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Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Na ï ve Patients

View Article: PubMed Central - PubMed

ABSTRACT

Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

No MeSH data available.


PCR primers positions and amplicons lenght for NS3 and NS5B HCV genomic regions, according to the HCV1b reference sequence (Con1 AJ238799).
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ijms-17-01416-f002: PCR primers positions and amplicons lenght for NS3 and NS5B HCV genomic regions, according to the HCV1b reference sequence (Con1 AJ238799).

Mentions: Viral RNA was extracted from 140 µL serum using the QIAmp viral RNA extraction kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kits protocol (Applied Biosystems, Foster City, CA, USA). The 2X reverse transcription Master Mix (2 µL 10× RT Buffer, 0.8 µL 25× dNTP Mix (100 mM), 2 µL 10× RT Random Primers, 1 µL MultiScribe™ Reverse Transcriptase, 1 µL RNase Inhibitor and RNase-free water) was added at 10 µL RNA to make up a final volume of 20 µL. Synthesized cDNA was amplified by PCR technique using in-house-developed primers specific for NS3 genomic region (650 bp), covering all NS3/4A positions involved in drug resistance using GoTaq® DNA Polymerase (Promega, Madison, WI, USA). The nested PCR amplification for both rounds was performed under the following conditions: 1.25 units of GoTaq® DNA Polymerase, 50 mM KCl, 30 mM Tris-HCl, 1.5 mM Mg2+, 200 µM of each dNTP, 0.2 µM sense primer, 0.2 µM antisense primer. Six microliters of cDNA was used for the first round of PCR. NS3 primers were the following: Forward1 (outer) 5′-GGAGGGAGATACATCTGG-3′; Reverse1 (outer): 5′-GTTCAGGACAAGCACCTTAT-3′. Thermocycling conditions consisted in a denaturation step at 95 °C for 5 min, followed by 35 cycle at 95 °C for 30 s; 62 °C for 30 s; 72 °C for 1 min; a final elongation cycle of 7 min at 72 °C and finally a hold at 4 °C. Five microliters of the first round of PCR products was used for nested-PCR, using the same thermocycling conditions. NS3 nested primers were the following: Forward2 (inner) 5′-ACTCCTCGCGCCTATTACG-3′; Reverse2 (inner) 5′-TTAGTGCTCTTGCCGCTACC-3′ (Figure 2). In order to obtain enough material for sequencing, the second PCR was performed in duplicate. These PCR products were quantified by semi-fluidic electrophoresis (Agilent® Bioanalyzer®, Santa Clara, CA, USA), using Agilent High Sensitivity DNA Kit and diluted to in order to obtain 100 ng of HCV cDNA in a final volume of 40 µL.


Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Na ï ve Patients
PCR primers positions and amplicons lenght for NS3 and NS5B HCV genomic regions, according to the HCV1b reference sequence (Con1 AJ238799).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037695&req=5

ijms-17-01416-f002: PCR primers positions and amplicons lenght for NS3 and NS5B HCV genomic regions, according to the HCV1b reference sequence (Con1 AJ238799).
Mentions: Viral RNA was extracted from 140 µL serum using the QIAmp viral RNA extraction kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kits protocol (Applied Biosystems, Foster City, CA, USA). The 2X reverse transcription Master Mix (2 µL 10× RT Buffer, 0.8 µL 25× dNTP Mix (100 mM), 2 µL 10× RT Random Primers, 1 µL MultiScribe™ Reverse Transcriptase, 1 µL RNase Inhibitor and RNase-free water) was added at 10 µL RNA to make up a final volume of 20 µL. Synthesized cDNA was amplified by PCR technique using in-house-developed primers specific for NS3 genomic region (650 bp), covering all NS3/4A positions involved in drug resistance using GoTaq® DNA Polymerase (Promega, Madison, WI, USA). The nested PCR amplification for both rounds was performed under the following conditions: 1.25 units of GoTaq® DNA Polymerase, 50 mM KCl, 30 mM Tris-HCl, 1.5 mM Mg2+, 200 µM of each dNTP, 0.2 µM sense primer, 0.2 µM antisense primer. Six microliters of cDNA was used for the first round of PCR. NS3 primers were the following: Forward1 (outer) 5′-GGAGGGAGATACATCTGG-3′; Reverse1 (outer): 5′-GTTCAGGACAAGCACCTTAT-3′. Thermocycling conditions consisted in a denaturation step at 95 °C for 5 min, followed by 35 cycle at 95 °C for 30 s; 62 °C for 30 s; 72 °C for 1 min; a final elongation cycle of 7 min at 72 °C and finally a hold at 4 °C. Five microliters of the first round of PCR products was used for nested-PCR, using the same thermocycling conditions. NS3 nested primers were the following: Forward2 (inner) 5′-ACTCCTCGCGCCTATTACG-3′; Reverse2 (inner) 5′-TTAGTGCTCTTGCCGCTACC-3′ (Figure 2). In order to obtain enough material for sequencing, the second PCR was performed in duplicate. These PCR products were quantified by semi-fluidic electrophoresis (Agilent® Bioanalyzer®, Santa Clara, CA, USA), using Agilent High Sensitivity DNA Kit and diluted to in order to obtain 100 ng of HCV cDNA in a final volume of 40 µL.

View Article: PubMed Central - PubMed

ABSTRACT

Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

No MeSH data available.