT CASE:
Infection with more than one subtype at the same time.
HIGH RESOLUTION HCV SUBTYPING
55
PATIENT CASE:
Infection with more than one subtype at the same time.
HIGH RESOLUTION HCV SUBTYPING
56
Infection with more than one subtype at the same time
G1a
HIGH RESOLUTION HCV SUBTYPING
57
PATIENT CASE:
Infection with more than one subtype at the same time.
HIGH RESOLUTION HCV SUBTYPING
58
Infection with more than one subtype at the same time.
G4d
HIGH RESOLUTION HCV SUBTYPING
59
PATIENT CASE:
Infection with more than one subtype at the same time = Mixed infection.
5
Patient 37 is infected by:
G1a (1506 reads -60.53%-) + G4d (982 reads -39.47%)
HIGH RESOLUTION HCV SUBTYPING
60
COMPARATIVE study
Sanger
sequencing
NS5B
High-resolution HCV
subtyping GS-Junior
NS5B
Versant HCV
Genotype 2.0
LiPA Siemens
5’-core
Real Time HCV
Genotype II. Abbott
5’-core
NS5B (1a/1b)
61
G1 samples
(n=82)
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 1a
P18-61 1b 1b 1b 1b
P62 1a 1 1 1a
P63 1a 1a 1 1a
P64 1a 1 1a 1a
P65 1a 1a 1 1a
P66 1a 1 1 1a
P67 1a 1 1 1a
P68 1a 1 1 1a
P69 1a 1a 1 1a
P70 1b 1 1b 1b
P71 1b 1 1 1b
P72 1b 1 1b 1b
P73 1b 1 1b 1b
P74 1b 1 1b 1b
P75 1b 1 1 1b
P76 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 1b
P81 1b 1b 1 1b
P82 1b 1b 1 1b
Table summary Untyped G1 samples
Number 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.
62
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
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
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%)
ND= Non determinedneg = non PCR amplification because of low sensitivity
* Abbott results 4+5 = Genotype 4 + reactivity with genotype 5
Ind= Indeterminate result. It can be interpretable by the facultative.
P-IND = Patient samples with Indeterminate genotype (by first generation Inno-LiPA)
Non-G1 samples
63
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
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
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%)
ND= Non determinedneg = non PCR amplification because of low sensitivity
* Abbott results 4+5 = Genotype 4 + reactivity with genotype 5
Ind= Indeterminate result. It can be interpretable by the facultative.
P-IND = Patient samples with Indeterminate genotype (by first generation Inno-LiPA)
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
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
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%)
ND= Non determinedneg = non PCR amplification because of low sensitivity
* Abbott results 4+5 = Genotype 4 + reactivity with genotype 5
Ind= Indeterminate result. It can be interpretable by the facultative.
P-IND = Patient samples with Indeterminate genotype (by first generation Inno-LiPA)
Non-G1 samples
* 4 or 5
64
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
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
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%)
ND= Non determinedneg = non PCR amplification because of low sensitivity
* Abbott results 4+5 = Genotype 4 + reactivity with genotype 5
Ind= Indeterminate result. It can be interpretable by the facultative.
P-IND = Patient samples with Indeterminate genotype (by first generation Inno-LiPA)
Mixed Infections
2,52 %
mixed infections in patients
visited in the Hospital
outpatient 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
Mixed infections
65
SPECIAL CASE:
PATIENT INFECTED with 1b + 3a + 2c + 4d + 1a.
HIGH RESOLUTION HCV SUBTYPING
66
RESISTANCE MUTATIONS TO NS3 INHIBITOR TELAPREVIR (TVR). TWO YEARS AFTER STOPPING
TREATMENT.
67
Patient treated two years ago with TVR: Alignment of the 48 different amplified nucleotide sequences
Here we show 150 from total 443 nts.
68 V36A = 0.2% Q80R = 0.27%
Patient treated two years ago with TVR: Alignment of 48 amino acid sequences
69
NS3 (67kDa): 631 aa 1893 nts PROTEINASE 189aa (567nts) / HELICASE 442aa (1326nts)
Faldaprevir=FDV Simeprevir=SMV
TVR
BOC
Sarrazin C, Hézode C, Zeuzem S & Pawlotsky JM J.Hepatol 2012; S88-S100
70
Kwong AD, Najera I, et al. Gastroenterology 2011 Mar;140(3):755-60. doi:
10.1053/j.gastro.2011.01.029
71
SUMMARY
1.- Massive sequencing allows high-resolution studies on viral infections. Variability
studies. 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 mixed
infections.
3.- High resolution HCV sequencing allows detection of resistance mutations.
Antonio Madejón
Javier García-Samaniego Francisco Rodríguez-Frías
María Buti
David García-Cehic
Rosario Casillas
María Blasi
María Homs
David Tabernero
Leo Nieto
María Cubero
Silvia Sauleda
Marta Bes
Andrea Caballero
Rafael Esteban
Jaume Guardia
Juan Ignacio Esteban
Sofía Pérez del Pulgar
Xavier Forns
Esteban Domingo
Celia Perales
Julie Sheldon
Carlos Briones
Manolo Romero-Gómez
Jose Antonio del Campo
Josep Gregori
Miguel Alvárez-Tejado
José Manuel Muñoz Jordi Gómez
Javier Salmerón
Paloma Muñoz de Rueda
Rosa Quiles-Pérez
Ángela Extremera
Ricardo Moreno
Paloma Sanz-Cameno
Rosa López-Rodríguez
73
THANK YOU !
74
Why genotypes have different sensitivity to pegIFN?
75
IL28B
(=IFNλ3)
rs12979860
(chr19:39738787)
IL28A
(=IFNλ2)
IL29
(=IFNλ1)
CC
SVR to
pegIFN
based
treatments
Favorable
rs368234815
(chr19:39739154)
-G
IL28B
(=IFNλ3)
rs12979860
(chr19:39738787)
IL28A
(=IFNλ2)
IL29
(=IFNλ1)
SVR to
pegIFN
based
treatments
Unfavorable CT / TT
rs368234815
(chr19:39739154)
TT
IFNλ4
IL28B (=IFNλ3), IL28A (= IFNλ2) i IL29 (=IFNλ1)
Carlos Fernández-Carrillo, P.González, G.Crespo, M.Coto-Llerena, M.Navasa, X.Forns, Sofia Pérez del Pulgar
XXXIX Congreso AEEH Madrid, 19-21 Febrero 2014
76
Chr19:39,681,230-39,813,463 = 132,234 bp
IFN- λ = IL28A, IL28B, IL29
IL28B (=IFNλ3), IL28A (= IFNλ2) i IL29 (=IFNλ1)
IL28B (uc010xuu.1) at chr19:39734273-39735646 - interleukin 28B
IL28B (uc010xut.1) at chr19:39734273-39735609 - interleukin 28B
IL28A (uc002oku.1) at chr19:39759157-39760732 - interleukin 28A precursor
IL29 (uc002okv.2) at chr19:39786965-39789311 - interleukin 29 precursor
Chromosome 19
ideogram
UCSC Genome Browser on Human 25 Jan. 2010 - New Custom Track type: Binary Alignment/Map (BAM)
C/C in rs12979860 strongly asociated with SVR. 3kb upstream of the IL28B gene
in the region 19q13.13 (position: 39738787) between IL28B and IL28A
77
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 <10 x
Medium level resistance 10-100 x
high level resistance >100 x
Not done
NS3 resistance mutation (protease-helicase)
78
Identification of non-primate hepaciviruses (NPHV)
• A novel hepacivirus identified by high-throughput sequencing of respiratory samples from dogs
• Animals from respiratory disease outbreaks in two shelters found 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 canine
viruses!!! • 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
Antiretrovirals 153.286.409 23,6
Citostàtics 150.838.716 23,3
Immunosupressors selectius 99.378.048 15,3
Altres medicaments d'ús hospitalari 83.793.753 12,9
Esclerosi múltiple 41.061.364 6,3
Eritropoetina 26.327.776 4,1
Hormona de creixement 18.723.454 2,9
Factors antihemofílics 18.724.676 2,9
Nutrició enteral (NE) per sonda a domicili més material NE 18.659.082 2,9
Tractaments hepatitis C 15.095.863 2,3
Immunoglobines humanes inespecífiques 11.891.718 1,8
Factors d'estimulants de colònies 6.731.840 1
Medicació estrangera 2.973.335 0,5
Medicació i solucions per a la nutrició parenteral (NP) intravenosa 656.824 0,1
Medicaments per al tractament de la fibrosi quística 223.953 0
Total 648.366.813 100
Font: Gerència d’Atenció Farmacèutica i Prestacions Complementàries.
Pagina 210. Memoria 2012 CatSalut.
Taula 102. Import dels medicaments dispensats a pacients en règim ambulatori als hospitals de Catalunya
concertats i de l’ICS, 2012