Olga Kalinina, Olga Znoiko Saint-Petersburg Pasteur Institute Moscow State University of Dentistry and Medicine CLINICAL AND EPIDEMIOLOGICAL FEATURES OF

Olga Kalinina, Olga ZnoikoOlga Kalinina, Olga Znoiko

Saint-Petersburg Pasteur Institute Saint-Petersburg Pasteur Institute Moscow State University of Dentistry and MedicineMoscow State University of Dentistry and Medicine

CLINICAL AND EPIDEMIOLOGICAL FEATURES OF CLINICAL AND EPIDEMIOLOGICAL FEATURES OF

THE NATURAL HEPATITIS C VIRUS RECOMBINANT THE NATURAL HEPATITIS C VIRUS RECOMBINANT

RF1_2k/1bRF1_2k/1b

Genome organization of Hepatitis C VirionGenome organization of Hepatitis C Virion

Lindenbach and Rice, 2005

IRES

Genetic diversity of Hepatitis C Virus Genetic diversity of Hepatitis C Virus based on based on a high mutation ratea high mutation rate

QuasispeciesQuasispecies

GenotypesGenotypes/ Clades/ Clades from 1 to 6

30-35%30-35%

Subtypes Subtypes varying numbers from a to …

20-25%20-25%

IsolatesIsolates

10%10%

Geographic Distribution of HCV Clades/Genotypes Geographic Distribution of HCV Clades/Genotypes

India

EuropaEuropa

North America

Latin America

Egypt

1b 1b 1a, 2a, 2b, 2c, 3a

1a, 1b 1a, 1b 2a, 2b, 3a

44

1a, 1b 1a, 1b 2a, 2b, 3a

5a5a

SouthAfrica

1b, 3a1b, 3a

1b, 3a1b, 3a

1b, 6, 3a, 2a1b, 6, 3a, 2a

Australia

China, Cambodia, Vietnam Japan

Russia

1b, 3a 1b, 3a 1a, 2a, 2b, 2c, 2k

1b1b

Cameroon

4, 14, 1

Genetic diversity of Hepatitis C Virion Genetic diversity of Hepatitis C Virion based on recombinationbased on recombination

PJ6CF USAPH77CV Chimera

HC-J6 Japan

778 St.Petersburg686 St.Petersburg300

St.Petersburg

BEBE1 Japan

HCJK081 Jakarta

HCJK139 Thailand611 R St.Petersburg

203 R St.Petersburg

747 R St.Petersburg796 R St.Petersburg


VAT-96 Moldova464 St.Petersburg

HCJK025 JakartaHCJK109 Jakarta

Md2b-1

VN004 Vietnam

VN405 Vietnam

VN235 VietnamHCJK046 Jakarta

2

65a

be

k

f

c

a

100

9175

91

CORE

HC16362 KoreaHCV-JS Japan

MD8-2 JapanHCV-L2 KoreaHC-C2 ChinaHPVHCVN JapanHCV-HB ChinaMD4-1 Japan

309 St.Petersburg576 St.Petersburg

204 St.PetersburgHCJTa JapanHCJTb Japan643 St.Petersburg788 St.Petersburg


615 St.PetersburgHD-1 Germany








732 St.PetersburgMD1-1 JapanMD1-2 Japan

MD5-1 JapanHPCPP Japan

HCV-H USAPH77CV Chimera

HC-J1 USA785 St.PetersburgEUH1480 South Africa

1

5aa

b100

95

NS5B

FlaviviridaeFlaviviridae

FlavivirusFlavivirusDengue virus (Holmes et al, 1999; Tolou et al, 2001)

PestivirusPestivirusBovine viral diarreheaBovine viral diarreheavirus virus (Ridpath et al, 2000)

HepacivirusHepacivirusHepatitis C virusHepatitis C virus

(Kalinina et al. 2002)(Kalinina et al. 2002)

SimPlot – Query: 687 R 2k/1b Saint-Petersburg

K1-S2 1b

VAT-96 2k

CJTA... CJTB... 198 04 105...

1170 105... 1152 105... 1766 105...

1192 105... HCV N... 1187 105... 450 105...

735 105... 1182 105... 380 105...

2055 Estonia... 644 105...

2191 105... MD4 1... HPCGENOM...

2246 105... HPCRNA... HPVHCVN...

1774 105... HCD85516...

HC16362... MD8 2... HCU01214...

1189 105... 643 105...

640 Estonia... 2187 105...

MD3 1... 2035 Estonia...

1155 Estonia... CJ483... HC J4...

455 105... 514 105... 187 04 105...

728 105... 480 105...

448 105... 1188 105...

2188 105... 607 Estonia... 2079 Estonia... 615 Estonia...

197 04 105... 201 04 105... 641 105...

1171 105... 1163 Estonia... 622 Estonia...

GQ418317 cirrhosis Indonesia 2007 1... 2077 Estonia...

1160 105... K1 R3... 2248 105...

383 105... 2178 105...

CON1... 1145 105...

589 105... 181 04 105... 197 03 105...

AF071987 1bSweden... MD2 1...

MD1 1... MD5 1... HPCPP... 377 105...

2180 105... GQ418304 indonesia...

177 04 105... 2051 Estonia... JF424774 USA pat 26 2008 1...

1178 105... D89815...

DQ345619 Madagascar 2004... 185 105...

2185 105... HM106914 Irlend 2011 non recom 1...

EU710293 australia... 568 105...

Рекомбинант RF_2k/1b

1777 105... J33...

1161 105... K1 S3...

K1 R2... K1 S2... HD 1...

678 105... 1196 105... DQ508480 Tunisia hemodialysis...

704 105... 1194 105...

892 105... 779 105...

2019 Estonia... K1 S1...

1146 105... 885 105...

2031 Estonia... 761 105...

HCV AD78... 1776 105...

HCVPOLYP... 1153 105...

576 105... 176 04 105... 2052 Estonia...

2022 Estonia... 1149 Estonia...

1159 105... 645 105...

1162 105... 730 105...

742 105... 732 105...

700 105... EF543254 Tailand 2007 1...

183 04 105... 200 04 105...

HPCP1... субтип 1а

Recently revelead inter-genotype HCV RecombinantsRecently revelead inter-genotype HCV Recombinants

2i/6p recombination

point within the NS2/NS3 region

from blood donor, Ho Chi Minh City

Vietnam

2005

2/5

recombination point within the NS2/NS3

region, France 2007

2b/1b recombination

point within the NS2/NS3 region

Metro Manila Philippines

2006

RF1_2k/1b

Russia, Siberia, Estonia, Uzbekistan, Sweden, Ireland, Holland, France,

Azerbaijan, Cyprus

2b/1a

recombination point within the NS2/NS3 region,

USA

2011

2b/6w recombination

point beginning the NS3 region

Taiwan

2010

Recently revelead intra-genotype HCV RecombinantsRecently revelead intra-genotype HCV Recombinants

1b/1a recombination

point within the NS5B

Peru

2004

1a/1c

two recombination points within the E1-E2

regions

Japan

2006

1a/1c

five recombination points within the core-

NS3 regions

India

2008

1b/1a recombination

point within the core

Uruguay

2009

CONCLUSIONS ICONCLUSIONS I

Currently, more than 40 recombinant RF1_2k/1b strains circulating in the human population are known; most of them from the former Soviet Union Countries where recombinant has likely emerged

At present, six natural inter-genotyping and four intra-genotyping recombinant HCV forms were identified in the world

Recombination plays a significant role in the HCV evolution by Recombination plays a significant role in the HCV evolution by creating genetic variants with new properties that has to be studiedcreating genetic variants with new properties that has to be studied

Genome organization of HCV Recombinant Genome organization of HCV Recombinant RF1_2k/1b RF1_2k/1b

Predicted amino acid sequences for HCV Recombinant

number of the amino acid residues genome

region genotype2

(HC-J6)

RF1-2k/1b

(N687 StP)

subtype1b

(HC-J4)

subtype 1b

(N589 StP)

ORF 3033 3014 3010 3010

Core 191 191 191 191

E1 192 192 192 192

E2 367 367 363 363

p7 63 63 63 63

NS2 217 217 217 217

NS3 631 631 631 631

NS4a 54 54 54 54

NS4b 261 261 261 261

NS5a 466 447 447 447

NS5b 591 591 591 591

ISDR region within NS5A2216 2226 2236 2246

A P S L K A T C T T H H D S P D A D L I E A N L L W R Q E M G G N I T R V E S E N K HC-J4 1b. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687 R . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . k1-s2 1b. . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . 589 1b. . . . R . . . . . . G K A Y . V . M V D . . . F - - - - . . . D V . . I . . . S . HCJ6 2a. . . . R . . . . A . A K N Y A V E M V D . . F F - - - - . . S D V . . I . . . T . Vat96 2k. . . . . . . . Q . . R P H . . . E . V N . . . . . . . . . . S . . . . . . . . T . CB 3a. . . . . . . . Q . . R P H . . . E . V D . . . . . . . . . . S . . . . . . . . T . NZL1 3a

658 668 678

R D R S E L S P L L L S T T E W Q T L P C HC-J4 1b

. . . . . . . . . . . . . . . . . I . . . k1-s2 1b

. . . . . . . . . . . . . . . . . I . . . 589 1b

. . . . Q . . . . . H . . . . . A I . . . 687 R

. . . . Q . . . . . H . . . . . A I . . . 674 R

. . . . Q . . . . . H . . . . . A I . . . 747 R

. . . . Q . . . . . H . . . . . A I . . . 796 R

. . . . Q . . . . . H . . . . . A I . . . Vat96 2k

. . . . Q . . . . . H . . . . . A I . . . HCJ6 2a. . . . . Q H . . . H . . . . L A I . . . CB 3a

. . . . . Q H . . . H . . . . L A I . . . NZL1 3a

PePHD region within E2 PePHD region within E2

Serine at position 2300 isSerine at position 2300 isassociated with resistance to associated with resistance to interferon + ribaverin therapyinterferon + ribaverin therapy

(Bouzgarrou et al., 2009)(Bouzgarrou et al., 2009)

2260 2270 2280 2290

I L D S F D P L R A E E D E R E V S V A A E I L R K S K K F P P A L P I W A R P D Y N P P L HC-J4 1bV . . . . . . I . P . . . . . . . . . P . . . . . . . R . . . R . M . . . . . . . . . . . . 687687 RF RF. . . . . . . . Q . . . . . . . . . . P . . . . . . . R . . . R . I . . . . . . . . . . . . M21M21 RF RF

. . . . . E . . . . . . . . . . . . L P . . . . . . . R . . . . . M . . . . . . . . . . . . k1-s2 1b

. . . . . E . . . . . . . . . . I . . P . . . . . . . R . . . . . . . . . . . . . . . . . . 589589 1b 1b

V . . . L . . M V E . R S D L . P . I P S . Y M L P K . R . . . . . . A . . . . . . . . . . HC-J6 2a. . . . L . . S V E . . . . . . P . . P S . Y . L P K . . . . Q . . . V . . . . . . . . . V VAT-96 2k. . . . . E . . . . . T . D A . L . . . . . C F K . P P . Y . . . . . . . . . . . . . . . . CB 3a

V . . . . E . . . . . T . D V . P . . . . . C F K . P P . Y . . . . . . . . . . . . . . . . NZL1 3a

23002300 2310 2320 2330 2340

L E S W K S P D Y V P P A V H G C P L P P T T G P P I P P P R K K R T V V L T E S T V S S A HC-J4 1b

. A . . . D . . . . H . V . . . . . . . . . K A . . . . . . . R . . . . . . . . . . . . . . 687687 RF RF

. . . . . D . . . . . . V . . . . . . . . . K A . . V . . . . R . . . . . . . . . . . . . . M21 M21 RRFF

I . . . . D . . . . . . V . . . . . . . . . K A . . . . . . . R . . . . . . . . . . . . . . k1-s2 1b. . P . . D . . . . . . V . . . . . . . . . K A . . . . . . . . . . . . . . . . . . . . . . 589589 1b 1bV . . . . R . . . Q . A T . A . . A . . . P K K T . T . . . . R R . . . G . S . . S I A D . HC-J6 2a

V . T . . R . . . D . . T . S . . A . . . R V T A . T . . . . R R . A L . . S Q . N . G E . VAT-96 2k. D R . . A . . . . . . T . . . . A . . . R G A . . V . . . . R . . . I Q . D G . N . . A . CB 3a

. D R . . A . . . . . . T . . . . A . . . R G A . . V . . . . R . . . I Q . D G . N . . A . NZL1 3a

2350 2360 2370 2380

L A E L A T K T F G S S - - - - - G S S A V D S G T A T A P P D Q T S D D G D K E S D V E S HC-J4 1b

. . . . . . . . . . . . - - - - - . . . . . . . . . . S . . Q N . . . N . . . T G . . . . . 687 687 RF RF

. . . . . . . . . . . . - - - - - . . . . . . . . . . . . . Y N . . . N . . . A G . . . . . M21 M21 RRFF

. . . . . . . . . . . . - - - - - E . . . A . . . . . . . . . . . P . N . . . A G . . . . . k1-s2 1b

. . . . . . . . . . . . - - - - - E . . . . . . . . . . . . . . . A . . . . . . . . . . . . 589589 1b 1b

. Q Q . . I . S . . Q P P P S G D S G L S T G A D A . D S G - S R . P P . E L A L . E T G . HC-J6 2a

. Q A . . I . S . . Q L P P S C D S G R S T G M D . T D . T - . . P A L K E S T D . E A G . VAT-96 2k

. R A . . E . S . P . L K P Q E E N N . S S G V D . Q S S T T S K V P P S P G G . . . S . . CB 3a

. . A . . E . S . P . . K P Q E E N . . S S G V D . Q S S T T S K V P P S P G G . . . S . . NZL1 3a

2210 2220 2230 2240 2250

L S A P S L K A T C T T H H D S P D A D L I E A N L L W R Q E M G G N I T R V E S E N K V V HC-J4 1b

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687687 RF RF

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M21M21 RF RF

. . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . k1-s2 1b

. . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . C . . . . . . . . . A . . . 589589 1b 1b

. . . . . . R . . . . . . G K A Y . V . M V D . . . F - - - - . . . D V . . I . . . S . . . HC-J6 2a

. . . . . . R . . . . A . A K N Y A V E M V D . . F F - - - - . . S D V . . I . . . T . . L VAT-96 2k

. . . . . . . . . . Q . . R P H . . . E . V N . . . . . . . . . . S . . . . . . . . T . . . CB 3a

. . . . . . . . . . Q . . R P H . . . E . V D . . . . . . . . . . S . . . . . . . . T . . . NZL1 3a

Interferon sensitivity-determining region Interferon sensitivity-determining region

ISDR/PKRISDR/PKR binding domain binding domain

ISDR/PKRISDR/PKR binding domain binding domain

V3V3 variable domain variable domain

Outcome of Hepatitis C InfectionOutcome of Hepatitis C Infection

HCV sensitivity to interferon therapy HCV sensitivity to interferon therapy mainly depends on genotype and is the mainly depends on genotype and is the poorest for HCV 1b.poorest for HCV 1b.

Genotype accounting for recombination Genotype accounting for recombination is important for selection the therapy is important for selection the therapy approach(es) and prognosis the responseapproach(es) and prognosis the response

Outcome of Recombinant RF1_2k/1b Hepatitis C Outcome of Recombinant RF1_2k/1b Hepatitis C InfectionInfection

Patients Patients

N 1763 men, 51 years old, chronic hepatitis C, revealed as a contact person N 1763 men, 51 years old, chronic hepatitis C, revealed as a contact person with patient N 1764with patient N 1764

N 1764 feminine, 34 years old, acute hepatitis C, icteric form, 20 daysN 1764 feminine, 34 years old, acute hepatitis C, icteric form, 20 days after onset biochemical level became normalafter onset biochemical level became normal

M21 (sampled in France) men, 30 years old, IVDU, chronic hepatitis C, M21 (sampled in France) men, 30 years old, IVDU, chronic hepatitis C, known as infected by HCV genotype 3a known as infected by HCV genotype 3a

((Morel et al., 2010)Morel et al., 2010)


Patients N 1764, Patients N 1764, feminine, 34 years old, acute hepatitis C, icteric form, feminine, 34 years old, acute hepatitis C, icteric form, 20 days after onset biochemical level became normal20 days after onset biochemical level became normal

During the following 12 months she was positive for RNA HCV, During the following 12 months she was positive for RNA HCV, had increasing level of liver enzyme and progressed to chronic hepatitishad increasing level of liver enzyme and progressed to chronic hepatitis

After one year from onset of disease patient received mono-therapy by After one year from onset of disease patient received mono-therapy by αα2a-i2a-interferon: 3 000 000 Units three times per weeknterferon: 3 000 000 Units three times per week

by week 12, it was observed early virological response. Following 36 weeks by week 12, it was observed early virological response. Following 36 weeks patient received the same therapy patient received the same therapy

by week 48, it was sustained virological and biochemical responses by week 48, it was sustained virological and biochemical responses (also after 5 years later)(also after 5 years later)


Patients N 1763, Patients N 1763, men, 51 years old, chronic hepatitis Cmen, 51 years old, chronic hepatitis C

patient received mono-therapy by patient received mono-therapy by αα2a-interferon: 3 000 000 Units three 2a-interferon: 3 000 000 Units three times per weektimes per week by week 12, it was observed only biochemical responses but not virological by week 12, it was observed only biochemical responses but not virological after that patient received after that patient received αα2a-interferon + RIBA2a-interferon + RIBA by week 16, it was still not virological responseby week 16, it was still not virological response no progression of fibrosis stage for 6 yearsno progression of fibrosis stage for 6 years elastography (Fibroscan Echosens) in 2007 Fibrosiselastography (Fibroscan Echosens) in 2007 Fibrosis score - F1,score - F1, in 2010 Elastography - F2in 2010 Elastography - F2 at 2010 patient received Peg IFN + RIBA, by week 24, it was not virological at 2010 patient received Peg IFN + RIBA, by week 24, it was not virological and biochemical responsesand biochemical responses


M21 (sampled in France) men, 30 years old, chronic hepatitis C, M21 (sampled in France) men, 30 years old, chronic hepatitis C, known as infected by HCV genotype 3a (Morel et al., 2010) known as infected by HCV genotype 3a (Morel et al., 2010)

patient received combination therapy by pegylated patient received combination therapy by pegylated iinterferon and ribavirinnterferon and ribavirin

by week 24, it was observed virological response. One month later by week 24, it was observed virological response. One month later HCV RNA didn’t also detected in the serumHCV RNA didn’t also detected in the serum

Three months post-treatment, it was noted virological relapse Three months post-treatment, it was noted virological relapse with increasing level of ALTwith increasing level of ALT

HCV strain belonged to genotype 3a was no longer found. HCV strain belonged to genotype 3a was no longer found. In the same time in the serum samples HCV strain was subtypes In the same time in the serum samples HCV strain was subtypes as recombinant RF1_2k/1b as recombinant RF1_2k/1b

CONCLUSIONS IICONCLUSIONS II

At present HCV recombinant RF1_2k/1b has to be considered as At present HCV recombinant RF1_2k/1b has to be considered as ““difficult-to-treat” genotypedifficult-to-treat” genotype

Further studies are emergency needed to define the long-term outcome Further studies are emergency needed to define the long-term outcome and strategy for antiviral therapy in the case of RF1_2k/1band strategy for antiviral therapy in the case of RF1_2k/1b

AcknowledgementsAcknowledgements

IN THE EPIDEMIOLOGY STUDY PARTICIPATED

Lars O Magnius, Helene Norder Swedish Institute for Infectious Disease ControlTatjana Tallo

Sergey Mukomolov St. Petersburg Pasteur Institute

Olga Znoiko Moscow State University of Medicine and Dentistry

Maria Isaguliants Swedish Institute for Infectious Disease Control Ivanovsky Institute, Moscow

Valentina Tchakharian State Centre for Epidemiological Surveillance Konstantin Zhdanov Medical Military Academy, St. PetersburgDenis Gusev Medical Military Academy, St. PetersburgTimophei Vetrov St. Petersburg Pavlov Medical UniversityPavel Kislii St. Petersburg Metchnikov Medical Academy

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Olga Kalinina, Olga Znoiko Saint-Petersburg Pasteur Institute Moscow State University of Dentistry and Medicine CLINICAL AND EPIDEMIOLOGICAL FEATURES OF