Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

Bae, Andrew; Ku, Karin S.; Miller, Michael D.; Mo, Hongmei; Svarovskaia, Evguenia S.

doi:10.1128/jcm.00274-11

Cited by 8 publications

(11 citation statements)

References 20 publications

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“…In contrast to other methods used to detect resistance-associated substitutions, which require separate specific primers for G1a and G1b[14-16] our technique can identify a single point mutation using one set of primers for all G1 samples. Simple hybridization is then carried out, in this case with the specific probe that differentiated Q from K at a position 80.…”

Section: Discussionmentioning

confidence: 99%

New real-time-PCR method to identify single point mutations in hepatitis C virus

Chen

Belmonte

Buti

et al. 2016

WJG

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AIMTo develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus (HCV).METHODSIn patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. LightCycler methods, based on real-time PCR with sequence-specific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest.RESULTSUsing nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10% (mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80K was detected in 14.6% of G1a patients and 0% of G1b in our setting.CONCLUSIONA fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.

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Section: Discussionmentioning

confidence: 99%

New real-time-PCR method to identify single point mutations in hepatitis C virus

Chen

Belmonte

Buti

et al. 2016

WJG

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“…Allele‐specific real‐time PCR was performed on PCR products as previously described . Briefly, NS5B PCR products were diluted to approximately 10 6 copies/ μ L. A standard curve was generated by preparing 100%, 50%, 10%, 5%, 1%, 0.5%, 0.1% and 0% wild‐type 448Y and mutant 448H clonal DNA mixtures.…”

Section: Methodsmentioning

confidence: 99%

Antiviral response and resistance analysis of treatment‐naïve HCV‐infected patients receiving single and multiple doses of GS‐9190

Hedskog

Svarovskaia

et al. 2016

Journal of Viral Hepatitis

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GS-9190 is a NS5B non-nucleoside analogue with demonstrated effectiveness in a Phase 1 monotherapy study and in combination with other DAAs for treatment of chronic HCV infection. Here, the resistance profile of GS-9190 monotherapy in a Phase 1b study was investigated. Resistance analysis was performed by population sequencing and allele-specific PCR (AS-PCR) for Y448H with an assay cut-off of 0.5%. Phenotypic susceptibility analyses were performed on patient isolates as well as site-directed mutagenesis of mutations selected during monotherapy. No resistance-associated variants were observed in patients before or after receiving single doses of GS-9190 by population sequencing. In contrast, in patients who received GS-9190 for 8 days, mutations Y448H and Y452H in NS5B were observed by population sequencing in 21/36 (58%) and 2/36 (5.6%) patients, respectively, at Day 8 or Day 14. Among the remaining 15 patients who had no detectable Y448H at Day 8 or Day 14 by population sequencing, low frequencies of Y448H ranging from 1.3 to 9.7% were detected in 14 of 15 patients by AS-PCR. By AS-PCR, Y448H remained detectable at reduced frequency in the majority of patients analysed through 4-6 months of follow-up. Chimeric HCV replicons constructed with the NS5B sequence from patients with Y448H and Y448H + Y452H/Y demonstrated 27-fold and 78.5-fold reduced susceptibility to GS-9190. In conclusion, Y448H was rapidly selected in the majority of patients receiving multiple doses of GS-9190 as monotherapy, despite undetectable levels in pretreatment samples. Y448H confers reduced susceptibility to GS-9190 and other NNIs and persisted in most patients for months post-treatment.

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“…Total RNA was extracted from HCV-infected patient plasma samples with the QIAamp viral RNA minikit (Qiagen, Valencia, CA) and HCV replicon cells with the RNeasy minikit (Qiagen, Valencia, CA) following manufacturer's instructions. cDNA synthesis was performed with the MonsterScript reverse transcriptase (Epicentre, Madison, WI) and the HCV-specific primers 1aNS5B-3'9386-5′ CTA AGA GGC CGG AGT GTT TAC-3′ and 1bNS5B-3'9459 -5′ CCT ATT GGC CTG GAG TGT TTA GCT C-3′ for genotype 1a and 1b respectively [29]. cDNA was used as a template for consecutive PCR amplification of the NS5B gene using cycling conditions previously described [29].…”

Section: Rna Extraction Cdna Synthesis and Ns5b Pcr Amplificationmentioning

confidence: 99%

“…The second primer set is MM2417-FAM-GGCCCTGTATTGTCAAATCT and MM2418-HEX-GGCCCTGTATTGTCAAATCC. AS-PCR was performed on a LightCycler 480 Real-Time PCR Instrument (Roche Applied Science, Indianapolis, IN) using cycling conditions previously described [29].…”

Section: Y448h Allele-specific Real-time Pcrmentioning

confidence: 99%

“…Population sequencing can only detect minor populations present at 20-25% of the population. As previously reported, a Y448H AS-PCR assay for HCV GT 1a and 1b has been developed with a 0.5% sensitivity [29]. In this study, we used this highly-sensitive detection method of AS-PCR to monitor the kinetics of the NS5B Y448H mutant in HCV-infected GT 1 patients and TGV-treated replicon cells.…”

Section: Introductionmentioning

confidence: 98%

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Viral Kinetics of the Y448H HCV NS5B Polymerase Mutant in Patients Treated with the HCV Inhibitor Tegobuvir (GS-9190)

Svarovskaia¹

2013

J Antivir Antiretrovir

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Tegobuvir (GS-9190) is a novel hepatitis C virus (HCV) non-nucleoside NS5B polymerase inhibitor that effectively inhibits HCV replication in HCV-infected genotype (GT) 1 patients. The NS5B Y448H mutation was the most frequent mutation selected in patients receiving tegobuvir. In this study, we employed allele-specific PCR (AS-PCR) to monitor Y448H kinetics and estimate the pre-existing Y448H levels in HCV-infected patients and replicon cells. Y448H AS-PCR was developed with a 0.5% assay cutoff to test replicon cells treated with tegobuvir and samples from HCVinfected GT 1 patients who received 8-days of tegobuvir monotherapy. By population sequencing, Y448H was not detected at baseline in serum samples from any of the 65 patients enrolled in the study. Using the more sensitive AS-PCR, Y448H was assessed in 62/65 patients at baseline and detected at >0.5% in 5/62 patients. Longitudinal patient samples were tested to monitor the Y448H replication kinetics during the 8-day tegobuvir monotherapy. The replication kinetics of the mutant virus were used to extrapolate a median baseline Y448H frequency of 0.025% (-3.6 log 10). For the in vitro selection of GT 1b tegobuvir-treated replicon cells, pre-existing Y448H levels were similarly estimated at 0.015% (-3.8 log 10). Pre-existing levels of the drug-resistant variant Y448H suggest a maximal 3.6 log 10 HCV RNA reduction during monotherapy with optimal doses of NS5B non-nucleoside polymerase inhibitors that select for Y448H. These results aid in the prediction of the patient viral response and study designs to best achieve maximal antiviral response.

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Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

Cited by 8 publications

References 20 publications

New real-time-PCR method to identify single point mutations in hepatitis C virus

New real-time-PCR method to identify single point mutations in hepatitis C virus

Antiviral response and resistance analysis of treatment‐naïve HCV‐infected patients receiving single and multiple doses of GS‐9190

Viral Kinetics of the Y448H HCV NS5B Polymerase Mutant in Patients Treated with the HCV Inhibitor Tegobuvir (GS-9190)

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