Pooled Prevalence of NS5A Resistance-Associated Substitutions in Chronic HCV Genotype 3 Infection: A Study Based on Deposited Sequences in GenBank

Abstract

Using direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) infection results in a high treatment response rate. However, several factors can significantly alter this outcome such as resistance-associated substitutions (RASs) in HCV NS5A gene. This study aimed to evaluate the prevalence of naturally occurring RASs of NS5A in HCV genotype 3 (HCV-3) sequences isolated from individuals with chronic HCV-3 infection. All the registered sequences in the GenBank under “NS5A” AND “Hepacivirus C” query were evaluated and screened, those which followed our inclusion criteria were enrolled in our pooled analysis. The retrieved sequences of included studies were evaluated for substitutions, RASs, and RASs conferring >100 resistance fold change (RASs >100 × ) in NS5A amino acid positions 24, 28, 30, 31, 62, 92, and 93. From 7 enrolled studies, a total of 370 HCV-3a isolates were retrieved and investigated. Forty-eight (13.0%, 95% CI = 9.9–16.8%) isolates harbored NS5A RASs. Moreover, Y93H was the only NS5A RAS >100 × observed in 13 (3.5%, 95% CI = 2.0–5.9%) retrieved sequences. The low frequency of naturally occurring NS5A RASs, especially those with clinical relevance (RASs >100 × ), among individuals with HCV-3 infection and the high rate of treatment response to DAAs suggest not to investigate every individual with HCV-3 infection for NS5A RASs before treatment.

Introduction

Hepatitis C virus (HCV) is a 9.7 kb positive-sense, single-stranded RNA structured virus. It has only one open reading frame (ORF), which encodes three structural proteins (Core, E1, and E2) and seven nonstructural proteins (P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B).^1–4 According to the WHO fact sheet updated in July 2018, about 71 million individuals are suffering from chronic hepatitis C globally. Cirrhosis and hepatocellular carcinoma (HCC) are the leading causes of mortality among these individuals.⁵ Due to the high replication rate and lack of proofreading feature of HCV NS5B polymerase, HCV shows an intrinsic predisposition to mutate. These mutations will result in the existence of slightly different viral variants of the same species known as quasispecies.⁶ This phenomenon has led to the circulation of very high genetically variant forms of HCV genotypes and subtypes worldwide.^7,8 These variants have different geographical distributions, pathogenesis, and response to antiviral treatments.⁹ There are seven different HCV genotypes: genotypes 1, 2, and 3 are distributed worldwide and is accounted for more than 85% of all recorded HCV infections worldwide,⁹ genotypes 4 and 5 are mainly found in Africa, and genotype 6 is endemic in Asia.¹⁰ The HCV genotype 7 was isolated from the patients coming from Central Africa, Democratic Republic of Congo.^11,12

The first approved therapy for HCV was the utilization of interferons (IFN), which had less than 20% rate of success.^13,14 Using alpha-IFN and ribavirin (RBV) simultaneously, it was possible to achieve higher sustained virological response (SVR) rates.¹⁵ However, response rate to IFN+RBV is highly unpredictable in different patients, and the treatment is accompanied with many side effects. This could be caused by the treatment regimens, disease-related factors, host factors, and viral factors.^16,17 Direct-acting antiviral agents (DAAs) are the latest treatment against HCV infection and can cure more than 95% of patients.^18,19 DAAs are a group of antiviral agents that directly act with HCV nonstructural proteins and prevent them from performing their roles.²⁰ Based on what protein they bind to, DAAs are categorized into three groups: NS3 protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors. The combination of DAAs, PegIFN, and RBV would result in a 90% chance of reaching SVR, and in case of combination of two or three DAAs as an IFN-free regimen, the treatment response would increase to more than 95%.^21,22 DAA failure is an unfortunate event, which occurs due to a variety of situations and is generally associated with the presence of resistance-associated variant (RAV).^23–25 RAV species are defined by the presence of one or more resistance-associated substitution (RAS) in their genome.²⁴ RASs are amino acid substitutions, which can adversely impact the treatment response rate to DAAs.^26–28

HCV genotype 3 (HCV-3) is one of the most problematic genotypes to be treated with DAA-based regimens. Currently, the most affected regions struggling with HCV-3 infection are South Asia, Russia, and Australia.²⁹ Also in Western countries, specific groups such as people who inject drug are more susceptible to this virus.^30,31 Individuals with HCV-3 infection show a higher SVR rate to PegIFN plus RBV than patients with HCV-1 and HCV-4 infections; however, the SVR rate in HCV-3 infection would not exceed more than 80%.^32,33 The SVR rate in HCV-3 infection significantly increases to >95% when DAAs are utilized as a course of IFN-free HCV antiviral therapy.³⁴ Reaching a high SVR rate in individuals with HCV-3 infection is highly dependent on NS5A RASs.^28,35 To the best of our knowledge, a few studies have investigated the prevalence of NS5A RASs in HCV-3 infection.^36–38 This could be due to reasons such as lower prevalence of HCV-3 infection than HCV-1 genotype and geographical limitations.

This pooled analysis aimed to retrieve NS5A sequences from published studies indexed in PubMed to evaluate the frequency of naturally occurring HCV NS5A RASs in DAA-naive patients with chronic HCV-3 infection based on the sequences obtained from direct Sanger population sequencing method.

Methods

Search strategy

The GenBank of nucleotide database of the National Center for Biotechnology Information (NCBI) at www.ncbi.nlm.nih.gov/nuccore was comprehensively searched for the following query “NS5A”[All Fields] AND “Hepacivirus C”[porgn] on April 22, 2018. The retrieved sequences were screened for the available annotated PubMed identifier (PMID) of the PubMed-indexed publication of the study. Then, the PMIDs were searched in PubMed for retrieval of the publications.

Study selection

The following criteria must have been met before the selection of any study: the studies must have investigated HCV-3, participants of each study must have been chronically infected with HCV-3, and the studies must have registered 10 or more sequences of HCV-3 isolates, which were obtained from a studied population. Moreover, studies investigated the nucleotide sequence span between NS5A amino acid positions 24–93 were eligible for the pooled analysis. The reported sequences of each study must have been obtained from direct Sanger population sequencing with cutoff higher than 20–30% for detection of the minor viral population.

All the studies that had either one or more of the following criteria were excluded: studies with history of treatment with NS5A inhibitors; studies that had clonal assessment of viral quasispecies; studies that had investigated HCV genotypes other than 3 (including those with mixed HCV genotypes); studies that have reported rare subtypes and viral recombinant variants; studies that performed sequential sampling, all case report, review, meta-analysis, and editorial articles; studies that have not covered the desired NS5A sequences; studies that had participants with acute HCV, HCC, or liver transplantation; studies with single-source infection; and studies that had performed deep sequencing with cutoff for detection <20% of minor variants.

All the inclusion and exclusion criteria were assessed separately by two individual authors (S.Gh. and A.H.) in three separate steps of title, abstract, and full-text screening. All discrepancies were resolved by consulting with the third author (H.Sh.).

Sequence extraction and analysis

All the included studies were screened for registered sequence accession numbers throughout the text. The selected sequences were transferred to molecular evolutionary genetics analysis software version 7.0 (MEGA 7.0). For each study, the sequences were aligned and subsequently trimmed for the desired NS5A sequence encoding amino acids 24–93 of HCV NS5A. As it was mentioned, the studies in which their isolates did not cover the desired region were excluded. The genotype of the included isolates was confirmed by phylogenetic analysis, and the isolates with genotypes other than 3 were excluded. Finally, the nucleotide alignments were translated to NS5A protein and investigated for three subsets of substitutions: (1) Any NS5A substitutions in the amino acid positions 24, 28, 30, 31, 62, 92, and 93; (2) NS5A RASs: the substitutions causing more than two resistance fold change to at least one of the NS5A inhibitors; (3) NS5A RASs >100 × : the substitutions causing more than 100 resistance fold change to at least one of the NS5A inhibitors. The consensus for the amino acid sequence of NS5A (Table 1) was obtained from the alignments. The criteria for selection of NS5A RASs and RASs >100 × were obtained from the European Association for the Study of the Liver recommendations on treatment of hepatitis C 2018³⁹ and the review article by Sorbo et al.,⁴⁰ respectively. The NS5A RASs and NS5A RASs >100 × are listed in Table 1.

Table 1.

The Included NS5A Amino Acid Positions, the Consensus Amino Acid for Each Position, NS5A Resistance-Associated Substitutions, and the NS5A RASs with >100 Resistance Fold Change

Amino acid position	Cons. ^a	NS5A RAS ^b	NS5A RAS >100 × ^c	Ref.
24	S	F	—	^39,40
28	M	T/K	—
30	A	K/S	—
31	L	F/I/M/V	I/V
62	S	L	—
92	E	K	—
93	Y	H	H

Consensus amino acid was selected based on the alignment of the included sequences.

Amino acid substitutions conferring >2 resistance fold change for at least one of the NS5A inhibitors. NS5A RASs were selected from European Association for the Study of the Liver recommendations on treatment of hepatitis C 2018.³⁹

Amino acid substitutions conferring >100 resistance fold change for at least one of the NS5A inhibitors. NS5A RASs >100 × were selected from the review article by Sorbo et al.⁴⁰

Cons., consensus; RAS, resistance-associated substitution; Ref., reference; A, alanine; T, threonine; V, valine; E, glutamate; H, histidine; I, isoleucine; K, lysine; Y, tyrosine; F, phenylalanine; M, methionine; S, serine; L, leucine.

Data extraction and statistical analysis

The following data were extracted from the included studies: publication date, study and sampling locations, HCV subtype, and accession numbers. The following data were extracted from sequence alignments: NS5A amino acid substitutions at positions 24, 28, 30, 31, 62, 92, and 93. All the data mentioned above were analyzed via SPSS version 20. The correlations between NS5A substitutions (including RASs and RASs >100 × ) were calculated using the Pearson correlation coefficient. A p-value of <0.05 was considered to be statistically significant.

Results

Study screening and selection

A total of 29,090 sequences were retrieved by searching the GenBank. We managed to obtain 259 PMIDs from retrieved sequences. Screening of titles and abstracts led to excluding 212 articles. Full-texts and eligibility of the remaining 47 articles were elaborately assessed. Finally, additional 40 articles were excluded from this study, and only 7 articles with a total of 370 isolates were enrolled in this study. The details of study selection and screening are summarized in Figure 1.

FIG. 1.

The details of study selection and screening. ^aThe retrieved isolates initially consisted of 374 sequences, including 4 HCV-3b isolates, which were excluded from final analysis due to their insignificant population.

Characteristics of the included studies

Seven studies were enrolled in this pooled analysis, all of which were published between 2002 and 2015. Five of the enrolled studies were conducted in European countries (France,⁴¹ United Kingdom,⁴² Germany,³⁸ Switzerland,⁴³ and Sweden³⁷), and the other two studies were conducted in the United States³⁶ and Thailand.⁴⁴ The pooled data from these seven studies were consisted of 374 retrieved isolates from patients who were chronically infected with HCV-3. We came across four HCV-3b isolates, due to their insignificant size, all of which were excluded from our analysis. Characteristics of the included studies are presented in Table 2.

Table 2.

The Characteristics of the Included Studies

Study	Ref.	Study location	Publication date	Sampling locations	No. of included patients with HCV-3a	Accession No.
Castelain et al.	⁴¹	France	2002	N/A	27	AF320787–AF3200813
Rauch et al.	⁴³	Switzerland	2009	Australia, Switzerland, UK	72	FJ931538–FJ931609
Humphreys et al.	⁴²	UK	2009	UK	18	GQ356200–GQ356217
Kumthip et al.	⁴⁴	Thailand	2010	Thailand	18	HM042064–HM042081
Hernandez et al.	³⁶	USA	2012	North America, Europe, Australia	104	JX674827–JX674930
Walker et al.	³⁸	Germany	2015	Germany	111	KR082016–KR082126
Lindstrom et al.	³⁷	Sweden	2015	Sweden	20	KP212069–KP212088

N/A, not available.

Prevalence of NS5A substitutions in amino acid positions 24, 28, 30, 31, 62, 92, and 93

Among 370 retrieved isolates, 164 (44.3%, 95%CI = 39.3–49.4%) had one or more substitutions at NS5A amino acid positions 24, 28, 30, 31, 62, 92, and 93. Three (0.8%) isolates of 370 enrolled isolates had 3 substitutions at the given positions, 24 (6.5%) isolates had 2 substitutions, and 137 (37.0%) isolates had only 1 substitution. Seventeen (4.6%) isolates had substitution at position 24, 3 (0.8%) isolates had substitution at position 28, 33 (8.9%) isolates had substitution at position 30, 126 (34.1%) isolates had substitution at position 62, 1 (0.2%) isolate had substitution at position 92, and 14 (3.7%) isolates had substitution at position 93. We did not observe any substitutions among retrieved isolates for position 31. Substitutions of positions 28 and 93 (p = 0.007), and 30 and 93 (p < 0.001) were correlated. Details of NS5A amino acid substitutions and their frequencies are given in Table 3.

Table 3.

The Frequency of NS5A Substitutions Including Resistance-Associated Substitutions and the RASs with >100 Resistance Fold Change

Amino acid position	NS5A non-RAS substitution		NS5A RAS 2 × –100 × ^a		NS5A RAS >100 × ^b
Amino acid position	Substitution	No. (%)	Substitution	No. (%)	Substitution	No. (%)
24	—	—	S24F	4 (1.1)	—	—
	S24L	5 (1.4)	—	—	—	—
	S24K	1 (0.3)	—	—	—	—
	S24T	1 (0.3)	—	—	—	—
	S24A	6 (1.6)	—	—	—	—
28	M28V	2 (0.5)	—	—	—	—
28	A28L	1 (0.3)	—	—	—	—
30	—	—	A30K	16 (4.3)	—	—
	—	—	A30S	4 (1.1)	—	—
	A30M	1 (0.3)	—	—	—	—
	A30T	8 (2.2)	—	—	—	—
	A30V	3 (0.8)	—	—	—	—
	A30L	1 (0.3)	—	—	—	—
62	—	—	S62L	14 (3.8)	—	—
	S62T	72 (19.5)	—	—	—	—
	S62P	15 (4.1)	—	—	—	—
	S62V	7 (1.9)	—	—	—	—
	S62I	5 (1.4)	—	—	—	—
	S62N	1 (0.3)	—	—	—	—
	S62A	5 (1.4)	—	—	—	—
	S62M	3 (0.8)	—	—	—	—
	S62Q	2 (0.5)	—	—	—	—
	S62F	2 (0.5)	—	—	—	—
92	E92A	1 (0.3)	—	—	—	—
93	—	—	—	—	Y93H	13 (3.5)
93	Y93F	1 (0.3)	—	—	—	—

NS5A amino acid substitutions conferring 2–100 resistance fold change for at least one of the NS5A inhibitors.

NS5A amino acid substitutions conferring >100 resistance fold change for at least one of the NS5A inhibitors.

No., number; A, alanine; T, threonine; V, valine; E, glutamate; H, histidine; I, isoleucine; K, lysine; Y, tyrosine; F, phenylalanine; M, methionine; N, asparagine; S, serine; L, leucine; P, proline; Q, glutamine.

Prevalence of NS5A RASs

Forty-eight (13.0%, 95%CI = 9.9–16.8%) retrieved isolates had one or two NS5A RASs. Three (0.8%) harbored 2 RASs, and 45 (12.2%) harbored only 1 RAS. There was no RAS among retrieved isolates at positions 28, 31, and 92. We observed 4 (1.1%) isolates with RAS at position 24, 20 (5.4%) isolates with RAS at position 30, 14 (3.8%) isolates with RAS at position 62, and 13 (3.5%) isolates with RAS at position 93. None of the observed NS5A RASs were correlated. The details of NS5A RASs in studied positions are given in Table 3.

Prevalence of NS5A RAS conferring more than 100 fold change

The only NS5A substitution at amino acid 93 (Y93H) had more than 100 resistance fold change in this study. Thirteen (3.5%, 95%CI = 2.0–5.9%) isolates harbored this NS5A RAS >100 × .

Discussion

Although DAAs have proven to be a formidable treatment for HCV infection, less than 5% of individuals who have gone under DAA-containing regimens do not achieve SVR.^45–50 To improve response rate, it has been proposed to individualize approach for each treatment course by identification of treatment predictors such as RAS predispositions, advanced liver fibrosis,⁵¹ HIV coinfection,⁵² and infection with HCV genotype 3.⁵³

The exact working mechanism of NS5A inhibitors has not been clarified. However, it is presumed that NS5A inhibitors will bind to domain I of NS5A protein and severely diminish its capacity to become hyperphosphorylated and dimerized.⁵⁴ The linker region on NS5A (amino acids 24–93) is a hot zone for occurring RASs. The most commonly observed NS5A RASs in patients with treatment failure were in positions 24, 28, 30, 31, 92, and 93, although the prevalence and frequency of which is different among HCV genotypes.^25,26,55 By binding to domain I, these NS5A inhibitors adversely affect three critical steps in HCV life cycle. First, they will prevent the formation of replication complex, second, the virion assembly and release will be reduced, and third, RNA degradation will be accelerated.⁵⁶

In this study, NS5A and its relative RASs were evaluated from nucleotide sequences retrieved from the GenBank. Our data revealed that around 44.3% of enrolled DAA-naive patients chronically infected with HCV-3 had one to three NS5A amino acid substitutions; among included isolates, only 13.0% had NS5A RASs. In this study, Y93H with a frequency of 3.5% was the only RAS, which has more than 100-fold change effect on NS5A inhibitors activity. Previous studies had similar findings as well. Malta et al.⁵⁷ investigated 31 individuals who were infected with HCV-3, they reported that 16.1%, 6.5%, and 3.2% of the enrolled population harbored A30K, Y93H, and A30S substitutions, respectively. On a similar study, Smith et al.⁵⁸ reported that 8.9% of their studied population had A30K and 12.3% had Y93H substitutions. They also observed L31M in 1.8%, P58S in 2.4%, M28T in 0.6%, and Q54H in 0.2% of their samples. Finally, in a study conducted by Bertoli et al.,⁵⁹ the following frequency for NS5A substitutions was reported: A30K (4.0%), A30V (0.6%), L31P (0.6%), A62L (8.5%), and Y93H (4.5%).

Even with new DAA-based treatment regimens, individuals who are infected with HCV-3 have proven to be more challenging to reach SVR than other HCV genotypes.^18,19 The existence of specific NS5A RASs and RASs >100 × such as Y93H is a significant contributor in failure to reach SVR. In a real-life study using sofosbuvir+daclatasvir, the response rate was moderately lower in patients with baseline Y93H than those without Y93H (75% vs. 96%).³⁵ Another study utilized sofosbuvir/velpatasvir as an alternative approach to treat HCV-3 patients with Y93H RAS and found that this approach could result in a higher SVR rate of 84%.⁶⁰ American Association for the Study of Liver Diseases (AASLD) guidelines for management of hepatitis C have recommended that individuals who are infected with HCV-3 and have cirrhosis should be checked for Y93H substitution before taking any DAA-based regimen combinations, to clarify whether extended treatment period or additional antivirals such ribavirin is necessary.⁶¹

This study had several limitations. First of all, few studies have investigated HCV-3. This has led to ambiguity regarding resistance to antiviral therapy in HCV-3 infection, NS5A RASs frequency, and best strategy to tackle this disease. Furthermore, in this study, we were unable to identify a standard sequence analysis method for those studies that have been included in this pooled analysis. Moreover, studies that were investigated and reported HCV-3 infection was probably suffering from a mixture of HCV genotypes and subtypes at the same time, but due to fluctuation of HCV population in that individuals, the sequencing method might have only detected a portion of these populations and the rest of them might have been interoperated as background noise in sequence analysis. Finally, in our search protocol to obtain relative sequences from the GenBank, we used the NS5A query as a keyword. The annotation of a region before uploading a sequence is not mandatory in the NCBI; this could result in losing sequences of this region that have not had annotation of word “NS5A.” Although these limitations are significant, to the best of our knowledge, we have raised HCV-3 investigated population from 110 to 370. By considering the absence of data on this criterion, this study has reached significant findings regarding HCV-3. Furthermore, we set strict inclusion and exclusion criteria to assure that only validated data would be enrolled in this pooled analysis. Finally, we have analyzed the data via SPSS software, which enabled us to calculate the correlation of substitutions, a feature that is missed in meta-analysis studies.

In conclusion, the naturally occurring NS5A RASs Y93H, A30K and S62L were present in 3.5%, 4.3%, and 3.8% of the total enrolled population. The above-mentioned NS5A RASs, specially clinically relevant Y93H with >100 resistance fold change, were observed in small number of isolates, and with consideration of the high SVR rate to newly developed DAAs, it is not cost-effective to screen all individuals with HCV-3 infection for NS5A RASs. Screening for NS5A substitutions will be valuable in case of failure following the treatment with NS5A inhibitor-containing regimens, which probably resulted by the presence of such RASs. Information on the frequency of NS5A RASs is very important as it can guide therapeutic strategies and diagnostic decisions by assessing resistance in candidates when treating hepatitis C. Standardization of definitions for NS5A RASs is needed to improve resistance analyses and facilitate comparisons of substitutions between studies worldwide.

Footnotes

Disclosure Statement

The authors have no conflicts of interest relevant to this article.

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