Genetics and Molecular Biology
Sociedade Brasileira de Genética
Resistance mutations of NS3 and NS5b in treatment-naïve patients infected with hepatitis C virus in Santa Catarina and Rio Grande do Sul states, Brazil
Volume: 43, Issue: 1
DOI 10.1590/1678-4685-GMB-2018-0237
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Hepatitis C virus (HCV) infection is a worldwide health problem. Nowadays, direct-acting antiviral agents (DAAs) are the main treatment for HCV; however, the high level of virus variability leads to the development of resistance-associated variants (RAVs). Thus, assessing RAVs in infected patients is important for monitoring treatment efficacy. The aim of our study was to investigate the presence of naturally occurring resistance mutations in HCV NS3 and NS5 regions in treatment-naïve patients. Ninety-six anti-HCV positive serum samples from blood donors at the Center of Hematology and Hemotherapy of Santa Catarina State (HEMOSC) were collected retrospectively in 2013 and evaluated in this study. HCV 1a (37.9%), 1b (25.3%), and 3a (36.8%) subtypes were found. The frequency of patients with RAVs in our study was 6.9%. The HCV NS5b sequencing reveled 1 sample with L320F mutation and 4 samples with the C316N/R polymorphism. The analysis of the NS3 region revealed the D168A/G/T (3.45%), S122G (1.15%), and V55A (2.3%) mutations. All samples from genotype 3a (36.8%) presented the V170 I/V non-synonymous mutation. In conclusion, we have shown that mutations in NS3 and NS5b genes are present in Brazilian isolates from therapy-naïve HCV patients.

Andrade, Rocha, Fontana-Maurell, Costa, Ribeiro, de Godoy, Ferreira, Tanuri, Brindeiro, and Alvarez: Resistance mutations of NS3 and NS5b in treatment-naïve patients infected with hepatitis C virus in Santa Catarina and Rio Grande do Sul states, Brazil


Hepatitis C virus (HCV) infection is a worldwide health problem. According to the Global Hepatitis Report, from the World Health Organization (WHO), approximately 71 million people have chronic HCV infection, and nearly 399,000 people die each year, mostly due to cirrhosis or hepatocellular carcinoma (WHO, 2017). HCV has a high genetic heterogeneity and is classified into seven genotypes (1 to 7) and 67 subtypes (Smith et al., 2014). The genotype distribution depends on geographical location and risk groups (Cantaloube et al., 2005). Genotype 1 is the most frequent in Brazil, followed by genotypes 3 and 2 (Campiotto et al., 2005; Lampe et al., 2013).

There is no vaccine available for preventing HCV infections. The main antiviral treatment until 2011, was PEGylated interferon-alfa (αPeg-IFN) alone or in combination with ribavirin, leading to a sustained virological response (SVR) in 50% of treated patients, depending on the virus genotype causing the HCV infection (Peres-da-Silva et al., 2012; Paolucci et al., 2013; Gross et al., 2018). Nowadays, direct-acting antiviral agents (DAAs) have been approved for HCV infection treatment, with an average SVR above 95%, at least for genotypes 1 and 4 (Leuw and Stephan, 2018). In Brazil, DAAs were incorporated by the Ministry of Health for the treatment of hepatitis C under the Unified Health System (SUS) since 2015 (Ministério da Saúde, 2018). Unfortunately, there is little data about the efficacy of DAAs in Brazil, with some information found in the study by Sette Jr et al. (2017).

The primary targets of DAAs are nonstructural proteins essentials for HCV replication, which include the NS3 protease, NS5B polymerase, and NS5A protein (Paolucci et al., 2013; Lontok et al., 2015). However, a challenge in HCV treatment is the emergence of viral resistance mutations that reduces susceptibility of the virus to DAA therapies (Hoffmann et al., 2015; Gededzha et al., 2017). The development of resistance-associated variants (RAVs) is due to the high level of virus variability, from the combination of the virus’ high replication rate, low RNA polymerase fidelity rate, and selective pressure for drug or immunomediated treatment (Peres-da-Silva et al., 2012; Paolucci et al., 2013; Gededzha et al., 2017).

The presence of RAVs in patients not yet under treatment has been reported previously in different countries (Peres-da Silva et al., 2010; Paolucci et al., 2013; Zeminian et al., 2013; Gededzha et al., 2017). In addition, a systematic review regarding HCV resistance-associated substitutions and their clinical relevance was published recently (Sorbo et al., 2018). Therefore, assessing RAVs in infected patients is important for monitoring the efficacy of therapy (Loggi et al., 2017) and the epidemiology of HCV in Brazil. Thus, the aim of our study was to investigate the presence of naturally occurring resistance mutations in HCV NS3 and NS5 regions in treatment-naïve patients.

Materials and Methods

The Center of Hematology and Hemotherapy of Santa Catarina State (HEMOSC) is currently responsible for the nucleic acid testing for HIV, HCV, and HBV in samples from blood donors from Santa Catarina and Rio Grande do Sul states. Annually, HEMOSC receives around 300 thousand blood donations. A total of 96 samples that were positive for HCV in 2013 were used for this study, retrospectively.

HCV RNA was extracted from plasma previously conserved at -80 °C using a molecular biology workstation (BioRobot MDx, Qiagen), with the Qiamp one-for-all nucleic acid kit (Qiagen), according to the manufacturer’s instructions. Plasma HCV RNA was quantified using COBAS/Taqman HCV Test v2.0 (Roche).

Genotyping/subtyping was performed by amplifying and sequencing a 339-bp amplicon of the NS5b region, according to Cantaloube et al. (2005). The nucleotide sequences obtained were analyzed in the Geno2pheno [HCV] (Kalaghatgi et al., 2016) for genotypes and subtypes, and possible resistance against licensed DAAs.

The amplification of the entire NS3 region of the HCV genome, followed by a second PCR was performed as described previously (Peres-da-Silva et al., 2010), using primers specific to subtypes 1a, 1b, and 3a. The nucleotide sequences obtained from each subtype were analyzed for drug resistance in the Geno2pheno [HCV] (Kalaghatgi et al., 2016).

The statistical program SPSS (IBM SPSS Statistics Base 22.0) was used. Multivariate analysis of variance (ANOVA) was applied to compare means of continuous variables with normal distribution (p<0.05).

Results and Discussion

From the 96 HCV-positive samples collected, nine did not have enough material to perform the assays and were excluded from the analysis. Eighty-seven samples were used for genotyping and analysis of NS3 and NS5b regions.

From the 87 samples evaluated, 33 (37.9%) were of genotype 1a, 22 (25.3%) were of genotype 1b, and 32 (36.8%) were of genotype 3a. Genotype 1 (1a plus 1b) was the most frequent, followed by genotype 3, a result that is in agreement with what was previously reported in Brazil (Campiotto et al., 2005; Lampe et al., 2013; Nishiya et al., 2014). We did not find genotypes 2, 4, and 5, known to be less frequent in Brazil. Sixty-three samples were successfully geno- and subtyped by the NS3 and NS5b region, and there was no disagreement between the HCV genotypes in both regions. Twenty-four samples had no amplification of the NS5b region, even with an alternative protocol (Sandres-Sauné et al., 2003), and were thus genotyped according to Peres-da-Silva (2010) protocol (NS3 region). This amplification divergence has already been discussed in Larrat et al. (2013), who reported a failure of some quantitative RT-PCR assays to detect or amplify correctly the NS5b region in some strains of HCV, even when using three sets of primers covering two different regions. This could be explained by the great variety of viruses, the use of primers not suitable for these peculiar strains, or by a mixed infection in the plasma sample.

The mean viral loads were 5.31 log IU/mL for genotype 1a, 5.18 log IU/mL for 1b, and 5.38 log IU/mL for 3a. There was no difference in viral load between the genotypes (p =0.6). The detected HCV genotypes and viral loads are both important predictors for therapeutic outcomes. It has been reported that patients infected with genotype 1 are more likely to have higher viral loads than those infected with genotype 2 and 3 (Scott et al., 2007; Soriano et al., 2008; Nishiya et al., 2014). In contrast to our results, in a study carried out with blood donors from São Paulo, the viral load from genotype 3a (5.22 log10 IU/mL) had a lower log mean than genotype 1a (5.99 log10 IU/ mL) (p =0.0002) and genotype 1b (6.35 log10 IU/mL) (Nishiya et al., 2014), in agreement with a previous report.

The frequency of patients with RAVs in our study (6.9%) was intermediate when compared with other Brazilian studies among HCV chronic carriers not treated with protease inhibitors (3.2% - 18.9%) (Hoffmann et al., 2013; Nishiya et al., 2014). Figure 1 present the frequency of specific NS3 and NS5b resistance-associated variants found in this study by HCV subtype.

Frequency of specific NS3 and NS5b resistance-associated variants found
in this study by HCV subtype.
Figure 1
Frequency of specific NS3 and NS5b resistance-associated variants found in this study by HCV subtype.

The HCV NS5b sequencing from 63 samples was analyzed. The L320F mutation was present in only one sample (1.59%) of genotype 1a. L320F is known to confer low resistance to sofosbuvir and sofosbuvir associated with mericitabine (Paolucci et al., 2013), which are associated to treatment failure in clinical trials (Constantino et al., 2015). In a previous study, L320F single mutation had no significant impact on the 50% effective concentration (EC50) and EC90 values for mericitabine (≤2.7 fold) (Tong et al., 2014). To our knowledge, this is the first time that mutation L320F is reported as naturally occurring in DAA treatment-naïve patients and it should be monitored due to treatment failures reported previously in clinical trials.

The polymorphism C316N/R was present in 4 samples (6.35%) of genotype 1b. C316N is reported to confer low level of resistance to sofosbuvir (Paolucci et al., 2013, Lontok et al., 2015). C316N mutation has been associated with a 10-fold increase in EC50 to a new experimental non-nucleoside drug, HCV796 (Castilho et al., 2011). Previous studies have found variable prevalence of C316N in Brazil, from 3.85% (Peres-da-Silva et al., 2017) to 11.6% (Castillho et al., 2011), 16.3% (Noble et al., 2017), and 24% (Castilho et al., 2011), and higher prevalence in North America (16.81%), Europe (7.47%), and Asia (49.71%) (Peres-da-Silva et al., 2017). The higher prevalence of mutations in genotype 1b has been reported previously and it was due to the presence of C316N (Paolucci et al., 2013; Peres-da-Silva et al., 2017).

Although it was not a goal of our study, we also observed the presence of D244N, Q309R, and A333E mutations conferring resistance to ribavirin and interferon in 42 samples (57.14%). Twenty samples (26.98%) presented Q309R, and three (1.58%) A333E. Seventeen were Q309R and D244N, two were Q309R and A333E, and two were triple positive. In a previous work, the most frequent mutation observed in Brazil was Q309R, present in all HCV subtypes (Castilho et al., 2011); in our study, it was present in 38 samples. No double mutations in the NS5b region conferring resistance to DAAs was observed in our samples. The emergence of double or triple-sites RAVs in the clinics is threatening the effectiveness of anti-HCV therapies, as published previously (Gane et al., 2016).

The analysis of the NS3 region revealed the mutations D168A/G/T (3.45%, 3/87), S122G (1.15%, 1/87), and V55A (2.3%, 2/87) that confer resistance to asunaprevir, boceprevir, grazoprevir, simeprevir, and paritaprevir (Zeminian et al., 2013; Lontok et al., 2015; Sorbo et al., 2018). V55A was observed at a higher frequencies in previous works, at 4.1% (Moreira et al., 2018) and 6% (Nishiya et al., 2014), from DAA naïve patients and blood donors, respectively, in São Paulo, and at 6% in Europe (Bartels et al., 2013). The V55A variant has been shown to confer 6.9-fold increase in EC50 to boceprevir (Vermehren et al., 2012). S122G was found in a higher frequency in Spain (6.23%) and China (85.48%) (Li et al., 2017). An in vitro study has shown that S122G did not reduce susceptibility to simeprevir (Izquierdo et al., 2014). However, another study showed that S122G reduced the susceptibility by 0.5-fold (Lenz et al., 2010). In São Paulo, the D168G mutation was found in one of the 125 HCV infected blood donors’ samples (Nishiya et al., 2014). In a transient susceptibility assay, D168G conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) for HCV genotype 1a. For genotype 1b, a higher level of asunaprevir-associated resistance was observed ranging from 170- to 400-fold relative to wild-type control. (McPhee et al., 2012).

No mutations were found that confer resistance to glecaprevir and voxilaprevir, drugs known to present a high barrier to resistance (Sorbo et al., 2018). However, this study found samples with mutations that decrease the susceptibility of HCV to these drugs, which reinforces the importance of monitoring HCV RAVs.

Samples from genotype 3a presented no mutations that confer or diminish resistance to glecaprevir and voxilaprevir, drugs recommended for treatment of patients infected with this genotype. This means that the standard protocol for treatment of patients with genotype 3 should be effective in Santa Catarina and Rio Grande do Sul. However, we found the non-synonymous mutation V170 I/V in all 32 samples of this genotype. In agreement with our results, Peres-da-Silva (2010) found that 100% (32/32) of the HCV 3a sequences contained the V170I substitution. Few data is available on effects of V170I substitution. The conservative substitution at this site was detected in up to 45% of patients infected with HCV genotype 1 (López-Labrador et al., 2008)

A different pattern of resistance associated with NS3 protease domain in therapy-naïve patients was previously reported in Brazil. V36L mutation was found in genotype 1a at a frequency of 5.6%, in 1b at 100% (Peres-da-Silva et al., 2010), and in genotypes 2, 3, 4, and 5 V36L mutation was found as a genetic signature with frequency of 99% (Vidal et al., 2016); in another work, V36L was found at a frequency of 4% in genotype 1a (Nishiya et al., 2014). T54S mutations were found in 4.1% of genotype 1a (Peres-da-Silva et al., 2010) and 100% in genotype 2 (Vidal et al., 2016). The samples investigated by our study presented none of these mutations. The Q80K, a common mutation in the USA (40%) (Bartels et al., 2013) that confers resistance to simeprevir, was not found in our study, but has previously been reported at prevalence ranging from 0.4% to 2.7% in Brazil (Nishiya et al., 2014; Vidal et al., 2015; Moreira et al., 2018). There is a strong geographic correlation regarding the frequency of the Q80K substitution (Moreira et al., 2018), and for this reason, studies from different geographic regions are of great importance, especially in a large country as Brazil.

Of all the samples evaluated, only one sample of genotype 1b showed mutation in the genes NS3 and NS5b, conferring resistance to sofosbuvir (C316N) and decreased susceptibility to gazoprevir (Y56F). This shows the importance of studying both NS3 and NS5 proteins when evaluating or choosing the therapy strategy for HCV-positive patients. Patients carrying combinations of resistance mutations are of particular interest, since they may increase the possibility of failure in the treatment with DAAs.

The frequency of resistance mutations and genotypes was twice as high among patients with subtype 1a compared to those with subtype 1b. A similar result was found in a study with blood donor’s samples from São Paulo (Nishiya et al., 2014). In addition, a higher frequency of virological failure for subtype 1a compared to 1b has been reported (Pawlotsky et al., 2011).

At last, several polymorphisms not associated with resistance to DAAs were observed in our study (Table 1), and previously reported by others (Constantino et al., 2015). Polymorphisms, prior to therapy, are part of the quasispecies population in infected individuals, and may not alter viral fitness (Peres-da-Silva et al., 2012; Paolucci et al., 2013; Nishiya et al., 2014).

Table 1
HCV polymorphic sites distribution according to genotype.
166A/S166 A/T/R459
* Some samples had more than one variant.

In conclusion, we have shown that mutations in NS3 and NS5b domains are present in Brazilian isolates from therapy-naïve patients, in this case, blood donors with unknown HCV infection. Monitoring the presence of RAVs is important for predicting the response to antiviral therapy, and regional discernment can help determine local policies for treatment. The results presented here will help ensure therapy strategies that are more successful for HCV-infected patients in Santa Catarina and Rio Grande do Sul states in Brazil.


The authors acknowledge financial support from Fundação Oswaldo Cruz, RJ.


Associate Editor: Carlos F.M. Menck

Competing interests

The authors declare that no competing interests exist.

Author contributions

PA, AT, RB, AGPF, EA designed the study; EA, DR, MFM, EC, MR conducted the experiments and analyzed the data; EA, DTG, data analysis and wrote the manuscript; AT, PA, critically analyzed the data and manuscript writing. All authors read and approved the final version of the manuscript.



    Bartels DJ, Sullivan JC, Zhang EZ, Tigges AM, Dorrian JL, Meyer S, Takemoto D, Dondero E, Kwong AD, Picchio G, 2013. . Hepatitis C virus variants with decreased sensitivity to direct-acting antivirals (DAAs) were rarely observed in DAA-naïve patients prior to treatment. J Virol 87: , pp.1544-1553


    Ministério da Saúde 2018. Protocolo Clínico e Diretrizes Terapêuticas para Hepatite C Ministério da SaúdeBrasilia , pp.108


    Campiotto S, Pinho JR, Carrilho FJ, Da Silva LC, Souto FJ, Spinelli V, Pereira LM, Coelho HS, Silva AO, Fonseca JC 2005. . Geographic distribution of hepatitis C virus genotypes in Brazil. Braz J Med Biol Res 38: , pp.41-49


    Cantaloube JF, Gallian P, Attoui H, Biagini P, Micco P, Lamballerie X 2005. . Genotype distribution and molecular epidemiology of hepatitis C virus in blood donors from southeast France. J Clin Microbiol 43: , pp.3624-3629


    Castilho MCB, Martins AN, Horbach IS, Perez RM, Figueiredo FAF, Pinto PTA, Nabuco LC, Lima DB, Tanuri A, Porto LC, 2011. . Association of hepatites C virus NS5B variants with resistance to new antiviral drugs among untreated patients. Mem Inst Oswaldo Cruz 106: , pp.968-975


    Constantino A, Spada E, Equestre M, Bruni R, Triarelli E, Coppola N, Sagnelli C, Sagnelli E, Ciccaglione R 2015. . Naturally occuring mutations associated with resistance to HCV NS5b polymerase and NS3 protease inhibitors in treatment-naïve patients with chronic hepatitis C. Virol J 12: , pp.186


    Gededzha MP, Mphahlele MJ, Blackard JT, Selabe G 2017. . Prevalence of NS5B resistance mutations in hepatitis C virus (HCV) treatment naïve South Africans. Hepat Mon 17: , pp.e14248


    Gross MJ, Härter G, Backhus J, Zizer E, Seufferlein T, Ludwig L, Dikopoulos N 2018. . Direct-acting antiviral agents in the treatment of chronic hepatitis C – “real-life” experience from an academic centre and two specialized clinical practices. Z Gastroenterol 56: , pp.351-360


    Hoffmann L, Ramos JA, Souza EV, Araujo Ramos AL, Villela-Nogueira CA, Urményi TP, Tanuri A, Rondinelli E, Silva R 2013. . Dynamics of resistance mutations to NS3 protease inhibitors in a cohort of Brazilian patients chronically infected with hepatitis C virus (genotype 1) treated with pegylated interferon and ribavirin: a prospective longitudinal study. Virol J 10: , pp.57


    Hoffmann L, Faffe DS, Lima JFC, Capitanio TA, Cabral BCA, Ürméndyi TP, Coelho HSM, Rondinelli E, Villela-Nogueira CA, Silva R 2015. . No correspondence between resistance mutations in the HCV-NS3 protease at baseline and early telaprevir-based triple therapy. BBA Clinical 3: , pp.146-151


    Gane E, Ben Ari Z, Mollison L, Zuckerman E, Bruck R, Baruch Y, Howe AY, Wahl J, Bhanja S, Hwang P, 2016. . Efficacy and safety of grazoprevir + ribavirin for 12 or 24 weeks in treatment-naive patients with hepatitis C virus genotype 1 infection. J Viral Hepat 23: , pp.789-797


    Izquierdo L, Helle F, François C, Castelain S, Duverlie G, Brochot E 2014. . Simeprevir for the treatment of hepatitis C virus infection. Pharmgenomics Pers Med 7: , pp.241-249


    Kalaghatgi P, Sikorski AM, Knops E, Rupp D, Sierra S, Heger E, Neumann-Fraune M, Beggel B, Walker A, Timm J, 2016. . Geno2pheno[HCV] – A Web-based interpretation system to support hepatitis C treatment decisions in the era of direct-acting antiviral agents. Plos One 11: , pp.e0155869


    Lampe E, Lewis-Ximenez L, Espirito-Santo MP, Delvaux NM, Pereira SA, Peres-da-Silva A, Martins RMB, Soares MA, Santos AF, Vidal LL, 2013. . Genetic diversity of HCV in Brazil. Antivir Ther 18: , pp.435-444


    Larrat S, Poveda JD, Coudret C, Fusillier K, Magnat N, Signori-Schmuck A, Thibault V, Morand P 2013. . Sequencing assays for failed genotyping with the versant hepatitis C virus genotype assay (LiPA), version 2.0. J Clin Microbiol 51: , pp.2815-2821


    Lenz O, Verbinnen T, Lin TI, Vijgen L, Cummings MD, Lindberg J, Berke JM, Dehertogh P, Fransen E, Scholliers A, 2010. . In vitro resistance profile of the hepatitis C virus NS3/4A protease inhibitor TMC435. Antimicrob Agents Chemother 54: , pp.1878-1887


    Leuw P, Stephan C 2018. . Protease inhibitor therapy for hepatitis C virus-infection. Expert Opin Pharmacother 19: , pp.577-587


    Li Z, Chen ZW, Li H, Ren H, Hu P 2017. . Prevalence of hepatitis C virus-resistant association substitutions to direct-acting antiviral agents in treatment-naïve hepatitis C genotype 1b-infected patients in western China. Infect Drug Resist 10: , pp.377-392


    Loggi E, Galli S, Vitale G, Donato RD, Vukotic R, Grandini E, Margotti M, Guarneri V, Furlini G, Galli C, 2017. . Monitoring the treatment of hepatitis C with directly acting antivirals by serological and molecular methods. PLOS One 12: , pp.e0187755


    Lontok E, Harrington P, Howe A, Kieffer T, Lennerstrand J, Lenz O, McPhee F, Mo H, Parkin N, Pilot-Matias T, 2015. . Hepatitis C virus drug resistance-associated substitutions: state of the art summary. Hepatology 62: , pp.1623-1632


    López-Labrador FX, Moya A, Gonzàlez-Candelas F 2008. . Mapping natural polymorphisms of hepatitis C virus NS3/4A protease and antiviral resistance to inhibitors in worldwide isolates. Antivir Ther 13: , pp.481-94


    McPhee F, Friborg J, Levine S, Chen C, Falk P, Yu F, Hernandez D, Lee M, Chaniewski S, Sheaffer A, 2012. . Resistance analysis of the hepatitis C virus NS3 protease inhibitor asunaprevir. Antimicrob Agents Chemother 56: , pp.3670-3681


    Moreira RC, Santos APT, Lisboa-Neto G, Mendes-Corrêa MCJ, Lemos MF, Malta FM, Santana RAF, Dastoli GTF, Castro VFD, Pinho JRR 2018. . Prevalence of naturally occurring amino acid substitutions associated with resistance to hepatites C vírus NS3/NS4A protease inhibitors in São Paulo state. Arch Virol 163: , pp.2757-2764


    Nishiya AS, Almeida-Neto C, Ferreira SC, Alencar CS, Di-Lorenzo-Oliveira C, Levi JE, Salles NA, Mendrone A, Sabino EC 2014. . HCV genotypes, characterization of mutations conferring drug resistance to protease inhibitors, and risk factors among blood donors in São Paulo, Brazil. PLoS One 9: , pp.e86413


    Noble CF, Malta F, Lisboa-Neto G, Gomes-Gouvêa MS, Leite AGB, Castro VFD, Santana RAF, Carrilho FJ, Mendes-Correa MC, Pinho JRR 2017. . Natural occurrence of NS5B inhibitor resisance-associated variants in Brazilian patients infected with HCV or HBV and HIV. Arch Virol 162: , pp.165-169


    Paolucci S, Fiorina L, Mariani B, Gulminetti R, Novati S, Barbarini G, Bruno R, Baldanti F 2013. . Naturally occurring resistance mutations to inhibitors of HCV NS5A region and NS5B polymerase in DAA treatment-naïve patients. Virol J 10: , pp.355


    Pawlotsky JM 2011. . Treatment failure and resistance with direct-acting antiviral drugs against hepatitis C virus. Hepatology 53: , pp.1742-1751


    Peres-da-Silva A, Almeida AJ, Lampe E 2010. . Mutations in hepatitis C virus NS3 protease domain associated with resistance to specific protease inhibitors in antiviral therapy naïve patients. Arch Virol 155: , pp.807-811


    Peres-da-Silva A, Almeida AJ, Lampe E 2012. . Genetic diversity of NS3 protease from Brazilian HCV isolates and possible implications for therapy with direct-acting antiviral drugs. Mem Inst Oswaldo Cruz 107: , pp.254-261


    Peres-da-Silva A, Brandão-Mello CE, Lampe E 2017. . Prevalence of sofosbuvir resistance-associated variants in Brazilian and worldwide NS5B sequences of genotype-1 HCV. Antivir Ther 22: , pp.447-451


    Sandres-Sauné K, Deny P, Pasquier C, Thibaut V, Duvelier G, Izopet J 2003. . Determining hepatitis C genotype by analyzing the sequence of the NS5b region. J Virol Methods 109: , pp.187-93


    Sette H, Cheinquer H, Wolff FH, Araujo A, Coelho-Borges S, Soares SRP, Barros MFA 2017. . Treatment of chronic HCV infection with the new Direct Acting Antivirals (DAA): first report of a real world experience in southern Brazil. Ann Hepatol 16: , pp.727-733


    Scott JD, Gretch DR 2007. . Molecular diagnostics of hepatitis C virus infection: a systematic review. JAMA 297: , pp.724-732


    Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, Simmonds P 2014. . Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 59: , pp.318-327


    Sorbo MC, Cento V, Di Maio VC, Howe AYM, Garcia F, Perno CF, Ceccherini-Silberstein F 2018. . Hepatitis C virus drug resistance associated substitutions and their clinical relevance: Update 2018. Drug Resist Updat 37: , pp.17-39


    Soriano V, Mocroft A, Rockstroh J, Ledergerber B, Knysz B, Chaplinskas S, Peters L, Karlsson A, Katlama C, Toro C, 2008. . Spontaneous viral clearance, viral load, and genotype distribution of hepatitis C virus (HCV) in HIV-infected patients with anti-HCV antibodies in Europe. J Infect Dis 198: , pp.1337-1344


    Tong X, Le Pogam S, Li L, Haines K, Piso K, Baronas V, Yan JM, So SS, Klumpp K, Nájera I 2014. . In vivo emergence of a novel mutant L159F/L320F in the NS5B polymerase confers low-level resistance to the HCV polymerase inhibitors mericitabine and sofosbuvir. J Infect Dis 209: , pp.668-675


    Vermehren J, Susser S, Lange CM, Forestier N, Karey U, Hughes E, Ralston R, Tong X, Zeuzem S, Sarrazin C 2012. . Mutations selected in the hepatitis C virus NS3 protease domain during sequential treatment with boceprevir with and without pegylated interferon alfa-2b. J Viral Hepat 19: , pp.120-127


    Vidal LL, Santos AF, Soares MA 2015. . Worldwide distribution of the NS3 gene 80K polymorphism among circulating hepatitis C genotype 1 viruses: implication for simeprevir usage. J Antimicrob Chemother 70: , pp.2024-2027


    Vidal LL, Soares MA, Santos AF 2016. . NS3 protease polymorphisms and genetic barrier to drug resistance of distinct hepatites C virus genotypes from worldwide treatment-naïve subjects. J Viral Hepat 23: , pp.840-849


    Zeminian LB, Padovani JL, Corvino SM, Silva GF, Pardini MI, Grotto RMT 2013. . Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors. Mem Inst Oswaldo Cruz 108: , pp.13-17

Internet Resources


    WHO 2017. . WHO Global hepatitis report. mutations of NS3 and NS5b in treatment-naïve patients infected with hepatitis C virus in Santa Catarina and Rio Grande do Sul states, Brazil&author=Elisabete Andrade,Daniele Rocha,Marcela Fontana-Maurell,Elaine Costa,Marisa Ribeiro,Daniela Tupy de Godoy,Antonio G.P. Ferreira,Amilcar Tanuri,Rodrigo Brindeiro,Patrícia Alvarez,&keyword=Direct-acting antivirals,resistance-associated substitutions,blood donors,NS3,NS5b,&subject=Human and Medical Genetics,