Structural polymorphism within entire HCV NS5A region

as a possible predictor of treatment response

in Estonian chronic HCV 1b patients

Tatiana Kuznetsova1, PhDTatjana Tallo1, PhD,

Vadim Brjalin2, MD,

Valentina Tefanova1, PhD1Department of Virology, NIHD, Tallinn, Estonia2West-Tallinn Central Hospital, Tallinn, Estonia

IX Annual Conference of the New VISBY Network

on Hepatitis C (HepC 2012)April 1- 4, 2012, Saint Petersburg, Russia

Background

• Up to 1% of Estonian population or round 14 000 of inhabitants are infected with HCV

• 75% of chronic hepatitis caused by HCV

• HCV subtypes 1b and 3a are predominating, based on study of materials collected during 1998-2007 (Tallo et.al 2000; 2007)

• Standard treatment is based on combination of Pegylated interferon-α and Ribavirin

HCV genotype 1 - a lower rate of response to therapy (<50%)

HCV genotypes 2 and 3 - a higher rate of response to therapy (70 - 80%)

Estonia Population 1.34 mln Area - 45,226 sq km

Methods

C E1 E2 p7 NS2 NS3 NS4A NS4B NS5A NS5B5’UTR 3’UTR

1341 bp447 aa

• Pretreatment serum from 29 patients with chronic HCV genotype 1b were analysed (12 SVR, 8 NR, 7 RL and 2 ST)

• RNA extraction, cDNA synthesis and 2 rounds of PCR with specific primers were performed

• Full-lenght NS5a sequenses were obtained and edited with BioEdit and DNAStar programs

• Similarity was analyzed by BLAST and Metapiga software

• Genetic distances were calculated with Kimura-2 parameter model

Relationship between NS5A amino acid sequences and antiviral treatment responses

SVR (n=12) Non-SVR (n=15) P-valueISDR 1.00 ± 0.74 1.27 ± 0.70 0.351

PKR-bd 5.42 ± 1.08 4.67 ± 1.40 0.129V3 3.25 ± 1.36 3.53 ± 1.36 0.595

Entire NS5a 29.08 ± 3.96 28.27 ± 3.53 0.582

Comparison of amino acid substitution rate in the NS5a region according to response to the combination therapy

1 237 276 302 384 407 447 aa

Full-lenght NS5a (HCV genotype 1b)

ISDR

PKR-bd

V3

No statistically significant associations

Phylogenetic analysis

Phylogenetic tree delineating full-length NS5a sequence similarity (HCV genotype 1b)

HCV-JSVR 4SVR 41SVR 5ST 30SVR 8SVR 16RL 121SVR 21SVR 71RL 76SVR 111NR69NR 14NR 141RL 17SVR 12ST 27SVR 35SVR 42RL109RL 40RL 67SVR 62NR 143RL 51NR 28NR 23NR 55NR 123

I

II

IIIa

III

IIIb

IV

III

groups

Sequences are found to be clustered mostly according to the treatment outcome

Essential nucleotide positions

Nt position

by NS5a 21 96 114 147 248 258 403 586 587 595 639 644

HCV-J T G A G C A A G T C G A

I - SVR T/C 1:1 a G G A C A A A C C G A

II - SVR T A A A C G A/G 1:1 G T C G A

III - SVR T/C 12:1 G A/G 12:1 A C A A G T C/G 12:1 G A

IIIa - NR T G A A C A A G T C G A

IIIb - RL T G A A T A A G T C G A

IV - NR G G C/T 6:1 G C/T 6:1 A G/T 6:1 G T G A G/A 6:1

аа 7 aa 83 aa 135 aa 196 aa 199 aa 215

Nt position

by NS5a 920 975 999 1004 1029 1070 762 1205 1207 1217 1218 1220

HCV-J G G C T T G C A G A A G

I - SVR G G C A A G C A G A A A

II - SVR G T A T A A C A G G A G

III - SVR G G C T A G C A G/A 11:2 G/A 12:1 C G

IIIa - NR G G C T G G C A A G/A 1:2 C G

IIIb - RL A G C T A G C A/C 1:1 G/A 1:1 G/A 1:1 C G

IV - NR G G C/T 6:1 T A G T G G G C G

aa 307 aa 357 aa 403 aa 406 aa 407

a T/С 1:1 means nucleotide substitution at position 21, 1 sequence from group I has T and 1 sequence has C.

Subgroup-specific Real-time PCR probes may be used for rapid genotyping of new HCV isolates

Рhylogenetic analysis revealed 24 subgroup-specific nucleotide signatures

Essential amino acid substitutions

a T/A 1:1 means substitution Thr/Ala at position 7 , 1 sequence from group II has Thr and 1 sequence has Ala.

HCV sub-group typing may be possible by immunoassay with specific single-epitope antigenic probes

Aa numeration

CRS             NLS V3 

by NS5a 7 83 135 196 199 215 307 357 403 406 407

HCV-J D T T V L K R R D K G

I - SVR D T T T L K R R D K E

II - SVR D T T/A 1:1a V L K R K D E G/R 1:1

III - SVR D T T/I 12:1 V L/V 12:1 K R R D/N 11:2 A/T 12:1 R

IIIa - NR D T T V L K R R N A/T 1:2 R

IIIb - RL D M T V L K K R D/S 1:1 A/T 1:1 R

IV - NR E T/M 6:1 A/S 6:1 V V R/K 6:1 R R D A R

Рhylogenetic analysis revealed 24 subgroup-specific nucleotide signatures

Conclusions

• No significant difference in the number of mutations in ISDR, PKR-bd and V3 domains between SVR and non-SVR HCV 1b patients was found

• Sustainably inherited polymorphisms exhibiting different resistance of HCV 1b strains towards combination therapy were identified

• Occurrence of nucleotide substitutions specific for HCV 1b subgroups (nt signatures) allows development of Real-time PCR-based approach to rapid HCV 1b genotyping in Estonian isolates

• Novel amino acid residues characteristic for each HCV 1b group (aa signatures) were identified. A question about involvement of these residues to regulation of HCV replication cycle should be studied

Thank you for your attention!