uso de la secuenciación masiva para el estudio del virus de la … · 2014-04-26 · 8 consensus...

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

josep.quer@vhir.org

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