Olga Kalinina, Olga Znoiko Saint-Petersburg Pasteur Institute

1. CLINICAL AND EPIDEMIOLOGICAL FEATURES OF THE NATURAL HEPATITIS C VIRUS RECOMBINANT RF1_2k/1b Olga Kalinina, Olga Znoiko Saint-Petersburg Pasteur Institute Moscow State University of Dentistry and Medicine

2. Genome organization of Hepatitis C Virion IRES Lindenbach and Rice, 2005

3. Genetic diversity of Hepatitis C Virus based on a high mutation rate 30-35% 20-25% Genotypes/ Clades from 1 to 6 10% Subtypes varying numbers from a to … Isolates Quasispecies

4. Geographic Distribution of HCV Clades/Genotypes 1b, 3a 1a, 2a, 2b, 2c, 2k 1b 1a, 2a, 2b, 2c, 3a Russia 1a, 1b 2a, 2b, 3a Europa 1b, 6, 3a, 2a 1b China, Cambodia, Vietnam North America 4 Egypt Japan 4, 1 1b, 3a Cameroon India 1a, 1b 2a, 2b, 3a 5a 1b, 3a Latin America South Africa Australia

5. Genetic diversity of Hepatitis C Virion based on recombination 91 PJ6CF USA HC16362 Korea a 75 PH77CV Chimera HCV-JS Japan MD8-2 Japan HC-J6 Japan HCV-L2 Korea HC-C2 China 91 778 St.Petersburg HPVHCVN Japan c HCV-HB China 686 St.Petersburg MD4-1 Japan 300 St.Petersburg 309 St.Petersburg 576 St.Petersburg f BEBE1 Japan 204 St.Petersburg HCJTa Japan HCJK081 Jakarta CORE HCJTb Japan HCJK139 Thailand 643 St.Petersburg 611 R St.Petersburg 788 St.Petersburg NS5B 779 St.Petersburg 203 R St.Petersburg 704 St.Petersburg 615 St.Petersburg k 747 R St.Petersburg HD-1 Germany 747 R St.Petersburg 796 R St.Petersburg 674 R St.Petersburg SimPlot – Query: 687 R 2k/1b Saint-Petersburg 796 R St.Petersburg 674 R St.Petersburg 687 R St.Petersburg 687 R St.Petersburg 611 R St.Petersburg 2 203 R St.Petersburg K1-S2 1b 100 VAT-96 Moldova 761 St.Petersburg e 185 St.Petersburg 464 St.Petersburg 783 St.Petersburg b 735 St.Petersburg HCJK025 Jakarta 589 St.Petersburg b 789 St.Petersburg HCJK109 Jakarta 1 728 St.Petersburg Md2b-1 700 St.Petersburg VAT-96 2k 732 St.Petersburg 100 VN004 Vietnam 6 MD1-1 Japan MD1-2 Japan VN405 Vietnam 5a MD5-1 Japan a VN235 Vietnam HPCPP Japan 95 HCV-H USA HCJK046 Jakarta PH77CV Chimera 5a HC-J1 USA 785 St.Petersburg EUH1480 South Africa • Flaviviridae • Flavivirus Dengue virus (Holmes et al, 1999; Tolou et al, 2001) • Pestivirus Bovine viral diarrehea virus (Ridpath et al, 2000) • Hepacivirus Hepatitis C virus (Kalinina et al. 2002)

6. 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

7. Recently revelead inter-genotype HCV Recombinants RF1_2k/1b Russia, Siberia, Estonia, Uzbekistan, Sweden, Ireland, Holland, France, Azerbaijan, Cyprus 2/5 recombination point within the NS2/NS3 region, France 2007 2b/6w recombination point beginning the NS3 region Taiwan 2010 2b/1a recombination point within the NS2/NS3 region, USA 2011 2b/1b recombination point within the NS2/NS3 region Metro Manila Philippines 2006 2i/6p recombination point within the NS2/NS3 region from blood donor, Ho Chi Minh City Vietnam 2005

8. Recently revelead intra-genotype HCV Recombinants 1b/1a recombination point within the core Uruguay 2009 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 NS5B Peru 2004

9. CONCLUSIONS 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 creating genetic variants with new properties that has to be studied

10. 658 668 678 HC-J4 1b R D R S E L S P L L L S T T E W Q T L P C k1-s2 1b . . . . . . . . . . . . . . . . . I . . . 589 1b . . . . . . . . . . . . . . . . . I . . . Genome organization of HCV Recombinant RF1_2k/1b 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 . . . . . A I . . . genome number of the amino acid residues CB 3a . . . . . Q H . . . H . . . . L A I . . . NZL1 3a . region . . . . Q H . . . H . . . . L A I . . . genotype2 RF1 - 2k/1b subtype1b subtype 1b (HC - J6) (N687 StP) (HC - J4) (N589 StP) ORF 3033 3014 3010 3010 PePHD region within E2 Core 191 191 191 191 2216 2226 2236 2246 HC-J4 1b 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 E1 192 192 192 192 687 R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E2 367 367 363 363 k1-s2 1b . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589 1b . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . p7 63 63 63 63 . . . . 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 NS2 217 217 217 217 . . . . . . . . Q . . R P H . . . E . V N . . . . . . . . . . S . . . . . . . . T . CB 3a NS 3 631 631 631 631 . . . . . . . . Q . . R P H . . . E . V D . . . . . . . . . . S . . . . . . . . T . NZL1 3a NS4a 54 54 54 54 Predicted amino acid sequences for HCV Recombinant NS4b 261 261 261 261 NS5a 466 447 447 447 NS5b 591 591 591 591 ISDR region within NS5A

11. ISDR/PKRbinding domain Interferon sensitivity-determining region ISDR/PKRbinding domain V3variable domain 2210 2220 2230 2240 2250 HC-J4 1b 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687 RF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M21 RF k1-s2 1b . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589 1b . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . C . . . . . . . . . A . . . HC-J6 2a . . . . . . R . . . . . . G K A Y . V . M V D . . . F - - - - . . . D V . . I . . . S . . . 2260 2270 2280 2290 . . . . . . R . . . . A . A K N Y A V E M V D . . F F - - - - . . S D V . . I . . . T . . L VAT-96 2k 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 1b V . . . . . . I . P . . . . . . . . . P . . . . . . . R . . . R . M . . . . . . . . . . . . 687 RF . . . . . . . . . . Q . . R P H . . . E . V N . . . . . . . . . . S . . . . . . . . T . . . CB 3a M21 RF . . . . . . . . Q . . . . . . . . . . P . . . . . . . R . . . R . I . . . . . . . . . . . . . . . . . . . . . . Q . . R P H . . . E . V D . . . . . . . . . . S . . . . . . . . T . . . NZL1 3a k1-s2 1b . . . . . E . . . . . . . . . . . . L P . . . . . . . R . . . . . M . . . . . . . . . . . . 589 1b . . . . . E . . . . . . . . . . I . . P . . . . . . . R . . . . . . . . . . . . . . . . . . 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 CB 3a . . . . . E . . . . . T . D A . L . . . . . C F K . P P . Y . . . . . . . . . . . . . . . . NZL1 3a V . . . . E . . . . . T . D V . P . . . . . C F K . P P . Y . . . . . . . . . . . . . . . . 2300 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 . . . . . . . . . . . . . . 687 RF . . . . . D . . . . . . V . . . . . . . . . K A . . V . . . . R . . . . . . . . . . . . . . M21 RF I . . . . D . . . . . . V . . . . . . . . . K A . . . . . . . R . . . . . . . . . . . . . . k1-s2 1b 589 1b . . P . . D . . . . . . V . . . . . . . . . K A . . . . . . . . . . . . . . . . . . . . . . V . . . . 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 Serine at position 2300 is associated with resistance to interferon + ribaverin therapy (Bouzgarrou et al., 2009) 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 RF . . . . . . . . . . . . - - - - - . . . . . . . . . . . . . Y N . . . N . . . A G . . . . . M21 RF k1-s2 1b . . . . . . . . . . . . - - - - - E . . . A . . . . . . . . . . . P . N . . . A G . . . . . 589 1b . . . . . . . . . . . . - - - - - E . . . . . . . . . . . . . . . A . . . . . . . . . . . . . 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 NZL1 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 . .

12. Outcome of Hepatitis C Infection • HCV sensitivity to interferon therapy mainly depends on genotype and is the poorest for HCV 1b. • Genotype accounting for recombination is important for selection the therapy approach(es) and prognosis the response

13. Outcome of Recombinant RF1_2k/1b Hepatitis C Infection • Patients • N 1763 men, 51 years old, chronic hepatitis C, revealed as a contact person with patient N 1764 • N 1764 feminine, 34 years old, acute hepatitis C, icteric form, 20 days after onset biochemical level became normal • M21 (sampled in France) men, 30 years old, IVDU, chronic hepatitis C, known as infected by HCV genotype 3a (Morel et al., 2010)

14. Outcome of Recombinant RF1_2k/1b Hepatitis C Infection • Patients N 1764, feminine, 34 years old, acute hepatitis C, icteric form, • 20 days after onset biochemical level became normal • During the following 12 months she was positive for RNA HCV, • had increasing level of liver enzyme and progressed to chronic hepatitis • After one year from onset of disease patient received mono-therapy by • α2a-interferon: 3 000 000 Units three times per week • by week 12, it was observed early virological response. Following 36 weeks patient received the same therapy • by week 48, it was sustained virological and biochemical responses (also after 5 years later)

15. Outcome of Recombinant RF1_2k/1b Hepatitis C Infection • Patients N 1763, men, 51 years old, chronic hepatitis C • patient received mono-therapy by α2a-interferon: 3 000 000 Units three times per week • by week 12, it was observed only biochemical responses but not virological • after that patient received α2a-interferon + RIBA • by week 16, it was still not virological response • no progression of fibrosis stage for 6 years • elastography (Fibroscan Echosens) in 2007 Fibrosisscore - F1, in 2010 Elastography - F2 • at 2010 patient received Peg IFN + RIBA, by week 24, it was not virological and biochemical responses

16. Outcome of Recombinant RF1_2k/1b Hepatitis C Infection • M21 (sampled in France) men, 30 years old, chronic hepatitis C, • known as infected by HCV genotype 3a (Morel et al., 2010) • patient received combination therapy by pegylated interferon and ribavirin • by week 24, it was observed virological response. One month later HCV RNA didn’t also detected in the serum • Three months post-treatment, it was noted virological relapse with increasing level of ALT • HCV strain belonged to genotype 3a was no longer found. In the same time in the serum samples HCV strain was subtypes as recombinant RF1_2k/1b

17. CONCLUSIONS II • At present HCV recombinant RF1_2k/1b has to be considered as • “difficult-to-treat” genotype • Further studies are emergency needed to define the long-term outcome and strategy for antiviral therapy in the case of RF1_2k/1b

18. Acknowledgements IN THE EPIDEMIOLOGY STUDY PARTICIPATED Lars O Magnius, Helene Norder Swedish Institute for Infectious Disease Control Tatjana 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. Petersburg Denis Gusev Medical Military Academy, St. Petersburg Timophei Vetrov St. Petersburg Pavlov Medical University Pavel Kislii St. Petersburg Metchnikov Medical Academy

19. Acknowledgements THANK YOU for YOUR ATTENTION