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Structural polymorphism within entire HCV NS5A region

Structural polymorphism within entire HCV NS5A region as a possible predictor of treatment response in Estonian chronic HCV 1b patients Tatiana Kuznetsova 1 , PhD Tatjana Tallo 1 , PhD, Vadim Brjalin 2 , MD, Valentina Tefanova 1 , PhD 1 Department of Virology, NIHD, Tallinn, Estonia

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Structural polymorphism within entire HCV NS5A region

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  1. Structural polymorphism within entire HCV NS5A region as a possible predictor of treatment response in Estonian chronic HCV 1b patients Tatiana Kuznetsova1, PhD Tatjana Tallo1, PhD, Vadim Brjalin2, MD, Valentina Tefanova1, PhD 1Department of Virology, NIHD, Tallinn, Estonia 2West-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

  2. Estonia Background • Up to 1% of Estonian population or round14000 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 andRibavirin HCV genotype1 - a lower rate of response to therapy (<50%) HCV genotypes 2 and 3- a higher rate of responseto therapy (70 - 80%) Population 1.34 mln Area - 45,226 sq km

  3. Methods • Pretreatment serum from 29 patients with chronic HCVgenotype 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 parametermodel 3’UTR 5’UTR NS5A NS4A NS5B NS4B E1 p7 NS2 E2 NS3 C 1341 bp 447 aa

  4. Relationshipbetween NS5A amino acid sequences and antiviral treatment responses Full-lenght NS5a (HCV genotype 1b) 1 237 276 302 384 407 447 aa ISDR V3 PKR-bd • Comparison of amino acid substitution rate in the NS5a region • according toresponse to the combination therapy • No statistically significant associations

  5. Phylogenetic analysis • Phylogenetic tree delineating full-length NS5a sequence similarity • (HCV genotype 1b) HCV-J SVR 4 SVR 41 SVR 5 ST 30 SVR 8 SVR 16 RL 121 SVR 21 SVR 71 RL 76 SVR 111 NR69 NR 14 NR 141 RL 17 SVR 12 ST 27 SVR 35 SVR 42 RL109 RL 40 RL 67 SVR 62 NR 143 RL 51 NR 28 NR 23 NR 55 NR 123 groups I II III IIIa III IIIb IV • Sequences are found to be clustered • mostly according to the treatment outcome

  6. Essential nucleotide positions • Рhylogenetic analysisrevealed24subgroup-specific • nucleotide signatures • a T/С 1:1 means nucleotidesubstitution at position 21, 1 sequence from group I has T and 1 sequence has C. • Subgroup-specific Real-time PCRprobes may be used • for rapid genotyping of new HCV isolates

  7. Essential amino acid substitutions • Рhylogenetic analysisrevealed24subgroup-specific • nucleotide signatures • 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

  8. Conclusions • No significant difference in the number of mutations in ISDR, PKR-bd and V3 domains between SVR and non-SVR HCV 1bpatients was found • Sustainably inherited polymorphisms exhibiting different resistance of HCV 1b strains towards combination therapy were identified • Occurrence of nucleotide substitutionsspecific for HCV1b 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

  9. Thank you for your attention!

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