Whole genome sequencing of Hepatitis C virus subtype 6xa and NS5A resistance mutations in a genotype 3b relapse case after direct-acting antiviral therapy in India

Whole genome sequencing of Hepatitis C virus subtype 6xa and NS5A resistance mutations in a genotype 3b relapse case after direct-acting antiviral therapy in India | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 14 August 2025 V1 Latest version Share on Whole genome sequencing of Hepatitis C virus subtype 6xa and NS5A resistance mutations in a genotype 3b relapse case after direct-acting antiviral therapy in India Authors : Shilpa J. Tomar 0000-0003-3850-2316 [email protected] , Abhranil Gangopadhayya , Bhupen Barman , Onkar Ghuge , Bhanu Teja Korra , Ciyona Bastin , Baiahunlang Lamare , Ashwini Y. Ramdasi , Wihiwot V. Lyngdoh , Kavita Lole , and Harpreet Kaur Authors Info & Affiliations https://doi.org/10.22541/au.175517118.80099254/v1 182 views 116 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Introduction and Objectives: Pangenotypic direct-acting antivirals (DAA) are effective against highly prevalent Hepatitis C virus (HCV) subtypes, but have been clinically validated almost exclusively in high-income countries. Unusual HCV subtypes may carry natural polymorphisms, potentially impacting DAA susceptibility. We conducted full-genome characterization and resistance analysis of unusual HCV subtypes in patients receiving DAA treatment. Patients and methods: In this prospective hospital-based study, eligible patients were screened for anti-HCV antibodies and active infection was confirmed by diagnostic 5’NCR-based HCV RNA detection. Genotyping was performed by core region sequencing, and viral load quantified by real-time PCR. For whole genome sequencing, multiplex primers were designed using alignments of global reference sequences. Sequencing was carried out using the Oxford Nanopore Technology platform. Phylogenetic analysis used multiple sequence alignment and the HCV-GLUE resource for resistance-associated substitution (RAS) analysis. Results: Predominant genotype was genotype 3 in 64.3% (n=45); genotype 6 in 21.4% (n=15); and genotype 1 in 14.2% (n=10). Unusual HCV subtype 6xa was detected in two patients and showed no NS5A resistance mutations. One genotype 3b patient relapsed at 24 weeks post-DAA treatment completion and carried NS5A resistance-associated substitutions 30K and 31M both at baseline and at relapse, conferring high-level resistance to NS5A inhibitors. Conclusion: This is the first report from India of whole genome sequencing of HCV subtype 6xa. The identification of NS5A resistance mutations in the 3b relapse case underscores challenges for global HCV elimination strategies . Whole genome sequencing of Hepatitis C virus subtype 6xa and NS5A resistance mutations in a genotype 3b relapse case after direct-acting antiviral therapy in India Shilpa Tomar#* 1 , Abhranil Gangopadhayya# 1 , Bhupen Barman* 2 , Onkar Ghuge 1 , Bhanu Teja Korra 1 , Ciyona Bastin 1 , Baiahunlang Lamare 2, Ashwini Ramdasi 1 , Valarie W Lyngdoh 2 , Kavita S. Lole 1 , Harpreet Kaur 3 #Equal authorship Department(s) and institution(s) 1 Hepatitis Group, ICMR- National Institute of Virology (NIV), Pune, India 2 North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences (NEIGRIHMS), Shillong, India 3 Indian Council of Medical Research, New Delhi, India *Corresponding Authors: Equal correspondence: Dr. Shilpa Tomar, Scientist C, Hepatitis Group, ICMR-National Institute of Virology, Microbial Containment Complex (MCC), Pune-411021, Maharashtra, India Email: [email protected] Dr. Bhupen Barman, Professor and Head, Department of General Medicine, All India Institute of Medical Sciences, Guwahati- 781101 Email: [email protected] Abstract Introduction and Objectives: Pangenotypic direct-acting antivirals (DAA) are effective against highly prevalent Hepatitis C virus (HCV) subtypes, but have been clinically validated almost exclusively in high-income countries. Unusual HCV subtypes may carry natural polymorphisms, potentially impacting DAA susceptibility. We conducted full-genome characterization and resistance analysis of unusual HCV subtypes in patients receiving DAA treatment. Patients and methods: In this prospective hospital-based study, eligible patients were screened for anti-HCV antibodies and active infection was confirmed by diagnostic 5’NCR-based HCV RNA detection. Genotyping was performed by core region sequencing, and viral load quantified by real-time PCR. For whole genome sequencing, multiplex primers were designed using alignments of global reference sequences. Sequencing was carried out using the Oxford Nanopore Technology platform. Phylogenetic analysis used multiple sequence alignment and the HCV-GLUE resource for resistance-associated substitution (RAS) analysis. Results: Predominant genotype was genotype 3 in 64.3% (n=45); genotype 6 in 21.4% (n=15); and genotype 1 in 14.2% (n=10). Unusual HCV subtype 6xa was detected in two patients and showed no NS5A resistance mutations. One genotype 3b patient relapsed at 24 weeks post-DAA treatment completion and carried NS5A resistance-associated substitutions 30K and 31M both at baseline and at relapse, conferring high-level resistance to NS5A inhibitors. Conclusion: This is the first report from India of whole genome sequencing of HCV subtype 6xa. The identification of NS5A resistance mutations in the 3b relapse case underscores challenges for global HCV elimination strategies . Keywords: Hepatitis C virus, whole genome sequencing, NS5A resistance mutations, genotype 6xa, genotype 3b relapse Abbreviations: HCV: Hepatitis C virus DAA: Direct-acting antiviral RAS: Resistance-associated substitutions SVR: Sustained virologic response SOF: Sofosbuvir VEL: Velpatasvir LDV: Ledipasvir DCV: Daclatasvir Introduction HCV is a major health challenge, estimated to infect 71 million people worldwide(1). It belongs to Flaviviridae family, with a positive-sense single-stranded RNA genome spanning approximately 9.6 kb. HCV exhibits profound genetic diversity, attributed mainly to lack of proofreading activity of its RNA-dependent RNA polymerase(2), resulting in Darwinian evolutionary process. Continuous viral population diversification leads to selection of fittest variants; permanent selection of variant strains in epidemiologically or geographically diverse populations, resulting in further diversification of HCV genotypes i.e. phylogenetic clades and subclades(3). Chronic HCV infection gradually progresses to liver fibrosis with cumulative probability of approximately 45% at 20 years to develop hepatocellular carcinoma(4). Traditionally, interferon (IFN) and ribavirin combination therapy was used to treat HCV, but in 2014, a major paradigm shift came with introduction of IFN-free treatment with DAAs, improving sustained virologic response (SVR) to >95%. Currently approved DAA pangenotypic therapy are designed to be effective against ‘usual’ or common HCV genotypes(3), however ‘unusual’ genotypes are prevalent in certain geographical regions. Though frequency of late relapse with DAA therapy is still largely unknown, there have been recent reports of the same(5–7). Among the 8 HCV genotypes, genotype 6 is the most genetically divergent, attributable to its inherent intra and inter-genotype recombination property(8). HCV genotype 3, representing 22-30% of all infections, leads to higher rates of steatosis, faster cirrhosis progression and higher conversion to hepatocellular carcinoma. In the current DAA era, cure rates achieved for genotype 3 have relatively lagged behind other genotypes, up until sofosbuvir and daclatasvir approval in 2015, and recent fixed dose combination of velpatasvir and sofosbuvir(9,10). In WHO regions of Southeast Asia, Africa and Western Pacific, approximately 30 million patients are living with HCV infection, with prevalence of unusual HCV genotypes representing a substantial proportion. In India, prevalence rate of HCV viremia, according to global estimates in 2015 was 0.5%, affecting approximately 4.7 to 10.9 million people(11). In a meta-analysis including 327 studies, HCV prevalence rate was estimated by anti-HCV antibodies positivity and found to be 0.85% in community studies, 0.88% in pregnant females and 0.44% in asymptomatic blood donors(12). Community-based study results were only available from three states and associated with considerable heterogeneity thus might not reflect true nationwide prevalence. In spite of these limitations, data suggests a huge burden of HCV disease in a country having more than 1.3 billion individuals(13). In contrast to the West, with genotype 1 predominance, in India HCV genotype 3 is predominant and associated with higher progression to cirrhosis, hepatocellular carcinoma and all-cause mortality(14). Of all globally available DAAs, only four are available and marketed in India i.e. sofosbuvir (SOF), velpatasvir (VEL), ledipasvir (LDV), and daclatasvir (DCV), thus limiting our armamentarium. In the current study, we conducted genotyping and viral load of HCV infected patients receiving DAA therapy, who were followed up at various time-points, post initiation of therapy and further conducted whole genome sequencing along with drug resistance analysis, in unusual genotypes identified. Materials and Methods Our study was a hospital based, cohort study conducted for a period of three years. Blood samples were collected from HCV infected, treatment-naïve patients presenting to the Medicine OPD of North Eastern Indira Gandhi Regional Institute of Health & Medical Sciences, (NEIGRIHMS), Shillong. Laboratory testing was conducted at the Hepatitis Group of ICMR-National Institute of Virology (NIV) Pune, India. This study was approved by reviewing board of the institutional human ethics committees (Reference ethics ID: NEIGR/IEC/M9/F13/19 and NIV/IEC/Oct/2019/D-1). All investigations were conducted according to ethical guidelines of Declaration of Helsinki. Patients were followed up and samples were collected at different time-points, as follows: Baseline (before initiating treatment), end of treatment (EOT), 12 weeks after completion of treatment (SVR12) and 24 weeks after completion of treatment (SVR12). All patients in the study received Sofosbuvir (SOF) (a nucleoside analogue inhibiting RdRp, i.e. NS5b) and Velpatasvir (VEL) (NS5A inhibitor) therapy for 3 months. HCV RNA extraction, RT-PCR, quantitation, sequencing and phylogenetic analysis RNA was extracted from 140 μL of serum using QIAamp viral RNA columns (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. For detection of HCV RNA, nested reverse transcriptase (RT)-PCR was carried out with 5’NCR primers as described by Bukh et al(15) (nucleotides [nt]-276 to -21; 256 bp). For quantitative detection (viral load) of HCV specific RNA, real time RT PCR test was done using artus® HCV RG RT-PCR Kit. For HCV genotyping, core region (405 bp) was amplified and sequenced using primers as decribed by Lole et al(16) by Sanger’s method. Briefly, RNA-positive PCR products were purified in a column with a gel extraction kit (Qiagen, Hilden, Germany) and used as templates for sequencing in Big-Dye Terminator cycle sequencing ready reaction kit (Applied Biosystems). Samples were analyzed on an automated sequencer (3130xl Applied Biosystems). The core region sequence from nt 1 to 360 was taken for analysis. HCV whole genome sequencing Subtype specific primers were designed by aligning HCV whole genome reference sequences available globally, and using Molecular Evolutionary Genetics Analysis (MEGA) software version 5.2. PrimalScheme version v.3.0.2. online software (17) was used for multiplex primer designing, keeping a high Guanine-Cytosine content setting, with preferred amplicon length of 1000 bp. Primers for each subtype were constituted into A and B pools, according to scheme given by PrimalScheme software. After RNA extraction, r everse Transcription was done to prepare complementary DNA (cDNA) from viral RNA using LunaScript® RT SuperMix (New England Biolabs, Massachusetts, USA), by mixing 4.0 µl of enzyme and 16.0 µl of viral RNA, with a thermal profile of 25°C for 2 min, 55°C for 10 min, 95°C for 1 min, and a 4°C hold. The prepared cDNA was used for PCR with enzyme Q5 Hot Start High-Fidelity 2X Master Mix using 30 picomoles of each primer represented by primer pools A and B and 5.0 µl of template cDNA. Cycling conditions were an initial denaturation of 98°C for 30 sec, 35 cycles of denaturation at 98°C for 15 sec & annealing/extension at 65°C for 5 min, and a final holding step at 4°C. The PCR products were analysed on a 2% agarose gel to check if amplicons of desired size were generated. PCR products were pooled together and purification of amplicons was done using AMPure XP Reagent magnetic beads (Beckman Coulter, California, USA). Quantification was done using Qubit dsDNA HS assay kit and Qubit Flex fluorometer (Invitrogen, Massachusetts, USA). Sequencing library preparation was done using Native Barcoding Kit SQK-NBD112.24 by Oxford Nanopore Technologies (ONT), following the manufacturer’s protocol. Briefly, 200 femtomoles (fmol) of purified product for each sample was subjected to DNA repair and end preparation using NEBNext Ultra II End repair/dA-tailing Module (New England Biolabs, Massachusetts, USA). Equimolar amounts of the repaired and end-prepped DNA were used for ligation to native barcodes supplied with Native Barcoding Kit, using Blunt/TA Ligase Master Mix (New England Biolabs, Massachusetts, USA). Barcoded DNA samples were pooled together and subjected to adapter ligation using native adapter provided and NEBNext Quick Ligation Module (New England Biolabs, Massachusetts, USA). About 20 fmol of the library was loaded onto a SpotON FLO-MIN111 R10.4 flow cell fitted on an Oxford MinION Mk1C device with MinKNOW interface. Sequencing run was performed with parameters of minimum read length 200 bp, Fast Basecalling option switched on, a q-score cutoff of 8 and maximum 5000 reads to be stored per FASTQ file. Demultiplexed and quality-controlled reads were stored into output files that were acquired in FASTQ format, without compression. For NGS data analysis, Commander-NGS software (Genotypic Technology Pvt. Ltd., Bangalore, India) was used. Individual FASTQ files for each barcode, representing each sample, were first merged and then run through “Variant Analysis” workflow, using reference bearing NCBI accession ID D49374 for assembling HCV 3b sequences, and reference bearing NCBI accession ID EU408330 for HCV 6xa sequences. Phylogenetic analysis of HCV whole genome sequences HCV subtype 3b and 6xa consensus sequences obtained were aligned with reference sequences using ClustalW algorithm of MEGA v.5.2. The best deduced model was used to construct a Maximum Likelihood phylogenetic tree with 1000 bootstrap replicates. For calculation of percent nucleotide identity (PNI), the alignment was submitted to Multiple Alignment using Fast Fourier Transform (MAFFT) server (18). The Clustal 2.1 algorithm of the server generated nucleotide identity values between all pairs of sequences submitted. In order to check if there are any known resistance mutations in sequences obtained, each consensus FASTA file was uploaded to HCV-GLUE online data resource (19). Recombination analysis using NCBI Genotyping Tool HCV 6xa sequences generated were run on NCBI Genotyping Tool (20) (https://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) to compare with closely phylogenetically related sequences as deduced from the phylogenetic tree. Results: Whole genome sequencing of HCV 6xa and NS5A resistance mutations in 3b relapse case During the study period, a total of 96 HCV infected patients were enrolled, of which 68 completed DAA therapy, 11 did not complete treatment and five were undergoing DAA therapy. Of the total patients, ten expired shortly after enrollment, one patient was lost to follow-up and remaining were yet to be initiated on treatment. Genotyping was performed for 70 HCV infected patients, of which the predominant genotype detected was genotype 3 in 45 HCV patients or 64.3% (Subtype 3b =28, 3a =12, 3i= 2, Genotype 3/not further typable =3), followed by genotype 6 (Subtype 6n=9, 6xa=2, 6c=1, 6e=1, 6u=1, not further typable=1, in 21.4%, n=15) and genotype 1(all subtype 1a) in 14.2% (n=10). We performed whole genome sequencing of two patients from whom the rare 6xa genotype (Sample ID HCV41 and HCV48) was detected and one patient who relapsed (Sample ID HCV17) at 24 weeks post-completion of DAA treatment. Phylogenetic analysis of HCV whole genome sequences For both subtype 3b and subtype 6xa samples there was 41X sequence depth with 100% genome coverage. Phylogenetic tree (Figure 1) shows evolutionary relationships among sequences obtained in this study with HCV whole genome reference sequences. One HCV subtype 7a isolate was used as outlier for tree construction, although it clustered closer to genotype 3 sequences. HCV 3b baseline 2023 WGS is the sequence obtained from baseline sample of an HCV patient (Sample ID HCV17), whereas HCV 3b relapsed 2023 WGS is obtained from the same patient after relapse at 24 weeks post-completion of DAA treatment, with viral load of 30,000 IU/ml. This patient had undetected viral load (<34 IU/ml) at both EOT and 12 weeks post-treatment completion. Both baseline and relapse sequences had negligible evolutionary distance (99.15% PNI), and appeared most closely related to a 1995 3b sequence from Japan (D49374.1). Figure. 1 . Maximum likelihood phylogenetic tree of the HCV whole genome sequences: The tree was constructed using MEGA software, version 5.2. using General Time Reversible model and 1000 bootstrap replicates. The node support values represent bootstrap proportions. The scale bar represents the number of nucleotide substitutions per site. The sequences reported here are highlighted in bold. Percent nucleotide identities of whole genome sequences with reference genomes Two whole genome sequences were obtained from patients infected by HCV subtype 6xa virus. Both sequences clustered together and had considerable evolutionary distance (93.41% PNI). They clustered together with HCV 6u sequences from China and USA, both from 2008 (EU408330.1 & EU408332.1), which was the previous nomenclature of 6xa subtype(22,23). Sequences generated in this study had considerable evolutionary distance with the US and Chinese 6u sequences. Closest subtype with least evolutionary distance to the two 6xa sequences reported here was 6v, a 2004 sequence from China (EU158186.1). Reference sequences of subtypes 6xb (KJ567645.1, Vietnam, 2014) and 6xc (KJ567651.1, Vietnam, 2014), were both found to have a large evolutionary distance with study 6xa sequences. Table 1 . Percent nucleotide identities Lowest percent nucleotide identity Highest percent nucleotide identity HCV 3b baseline 2023 WGS JF735126.1 (Somalia, 2011) ( 3h ) ( 72.4% ) D49374.1 (Japan, 1995) ( 3b ) ( 89.25% ) HCV 3b relapsed 2023 WGS JF735126.1 (Somalia, 2011) ( 3h ) ( 72.47% ) D49374.1 (Japan, 1995) ( 3b ) ( 88.7% ) HCV 41 6xa 2023 WGS Y12083.1 (UK, 1997) ( 6a ) ( 72.34% ) EU408330.1 (USA, 2008) ( 6u ) ( 96.34% ) HCV 48 6xa 2023 WGS Y12083.1 (UK, 1997) ( 6a ) ( 69.89% ) EU408330.1 (USA, 2008) ( 6u ) ( 93.45% ) Table 1 above shows PNI values of sequences obtained in this study, with reference sequences. Sequence of 3b relapsed sample had a slightly lower PNI with the 1995 Japan sequence, which may be due to nucleotide mutations that occurred over the course of DAA treatment. There were 75 nucleotide mutations detected in the relapsed sample ID HCV 17 compared to baseline sample, of which 56 were synonymous substitutions and 19 non-synonymous substitutions. The 6xa sequences bore lowest and highest PNI with the same references, but individual PNI values for both were quite different. Thus patients with sample ID HCV41 and HCV48, although had viruses of same subtype, still had considerable nucleotide differences. Recombination analysis of HCV 6xa sequences The 6xa sequences generated in our study were phylogenetically distant from 6xb and 6xc sequences, and closer to 6v sequence, thus a recombination analysis was done using NCBI Genotyping tool. This was done separately for HCV41 and HCV48 sequences, and graphical representations of both are shown in figures 2a and 2b. For both 6xa sequences, identity level did not cross 33% (score 100) for any of the reference sequences, except between positions 8800 and 9200 for 6v reference sequence, which is non-significant. Therefore, despite phylogenetic closeness of our study 6xa sequences with 6xb, 6xc and 6v reference sequences, there did not appear to have been any recombination events contributing to this. Figure 2a. Recombination analysis of HCV41 sample sequence Recombination analysis with HCV reference sequences of subtype 6v_China (EU158186.1), 6xb_Vietnam (KJ567645.1) and 6xc_Vietnam (KJ567651.1), to deduce possible recombination events between the 6xa sequences generated in our study and those used for phylogenetic analyses that clustered close to them. The bottom X-axis shows the nucleotide position in the HCV41 sequence, while the top X-axis shows the 300 base increment windows in which PNI is measured with each reference. The score on the Y-axis shows the extent to which identity is observed of our sequence with any reference sequence. A minimum score cutoff of 100 is needed for any significant level of identity. Figure 2b. Recombination analysis of HCV48 sample sequence Recombination analysis with HCV reference sequences of subtype 6v_China (EU158186.1), 6xb_Vietnam (KJ567645.1) and 6xc_Vietnam (KJ567651.1), to deduce possible recombination events between the 6xa sequences generated in our study and those used for phylogenetic analyses that clustered close to them. The bottom X-axis shows the nucleotide position in the HCV48 sequence, while the top X-axis shows the 300 base increment windows in which PNI is measured with each reference. The score on the Y-axis shows the extent to which identity is observed of our sequence with any reference sequence. A minimum score cutoff of 100 is needed for any significant level of identity. Resistance mutations against DAA by HCV-GLUE data resource On comparison of study subtype 3b sequences with HCV 3b reference D49374.1, a total of 1004 nucleotide mutations were found in baseline sample sequence, of which 206 were non-synonymous mutations. The relapsed sample sequence had a total of 1061 nucleotide mutations, 216 of them non-synonymous. Amino acid mutations analysis of 3b baseline and relapsed samples with reference D49374.1 revealed a block of residues changed at amino acid positions 2806 to 2809 (of whole genome) in relapsed sample compared to baseline sample, from Cysteine-Aspartic acid-Glycine-Alanine (CDGA) to Histidine-Glycine-Glutamic acid-Threonine (HGET). Comparison of subtype 6xa sequences with HCV 6xa reference EU408330.1 revealed 328 nucleotide mutations for sample 41, of which 93 were non-synonymous, and 316 nucleotide mutations for sample 48, of which 86 were non-synonymous. HCV-GLUE data resource revealed various kinds of resistance mutations to DAAs that were detected in the study sequences (Table 2). This system(19) detects presence of resistance-associated substitutions (RAS) and variants in viral genome sequences and summarises evidence that these confer resistance to DAAs. Polymorphisms are assigned to one of three categories according to strength of evidence for drug resistance. Category I polymorphisms have strongest evidence: either (a) in vitro resistance level ≥ 5 and found at baseline or treatment-emergent in vivo , or (b) both found at baseline and treatment-emergent. Category II: in vitro level ≥ 50 or found at baseline or treatment-emergent. Category III: in vitro level ≥ 5. Accordingly, resistance detection for a given drug is assigned to one of four categories i.e. category I polymorphisms: resistance detected, category II polymorphisms: probable resistance detected, category III polymorphisms: possible resistance and no significant resistance detected: none of the above. Table 2 . Resistance mutations against DAA detected by HCV-GLUE data resource in whole genome sequences obtained in this study. Numbers represent amino acid positions in the respective proteins. NS: Non-structural protein, GZR: Grazoprevir, EBR: Elbasvir, PIB: Pibrentasvir, OBV: Ombitasvir, Y: tyrosine, Q: glutamine, I: isoleucine, K: lysine, M: methionine, V: valine, S: serine, *category I mutation, #category II mutation, @category III mutation. NS3/4A protease inhibitors GZR 56Y+168Q+170I (#) [NS3] None found None found NS5A inhibitors EBR 30K (#) OBV 28V (#), 28V+30S (@) OBV 28M (@) PIB 30K (*), 31M (#) In our study, both subtype 3b sequences i.e., baseline and relapsed, showed identical resistance mutations for NS3/4A protease inhibitors i.e 56Y+168Q+170I and for NS5A inhibitors i.e 30K and 31M. For subtype 6xa sequences, none of the RAS were found for NS3/4A protease inhibitors in both the sequences. Sequence of sample 41 showed a combination of category II and III resistance mutations for NS5A inhibitors i.e. 28V and 28V+30S, respectively. Sample 48 sequence showed a category III 28M mutation against NS5A inhibitors. Discussion: Implications of HCV 6xa sequencing and NS5A resistance mutations in 3b relapse for DAA therapy We conducted genomic characterization of HCV infected patients before initiation of DAA treatment and followed them up for 12 and 24 weeks post-completion. One ‘unusual’ HCV subtype 6xa was obtained on sequencing of two HCV infected patients, and HCV 3b detected from a relapse case on DAA treatment. Complete genome sequencing of these HCV subtype 6xa was done for the first time in India, in our study. We carried out drug resistance analysis using HCV-GLUE online data resource revealing various non-synonymous RAS against NS5A inhibitors, which were present in sequences of all the samples tested. HCV genotype 3 We documented a patient with HCV genotype 3b, relapsing at 24 weeks post-completion of DAA treatment, though no HCV RNA was detected at EOT and 12 weeks post-treatment completion. Both 3b sequences i.e., baseline and relapsed, showed identical resistance mutations for NS3/4A protease inhibitors i.e. 56Y+168Q+170I and for NS5A inhibitors i.e. 30K and 31M. In previous studies, NS3 protease RASs V36L and D168Q have been reported in subtypes 3b and 3i(24). For NS5A inhibitors, presence of A30K RAS is more frequently seen in non-3a HCV subtypes in comparison to subtype 3a i.e. 84.6% vs 0.8%(25). A30K+L31M RAS combination, as seen in our study sequences, confers a high level of resistance to NS5A inhibitors, reported previously from a vast majority of patients infected with HCV subtypes 3b and 3g, including 94% patients from China infected with subtype 3b(26). Since these RAS were present in the 3b relapse case even in the baseline sample, it highlights an important issue i.e. presence of natural polymorphisms in viral genome leading to reduced susceptibility to DAAs, especially NS5A inhibitors, making these subtypes inherently resistant to therapy. Globally, it has been demonstrated that SVR rates are lower in patients infected with subtype 3b, even in various pangenotypic DAA combinations i.e. sofosbuvir/velpatasvir(27) where SVR rates of 3b versus 3a subtypes were 76% (32/42) and 95% (40/42) respectively, in glecaprevir/pibrentasvir (28) 70% (14/20) vs 95% (19/20), and with sofosbuvir plus coblopasvir (NS5A inhibitor available only in China) 89% (24/27) vs 91% (21/23)(29). Additionally, in subtype 3b patients without cirrhosis who received sofosbuvir/velpatasvir, SVR rate was 89% (25/28), but in those with compensated cirrhosis it was only 50% (7/14)(27). In cirrhotic patients of subtype 3b receiving glecaprevir/pibrentasvir, SVR rate of 58% (7/12) was demonstrated(28). In a Dutch cohort with subtype 3b, three of eight cases did not achieve SVR, who received sofosbuvir plus daclatasvir and sofosbuvir/velpatasvir, but all subtype 3k patients achieved SVR(30). HCV genotype 6 In our study, in subtype 6xa sequences, none of the RAS were found for NS3/4A protease inhibitors in both sequences. One of the sample sequence (HCV 6xa 41) showed a combination of category II and III resistance mutations for NS5A inhibitors i.e. 28V and 28V+30S, respectively, whereas sample HCV 6xa 48 sequence showed a category III 28M mutation against NS5A inhibitors. In Vietnam SEARCH study(31), prospective trial conducted on genotype 6-infected patients with advanced liver fibrosis or compensated cirrhosis, treated with SOF/DCV regimen for 12 weeks, they found distinct RAS to DCV i.e. F/L28V RAS in 100% of 6h, 6k and 6l subtypes and R30S RAS in 100% of 6e subtype. The study concluded that these RAS could represent wild-type (WT) amino acids in these genotypes, but may be able to provide some resistance to DCV(31). Not much clinical information is available on DAA susceptibility of many subtypes of genotype 6, which are circulating in Asia. There are regional differences in distribution and often the small numbers of patients from each subtype included, make it difficult to draw conclusions on true DAA susceptibility of various genotype 6 subtypes. In a recent study from India, 2.6% patients of chronic HCV infection (53 of 2052 patients), 81% co-infected with HIV, were infected with genotype 6. They identified nine different subtypes of genotype 6, with predominance of subtype 6xa (41%), followed closely by subtype 6n (40%). These patients had received combination DAA therapy of sofosbuvir and daclatasvir, with or without ribavirin and overall SVR rate was 81% (43/52)(8). In a study done at Myanmar, of 39 patients of genotype 6 treated with sofosbuvir/ledipasvir, 36 were subtype 6c-l, 1 was 6m and 2 with unassigned 6 subtype, and SVR rate was only 64% (25/39)(32). In contrast, in an open label study in US with cohort of Asian-born patients, where 43% were of subtype 6c-l and 22% 6a/b, treated with sofosbuvir/ledipasvir, 95% of cases achieved SVR (57/60)(33). Other studies done in various countries including France, USA and New Zealand, having diverse subtype distributions, also reported high SVR rates with different DAA combinations(3). Currently approved pangenotypic DAAs are effective against most common HCV subtypes i.e 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a and 6a. However, large African and Asian populations, often infected with unusual, non-epidemic HCV subtypes, maybe inherently resistant to current DAAs due to presence of natural polymorphisms at RAS positions(3). To be precise, subtypes 1l, 3b, 3g, 6u and 6v are found to be resistant to most NS5A inhibitors(3), with pibrentasvir showing a better efficacy than others, but may not be available in all the countries, including India. Data is also limited for other assigned or yet to be assigned HCV subtypes, which may have reduced DAA susceptibility. In low-to-middle-income countries (LMIC), having high prevalence of ‘unusual’ subtypes, currently only older-generation DAAs are available, commonly with generic combinations of sofosbuvir/daclatasvir or sofosbuvir/ledipasvir, challenging WHO HCV elimination goals (3). Even with recent pangenotypic combinations of sofosbuvir/velpatasvir and glecaprevir/pibrentasvir, treatment failures have been reported. EBR/GZR combination has a high barrier to RASs, effective in HCV genotype 1 and 4 infections, and has an excellent SVR rate even in challenging chronic kidney disease stage 4-5, compensated cirrhosis, HIV coinfection and treatment experienced patients(34). HCV GT1a patients may show lower SVR with EBR/GZR, and checking NS5A RASs might be necessary before receiving treatment. In 2016, European Association for the Study of the Liver (EASL) recommended that those patients with HCV genotype 1a-infection with baseline viral load > 800 000 IU/mL and NS5A RAS conferring resistance to EBR, or with a viral load should receive combination of EBR/GZR for a period of 16 weeks, along with daily weight-based ribavirin(35). In 2018, updated EASL guidelines recommended that resistance testing before treatment is not needed, implying that all patients with genotype 1a infection and high viral-load should receive prolonged EBR/GZR treatment with ribavirin in addition(36). In its latest updated, EASL does not recommend EBR/GZR use altogether for treatment of HCV genotype 1a, though referring earlier versions for guidance in case needed as per regional conditions. In 2020, EASL recommended that if sequencing of NS5A regions is available and affordable, patients infected with subtypes 1l, 3b, 3g, 6u and 6v or with unusual subtypes, documented to have more than one RAS conferring resistance to NS5A inhibitors, should be considered for first-line treatment with triple combination of sofosbuvir, velpatasvir and voxilaprevir(37). However, this recommendation can only be applicable to a very limited number of specialised referral centres, as vast majority of patients may be treated in basic clinical settings. There is need to characterise distribution of HCV subtypes circulating in each country, more so in LMICs, to help characterise prevalence of natural polymorphisms; which may influence response to DAA therapy and help tailor first-line treatment. Patients with unusual HCV subtypes may not achieve SVR, due to persistence of natural polymorphisms, often resulting in complex resistance patterns, difficult to even retreat with alternate drugs(3). Since routine sequencing in not feasible on a large-scale, there is a need to make available cheap, generic first-line therapy of sofosbuvir/velpatasvir/voxilaprevir or sofosbuvir plus glecaprevir/pibrentasvir, wherever there is prevalence of inherently resistant HCV subtypes(3). In countries, like India, where triple combination DAA therapy might not be available or not affordable, SVR at 12 and 24 weeks post-treatment must be monitored systematically, so that the patient can be retreated with triple combination, as second-line therapy. If not done, a substantial proportion of infected individuals might fail to clear the virus and transmit infection further to others. In view of WHO hepatitis C elimination strategy, well-tolerated and effective HCV treatment regimens are demanded, having a low pill burden and also low cost. This would help target highly vulnerable populations like people who inject drugs, in terms of initiation and adherence to treatment(38). Though pangenotypic treatment options are now available, there could be a need for genotype-specific options like EBR/GZR, but significant barriers such as elbasvir resistance testing, prolonged treatment with multi-tablet therapy and associated side-effects exist, leading to delay in initiating treatment. Despite, paucity of data on unusual subtypes, retreatment recommendation after DAA failure is same for common subtypes i.e. triple combination of sofosbuvir/velpatasvir/voxilaprevir or sofosbuvir plus glecaprevir/pibrentasvir), latter being preferred in subtype 3b(3). Conclusion Extremely high genetic variability of hepatitis C virus has led to the current genotypes, and into a large number of subtypes in various geographical areas. Currently approved pangenotypic DAA regimens are designed to be effective against majority of common subtypes. In Asia and Africa large populations maybe infected with unusual, non-epidemic HCV subtypes, which maybe inherently resistant to currently available DAAs. These unusual HCV subtypes are currently underestimated, but are a threat to global efforts being undertaken to eliminate viral hepatitis, warranting prompt and robust action. Funding: This study was funded by Indian Council of Medical Research by providing extramural funding. Declaration of interest: There are no conflicts of interest. Author Contributions: Conceptualization, ST; study design, ST, BB; performance of the work, S.T, A.G, O.G, B.T, CB, A.R; resource person for samples B.B, BL; statistical analysis, S.T, A.G; writing of the paper, S.T, A.G; and all the co-authors edited and approved it. Acknowledgments: We thank the Indian Council of Medical Research, New Delhi for providing funding for the project. We also thank the Director ICMR-NIV Pune and NEIGRIHMS Shillong for their support during conduct of the study. 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Tomar 0000-0003-3850-2316 [email protected] ICMR - National Institute of Virology View all articles by this author Abhranil Gangopadhayya ICMR - National Institute of Virology View all articles by this author Bhupen Barman North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences View all articles by this author Onkar Ghuge ICMR - National Institute of Virology View all articles by this author Bhanu Teja Korra ICMR - National Institute of Virology View all articles by this author Ciyona Bastin ICMR - National Institute of Virology View all articles by this author Baiahunlang Lamare North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences View all articles by this author Ashwini Y. Ramdasi ICMR - National Institute of Virology View all articles by this author Wihiwot V. Lyngdoh North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences View all articles by this author Kavita Lole ICMR - National Institute of Virology View all articles by this author Harpreet Kaur Indian Council of Medical Research View all articles by this author Metrics & Citations Metrics Article Usage 182 views 116 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shilpa J. Tomar, Abhranil Gangopadhayya, Bhupen Barman, et al. Whole genome sequencing of Hepatitis C virus subtype 6xa and NS5A resistance mutations in a genotype 3b relapse case after direct-acting antiviral therapy in India. Authorea . 14 August 2025. DOI: https://doi.org/10.22541/au.175517118.80099254/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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