Natural prevalence of NS3 gene resistance-associated substitutions (RASs) in patients with chronic hepatitis C from the state of Para/Brazil

Desir´ee Lopes da Silva, Heloisa Marceliano Nunes, Pedro Eduardo Bonfim Freitas*

Abstract

The resistance of hepatitis C virus (HCV) to direct-acting antiviral agents, used in chronic hepatitis C treatment, consists of a natural process resulting from resistance-associated substitutions (RASs) at specific amino acid regions. To identify and establish the natural prevalence of RASs in the NS3 gene in patients with chronic hepatitis C in the state of Para, northern Brazil. Molecular analysis was performed on a total of 35 patients ´ infected with HCV genotype 1, who were treatment-naive to protease inhibitors. HCV RNA was extracted from plasma and the NS3 region was amplified and submitted to DNA sequencing (Sanger). The general natural prevalence of RASs in the NS3 gene was 37.5 % (Y56F and S122T). The substitutions Y56F (34.3 %), S122T (3.1 %), V132I (15.6 %) and V170I (9.3 %) were identified. Y56F and S122T provide resistance to the protease inhibitors grazoprevir and simeprevir, respectively. All amino acid substitutions in the NS3 gene, including RASs, identified in patients from the state of Para were present in other Brazilian studies. The natural presence of RASs ´ in this study reflects the elevated genetic variability of HCV.

Keywords:
HCV
Direct acting antivirals Protease inhibitors
Antiviral drug resistance

1. Introduction

The hepatitis C virus (HCV) belongs to the family Flaviviridae, genus Hepacivirus, species Hepacivirus C and has an envelope and a genome formed by a simple RNA strand of approximately 9.6 kb with positive polarity that encodes a single polyprotein that is subsequently cleaved by cellular and viral proteases (Li and Chung, 2019). There are at least eight genotypes and several subtypes of the virus (Borgia et al., 2018). In 2019, the HCV classification into 8 genotypes and 86 subtypes was extended with the identification of 19 new subtypes in genotypes 2(9), 4 (5), 6(2), 1(1), 3(1) and 5(1). HCV genotype 1 is the most prevalent (46 %) genotype globally. (Hedskog et al., 2019)
In Brazil, genotypes 1a (40.9 %), 1b (30.2 %) and 3 (23.8 %) are considered the most prevalent, with the prevalence of other genotypes at less than 4%. In the South, Southeast and Central regions there is a higher prevalence of genotype 1a, while in the North region genotype 1b is the most prevalent (Nutini et al., 2020).
The World Health Organization estimates that approximately 71 million people are chronically infected with HCV worldwide and that approximately 400,000 individuals will die annually from chronic complications of this disease, mainly from cirrhosis and hepatocarcinoma (Geneva: World Health Organization, 2018).
The development of novel drugs, collectively now called direct- acting antiviral agents (DAAs), has created new approaches for the treatment of chronic hepatitis C. Preclinical and clinical studies have shown that the combined use of DAAs with pegylated interferon and ribavirin resulted in an increase in sustained virological response rates (SVR) (Asselah and Marcellin, 2012). Currently, the standard treatment for patients with chronic hepatitis C consists of the combined use of different classes of DAAs and is associated with high rates of SVR (Sarrazin, 2016; Asselah et al., 2018). The main targets of DAAs are key proteins in the HCV life cycle, in particular, the NS3/4A protein, the NS5A protein or the RNA-dependent NS5B polymerase (Asselah and Marcellin, 2011). Viral resistance to DAAs consists of a natural or adaptive process to HCV in response to unfavorable replication conditions. The reduction in HCV susceptibility to the inhibitory effect of direct-acting drugs results from mutations and amino acid substitutions able to alter the drug activity in its target protein region (Pawlotsky, 2011; Schneider and Sarrazin, 2014). The presence of natural polymorphism in an important region for the antiviral effect may confer reduced susceptibility to a specific DAA or a class of DAAs. The amino acid substitutions that confer resistance are called resistance-associated substitutions (RASs) (Pawlotsky, 2016; Feld, 2017).
The NS3 protein is a multifunctional enzyme essential for the HCV replication. It has serine protease activity on the amino-terminal domain NTPase/RNA helicase function in the carboxy-terminal domain. NS3 is a heterodimer along with the NS4A cofactor. This complex subsequently acts in the nonstructural region of the polyprotein, cleaving at four specific sites (Halfon and Locarnini, 2011). The NS3 protease peptidomimetic inhibitors of are able to block cleavage of viral polyprotein, competing with the natural substrate (Sorbo et al., 2018). In Brazil, the profile of natural resistance-associated substitutions to NS3 protease inhibitors in patients with chronic hepatitis C is still poorly understood. To date, most of the studies are concentrated in the southern regions. The present study aimed to identify and establish the natural prevalence of RASs in the NS3 gene in patients with chronic hepatitis C in state of Para, located in the western Amazon, northern Brazil. ´

2. Material and methods

2.1. Study location and ethical aspects

This was a prospective, observational-analytical and cross-sectional study, conducted on patients from the Santa Casa de Misericordia do ´ Para Foundation (FSCMP), a referral center for liver disease in the state. ´ Serum/plasma samples were collected at the Hepatology Department of the Evandro Chagas Institute (IEC) and subsequently stored at − 20 ◦C until molecular analysis. All molecular analyses were performed in the molecular biology laboratory of the IEC’s Hepatology Department. This study was approved by the Research Ethics Committee of the Evandro Chagas Institute, with approval number 1.815.535.

2.2. Patients

The fundamental criterion for the inclusion of patients in the sample was the confirmed diagnosis of chronic hepatitis C (positive anti-HCV and HCV-RNA antibodies for more than 6 months). This study sample includes the age group between 20 and 60 years of age. All patients included had HCV genotype 1 (1a and 1b) and were naive to treatment with NS3 protease inhibitors. Patients with positive samples for hepatitis B virus surface antigen (HBsAg) and/or antibodies to the HIV virus were excluded. This study was performed on a total of 35 pretreatment plasma samples, selected from patients diagnosed with chronic HCV infection.

2.3. RNA extraction

RNA extraction from serum samples (140 μl) was performed using a QIAamp Viral RNA Kit (Qiagen, Hilden, Germany), following the manufacturer’s recommendations. The extracted RNA was eluted at 60 μl and stored at − 70 ◦C.

2.4. Reverse transcription and PCR amplification

The HCV NS3 gene was partially amplified by a reverse transcription (RT) step attached to polymerase chain reaction (PCR), accompanied by a second PCR amplification (nested PCR), using specific primers for each subtype, which have already been previously described (Peres-da-Silva et al., 2010).
For RT-PCR, the Superscript III One-Step RT-PCR system (ThermoFisher, Massachusetts, USA) was used. The RT-PCR mix was formed by 10 μM of specific sense and antisense primers, 2X reaction mix, Super-Script ™ III RT / Platinum® Taq DNA Polymerase (4U/μL), nuclease- free ultra-pure water and 5 μL of viral RNA. The RT-PCR conditions were as follows: 50 ◦C for 30 min for reverse transcription, followed by an initial activation of DNA polymerase at 94 ◦C for 2 min. Subsequently, reaction conditions included 35 cycles of 94 ◦C for 15 s, 56 ◦C for 30 s, 68 ◦C for 90 s and a final extension of 68 ◦C for 5 min. Five microliters of the first PCR product was used in a second PCR, consisting of 10 μM of specific sense and antisense primers, 10x PCR buffer, 1.25 mM dNTP, 50 mM MgSO4 and Platinum® Taq High Fidelity (5 U/ μL). After an initial denaturation at 94 ◦C for 3 min, the DNA was amplified for 30 cycles of 94 ◦C for 30 s, 60 ◦C for 30 s, 72 ◦C for 90 s and a final extension of 72 ◦C for 7 min.
The PCR products, with an expected size of 765 base pairs (for both subtypes) were separated by electrophoresis on a 2% agarose gel, stained with SYBR® Safe DNA Gel Stain (ThermoFisher, Massachusetts, USA), and visualized using ultraviolet light

2.5. Nucleotide sequencing

The nested PCR products were purified using ExoSAP-IT ™ PCR Product Cleanup Reagent (Thermo Fisher, Massachusetts, USA). The purified PCR products were subjected to nucleotide sequencing reactions by the Sanger method, in both directions of the NS3 region using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s instructions. The sequencing reactions were performed on the ABI 3500 automatic DNA sequencer (Applied Biosystems).

2.6. Mutation analysis

The DNA sequences obtained were edited and aligned using the Geneious version 9.1 program and the amino acid sequences were deduced and obtained through the same program. These sequences (amino acids 1–181 from the NS3 region) were compared with the NS3 protease reference sequence, available in the NCBI database (GenBank). To check for the presence of resistance mutations for NS3 inhibitors, the sequences were analyzed to identify substitutions in amino acid residues associated with resistance and compare with those already described in the literature. The positions analyzed were: V36, Q41, F43, T54, V55, Y56, Q80, S122, V132, R155, A156, V158, D168, V170 and M175.

3. Results

According to the inclusion and signing criteria of the Free and Informed Consent Form, 35 patients chronically infected with HCV were selected. In relation to the sample studied, 57.1 % of the patients were male, and 42.9 % were female. The mean age was 59.9 years (± 9.2), varying between 39 and 77 years. The clinical and demographic characteristics of the patients are shown in Table 1. Analysis of RAS was performed only in those patients in which it was possible to amplify the NS3 region by nested PCR (n = 32). Subsequently, 25 samples of subtype 1b and 7 samples of subtype 1a were submitted to DNA nucleotide sequencing. Fifteen positions presenting clinically important RASs were analyzed and amino acid substitutions were observed in 4 positions (positions Y56, S122, V132 and V170). In positions 56, 122, 132 and 170, the substitutions Y56F, S122T, V132I and V170I were identified. Table 2 shows the natural prevalence of the identified substitutions and the association with resistance to DAAs. These substitutions were identified only in subtype 1b. The general natural prevalence of RASs was 37.5 % (12/32), and the presence of amino acid substitutions (with or without association with resistance to NS3 protease inhibitors) was observed in 62.5 % (20/32) of the sequences analyzed.

4. Discussion

The aim of the present study was to identify and establish the natural prevalence of RASs in the NS3 gene in patients with chronic hepatitis C who were not treated with DAAs from the state of Para. In 62.5 % of the ´ patients who were analyzed, the identified amino acid substitutions. A study carried out in Rio de Janeiro/Brazil by Costa et al. involving 73 patients before treatment with the DAAs simeprevir, daclastasvir and/or sofosbuvir revealed a general prevalence of RAS of 13.7 % in the NS3 gene (Costa et al., 2019). In the present study, the prevalence of RAS before treatment with protease inhibitors was 37.5 %. Moreira et al. carried out a study with 859 samples of treatment-naive patients to DAAs living in the state of Sao Paulo/Brazil and identified at least one ˜ RAS in the NS3 region in 9.4 % of the samples (Moreira et al., 2018).
The natural polymorphism Q80 K, frequently observed in patient genotype 1a, reduces the efficiency of the simeprevir DAA in patient genotype 1a cirrhotic treated with simeprevir and sofosbuvir for 12 weeks (Sorbo et al., 2018). The prevalence of the Q80 K polymorphism in patients infected with genotype 1 is quite varied geographically (Sarrazin et al., 2015). In the European and American populations, for example, the Q80 K resistance variant in genotype 1a, which confers resistance to simeprevir, was found in 25 % and 35 % of pretreatment patients with simeprevir, respectively (Pessoa et al., 2014ˆ ). Studies in the Brazilian population revealed a low prevalence of the resistant variant Q80 K (Pessoa et al., 2014ˆ ; Vidal et al., 2015), providing epidemiological data that may be related to the nondetection of this polymorphism in the present study.
In a Brazilian study, Zeminian et al. identified the presence of resistance mutations in 18.9 % of 37 patients (7 patients) with HCV genotype 1a without previous treatment with protease inhibitors. (Zeminian et al., 2013) Similar to the present study, amino acid substitutions (T54A, T54S, V55A and R155K and A156T) were identified in 4 different positions (codons 54,55,155 and 156) in the NS3 gene (Zeminian et al., 2013). In contrast, in the present study, resistance mutations in patients infected with subtype 1a were not identified. The absence of substitutions associated with resistance may be related to the small number of subtype 1a sequences (n = 7) that were analyzed.
In one of the first studies carried out in Brazil on the genetic variability in the NS3 region of HCV conducted by Peres-da-Silva et al. 114 patients with chronic hepatitis C who were treatment-naive to protease inhibitors and infected with subtype 1a (n = 48), 1b (n = 53) or 3a (n = 13) were analyzed (Peres-da-Silva et al., 2010). In the study by Peres-da-Silva et al. the V170I substitution was present in 46 of the 48 isolates of subtype 1a, in 19 of the 53 isolates of subtype 1b and in all isolates of subtype 3a (Peres-da-Silva et al., 2010). Although not associated with resistance to protease inhibitors, the V170I substitution was observed in the present study, with a prevalence of 9.3 %.
Grazoprevir (MK-5172) is a second generation pangenotype NS3/4A protease inhibitor approved for the treatment of chronic hepatitis C. In patients who failed to treat with grazoprevir, the RASs mainly identified in the NS3 region were V36L/M, Y56F/H, Q80K/L, R155I/K/L/S, A156G/M/T/V, V158A and D168A/C/E/G/K/N/V/Y, particularly in patients infected with genotypes 1a and 1b (Sorbo et al., 2018). In the present study, RAS Y56F, which confers resistance to grazoprevir, had a natural prevalence of 34.3 %.
In a study by Kliemann et al., 798 sequences were analyzed to assess the occurrence of resistance mutations in pretreatment sequences of the HCV NS3 region, stored in a European database (euHCVdb), and the Q80 K variant was the mutation most prevalent (Kliemann et al., 2016). Among the most prevalent mutations, variants Y56 F (15.93 %) and V132I (23.28 %) were also observed in subtype 1b (Kliemann et al., 2016). The V132I mutation was identified in the present study, with a prevalence of 15.6 %, and is not associated with resistance to protease inhibitors. Aguiar et al., in a study involving pretreatment samples of 144 Brazilian patients, identified mutations Y56F and V132I, with a prevalence of 17.3 % and 14.7 %, respectively (Aguiar et al., 2019).
In addition to the RAS Y56F, the present study identified the S122T mutation, with has a natural prevalence of 3.1 % and is associated with resistance to simeprevir. Simeprevir (TMC-435) consists of a first generation NS3/4A protease inhibitor, used to treat patients chronically infected with HCV genotypes 1 and 4 (Sorbo et al., 2018). Patients with genotype 1b who failed treatment with simeprevir had RASs mainly in positions 168 (D168A/E/F/H/N/T/V) and 122 (S122R/T) (Sorbo et al., 2018).
Chen et al. reported the global prevalence of RASs for DAAs, using information recorded in the GenBank database (NCBI Nucleotide Database) (Cheng et al., 2016). A total of 1459 sequences from patients chronically infected with HCV and treatment-naive to DAA were analyzed. Of this total, 1048 sequences were from patients with genotype 1 (687 with subtype 1a and 361 with subtype 1b) (Cheng et al., 2016). The occurrence of RASs in the NS3 region was 40 % of the total sequences analyzed, and was associated with resistance to boceprevir (12.1 %), telaprevir (5.5 %), simeprevir (29.8 %) and paritaprevir (2.5 %) (Cheng et al., 2016).
In the NS3 region, the variant Q80 K (which confers resistance to simeprevir) was the RAS most frequently observed in sequences with genotype 1a (37.6 %; 258/687) (Cheng et al., 2016). In sequences with genotype 1b, the S122T variant was the most frequently observed RAS (5.5 %; 20/361) (Cheng et al., 2016). In a study involving five different reference centers for infectious diseases in Brazil, 247 patients infected with HCV virus treatment-naive to protease inhibitors (monoinfected and coinfected HCV-HIV-1) were analyzed for the occurrence of resistance mutations in the NS3 region (Neto-Lisboa et al., 2015). In both groups, variants in the S122 position (S122G, S122N and S122T) were identified (Neto-Lisboa et al., 2015).
In this study all RASs were identified only in subtype 1b. Regarding treatment, studies show that in addition to the 12-week therapy with DAAs, previously untreated patients with genotype 1b without cirrhosis can be treated with ombitasvir, paritaprevir and ritonavir plus dasabuvir for 8 weeks (Welzel et al., 2017). Another alternative for reducing the duration of treatment is the use of grazoprevir-elbasvir for 8 weeks in naïve patients with genotype 1b and non-severe fibrosis (Abergel et al., 2020).
In addition to genotype 1, recently pangenotypic DAAs regimens also provide safe and effective treatment for patients infected with HCV genotype 2, 3, 4, 5 or 6. Infected patients with genotypes 4, 5 and 6 have been neglected in the past, for many reasons, including the low prevalence in high-income countries where the DAAs have been developed (Asselah et al., 2017). In the era of DAAs, the investigation of RASs in specific patient populations, such as those infected with genotypes 4, 5 and 6, becomes important for the definition of the molecular epidemiology of RASs (Asselah et al., 2017).

4.1. Conclusions

In summary, the molecular analysis carried out in patients with chronic hepatitis C who were not treated with protease inhibitors revealed substitutions of amino acids in the NS3 gene, showing a correlation of that the natural presence of RASs with the present study corresponds to a reflection of the high genetic variability of HCV. All mutations identified in patients in the state of Para were present in other ´ Brazilian studies. Subsequent studies with a greater number of patients, in particular for infected with subtype 1a, become necessary for the identification of other RASs and for a better definition of the natural prevalence of RASs in these patients. In addition to providing information on the natural prevalence of RASs in a state in northern Brazil, the analysis of RASs naturally produced during the HCV life cycle also gains importance in the context of current treatment of chronic hepatitis C, as it could assist in determining the best therapeutic strategy with DAAs adopted by certain patients.

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