A novel simple assay system to quantify the percent HCV-RNA levels of NS5A Y93H mutant strains and Y93 wild-type strains relative to the total HCV-RNA levels to determine the indication for antiviral therapy with NS5A inhibitors

Abstract

Aim: Oral treatment with asunaprevir and daclatasvir has been reported to yield a SVR ratio of 80% in patients with genotype 1b HCV infection, however, treatment failure has been reported, especially in patients with HCV strains showing the NS5A-Y93H mutation at baseline. An assay system to detect such strains was established to facilitate selection of appropriate candidates for this antiviral therapy.

Methods: Primer sets and 2 types of cycling probe mixtures were designed, and real-time PCR was performed with HCV-RNA purified from 332 patients with genotype 1b HCV infection, and the results were compared with those obtained by direct sequencing.

Results: Both the wild-type and mutant strains were quantified, with a threshold of 4.0 Log copies/mL, in 295 of the 332 patients (88.9%), and the percentage of the mutant strains relative to the total HCV-RNA level in the serum was calculated. The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing. Both wild-type and mutant strains were detected in the remaining 35 patients (11.9%), at levels between 1% and 99%, despite the mutant strains having been undetectable by direct sequencing in 11 patients with percentages of these strains of less than 25%.

Conclusion: A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established. This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.

Figure 1. Real-Time PCR Analysis Using a Synthesized Oligonucleoside Following Adjustment for a 100-fold Dilution Series from 1×10² to 1×10⁸ copies/mL.

(A) Amplification plots using SYN-NS5A_Ycac. (B) The standard curve for SYN-NS5A_Ycac generated from the amplification plots displayed in Fig 1A. The correlation coefficient was 0.998. (C)Amplification plots using SYN-NS5A_Hcac. (D) The standard curve for SYN-NS5A_Hcac. The correlation coefficient was 0.998. All figures were generated by the ABI Psism 7900 Sequence Detection Software (SDS), version 2.4.

Figure 2. Real-Time PCR Analysis Using Mixtures of Synthesized Oligonucleoside Following Adjustment to Different Percent Volumes of Both Oligonucleosides.

SYN-NS5A_Hcac/SYN-NS5A_Ycac and SYN-NS5A_Hcgc/SYN-NS5A_Ycgc were used as the mixtures and the percent volumes of both oligonucleosides were 0%, 5%, 10%, 30%, 50%, 70%, 90% or 100%. Ycac/Hcac or Ycgc/Hcgc was used as the cycling probe. The numbers on the left indicate the percent volumes of the SYN-NS5A_Hcac/SYN-NS5A_ Hcgc in each sample.

Figure 3. Percent RNA Levels of Y93H Mutant HCV Strains relative to the Total HCV-RNA levels in Patients with Genotype 1b HCV Infection.

White and black bars indicate the percentages of Y93 wild strains and Y93H mutant strains, respectively. Y93H mutant HCV strains were undetectable in 257 patients (80.3%), while were detected in 58 patients (19.7%) with relative percentage to total HCV strains from 1.4% to 100% by Real-time PCR analysis using cycling probe mixtures Bars within the arrow and dotted arrow indicate mixed HCV strains based on real-time PCR analysis (n = 35) and direct-sequencing (n = 25), respectively.

Figure 4. Nucleotide (nt265–285) and Amino Acid (aa89–95) Sequences in the NS5A Region of 647 Genotype 1b HCV Strains in the Database.

The figures in the lower panel denote the number of HCV strains showing each nucleotide (nt273, 276, 277, 279) among the 647 genotype 1b HCV strains.