Infection and Drug Resistance Dovepress open access to scientific and medical research Open Access Full Text Article C A S E R E P O RT Whole-genome characterization and resistance-associated substitutions in a new HCV Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 genotype 1 subtype This article was published in the following Dove Press journal: Infection and Drug Resistance Georg von Massow 1 Abstract: Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 Damir Garcia-Cehic 1,2 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high- Josep Gregori 1–3 resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free Francisco Rodriguez- classification of the virus using phylogenetic analysis and analysis of genetic distances in For personal use only. Frias 2,4 sequences from patient samples compared to reference sequences. During routine diagnostic, a sample from an Equatorial Guinea patient could not be classified into any of the existing María Dolores Macià 5 subtypes. The whole genome was analyzed to confirm that the new isolate could be classified Ana Escarda 6 as a new HCV subtype. In addition, naturally occurring resistance-associated substitutions Juan Ignacio Esteban 1,2,7 (RAS) were analyzed by NGS. Whole-genome analysis based on p-distances suggests that Josep Quer 1,2,7 the sample belongs to a new HCV genotype 1 subtype. Several RAS in the NS3 (S122T, 1 Liver Unit, Liver Diseases – Viral D168E and I170V) and NS5A protein (Q(1b)24K, R(1b)30Q and Y93L+Y93F) were found, Hepatitis, Vall d’Hebron Institut of which could limit the use of some inhibitors for treating this subtype. RAS studies of new Research (VHIR) – Hospital Universitari Vall d’Hebron (HUVH), Barcelona, Spain; subtypes are of great interest for tailoring treatment, as no data on treatment efficacy are 2 Centro de Investigación Biomédica en reported. In our case, the patient has not yet been treated, and the RAS report will be used to Red de Enfermedades Hepáticas design the most effective treatment. y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; 3Roche Keywords: subtype, direct-acting antivirals, HCV, genotype 1 Diagnostics S.L., Sant Cugat del Vallès, Barcelona, Spain; 4Biochemistry and Microbiology Department, VHIR-HUVH, Barcelona, Spain; 5Unidad de Microbiología Molecular, Servicio de Microbiología, Instituto de Investigación Background Sanitaria de les Illes Balears (IdISBa), Hepatitis C virus (HCV) is a blood-borne virus affecting an estimated 71 million Hospital Universitario Son Espases, Mallorca, Spain; 6Digestive Department, people worldwide resulting in a global prevalence of 1%-3%.1 Hospital Universitario Son Espases, HCV is a single-stranded, positive-sense RNA virus containing approximately Mallorca, Spain; 7Medicine Department, Universitat Autònoma de Barcelona, 9600 nucleotides and one open reading frame. Eight genotypes and 86 subtypes Barcelona, Spain have been reported according to the International Committee on Taxonomy of Viruses (ICTV).2 A genotype is assigned when its genetic distance to all other genotypes is >30%, whereas subtypes are defined as having a genetic distance of >15% to other subtypes of the same genotype.3 In a single patient, HCV can be present as a complex mixture of viral variants having small genetic differences (1–3%), known as a quasispecies.4 Because of this, HCV has high genetic hetero- Correspondence: Josep Quer Liver Unit, Liver Disease Laboratory- geneity, which has important implications for the diagnosis and treatment of Viral Hepatitis, Internal Medicine infected patients, and has impaired the development of an effective vaccine. Department, Vall d’Hebron Institut Recerca (VHIR-HUVH), Barcelona However, with the recent advances in HCV therapy, direct-acting antiviral (DAA) 08035, Spain treatment in chronically infected patients leads to a sustained virologic response Tel +3 493 489 4034 Email josep.quer@vhir.org (SVR) rate greater than 90%. However, drug resistance-associated substitutions submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 947–955 947 DovePress © 2019 von Massow et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/ terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing http://doi.org/10.2147/IDR.S195441 the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Powered by TCPDF (www.tcpdf.org) von Massow et al Dovepress (RAS) can emerge during this therapy and lead to treat- characterization using an HRCS technique, recently ment failure in 2%–10% of patients.5 In addition, RAS adapted to the MiSeq platform. HRCS is based on study- have been detected in some HCV subtypes as natural ing a short, highly variable region from the NS5B gene occurrences.6 Therefore, as HCV genotype and subtype flanked with highly conserved primers.10 in an infected patient may have an impact on the outcome In this study, we used next-generation sequencing of the treatment received, the therapy should be adjusted to (NGS) to subtype HCV and characterize the RAS profile these characteristics of the virus for it to be effective.7–9 In of the proteins NS3, NS5A and NS5B, targeted by DAA Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 this scenario, accurate identification of HCV genotype and inhibitors. Sanger sequencing was used to sequence the subtype is of paramount importance for proper patient whole genome. management, especially in countries where pan-genotypic The complete HCV genome was obtained by nested drugs are not available. Additionally, the degree of liver RT-PCR with 3 external primer pairs and 11 overlapping fibrosis and previous exposure to DAA and/or interferon, internal primer pairs (Table S1). The first externals (1, 2) together with the infecting genotype, are of pivotal impor- and the internals (1, 2, 3, 5, 6, 7) were the primers reported tance for the treatment strategy (including the duration of by Ordeig et al in 2016.11 The remaining external and therapy). internal primer pairs were newly designed, using the clo- A high-resolution HCV subtyping (HRCS) method sest subtype sequences (1l, 1h, 1c and 1e), identified dur- based on deep sequencing has been implemented in our ing the subtyping test performed by HRCS after 200 routine clinical laboratory.10 This method involves phylo- bootstrapping cycles. All primers were numbered accord- For personal use only. genetic analysis and genetic distance analysis of the new ing to isolate H77 (accession number AF009606).13 The template compared to the confirmed HCV subtypes.11,12 external fragments were amplified using the Transcriptor Because of the large number of reads obtained with this One Step RT-PCR Kit (#04655885001 Sigma, Roche), and method (>2,000), new subtypes can be detected, as well as for the internals, the FastStart High Fidelity PCR System, mixed infections in patients with more than 1 genotype or dNTPack (#04738292001 Sigma, Roche), adapting the subtype. various PCR programs to the lengths and temperatures of each specific primer pair. Due to the high variability of the Objectives region corresponding to internal fragment number 11, a A genotype 1 sample from an untreated patient from random priming (#11034731001 Sigma, Roche) PCR was Equatorial Guinea could not be classified during routine necessary to perform between the RT-PCR and the nested genotype-subtype analysis at the diagnostic laboratory. PCR with the specific primers, to obtain enough DNA for The aim of this study was to characterize the sample analysis. All amplifications were checked in 1.5% agarose through complete analysis of the HCV genome sequence gel to ensure that there was no contamination, and are and investigation of baseline RAS. purified in columns using the QIAquick PCR Purification Kit (#28106 QIAGEN). All fragments were Sanger- Methods sequenced, and were assembled, verified and edited using The original serum sample was obtained from an HCV- the GeneDoc 2.7.000 (2006) software. infected woman from Equatorial Guinea diagnosed at the Twenty-two complete reference genomes, corresponding Molecular Microbiology Department of the Instituto de to all the confirmed genotype 1 (G1) subtypes (1a, 1b, 1c, Investigación Sanitaria de les Illes Balears (IdISBa), 1d, 1e, 1g, 1h, 1i, 1j, 1k, 1l, 1m, 1n) and 8 previously Hospital Universitario Son Espases (Spain) in May 2017. unassigned isolates, were used to classify the sequence Patient signed an informed consent to have the case details under study;2 phylogenetic trees and a heatmap were con- published. As this study has no legal implications for the structed. The sequence was analyzed using a sliding window institution, special approval from it was not required to procedure to investigate possible viral recombination of publish the case details. As it was impossible to classify different subtypes. The scripts in R language used to obtain HCV subtype of this patient during the routine diagnostic the figures are provided The scripts in R language used to procedure and because HCV sequencing for further clas- obtain the figures and provided elsewhere.11,14 The sification is a requirement during the diagnostic and treat- R libraries included Biostrings, ape, stringr, RColorBrewer ment procedure as the data sheet supports, a sample taken and psych. MUSCLE was used for the required multiple in June 2018 was delivered to our laboratory on dry ice for alignments.15 To characterize any naturally occurring submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 948 DovePress Powered by TCPDF (www.tcpdf.org) Dovepress von Massow et al baseline RAS in the putative new subtype taking into 1 cluster (Figure S1). However, it could not be classified account that any isolate is composed by a population of into any of the genotype 1 subtypes, with subtype 1h being sequences,4 four new internal primers pairs were designed, the closest. The next step was to design primers to sequence corresponding to the three DAA-targeted regions of the and characterize the whole genome. HCV genome (1 NS3, 1 NS5A and 2 NS5B); using this time the isolate's full genome sequence, obtained with Phylogenetic analysis and pairwise Sanger ssequencing, was used as reference (Table S1). To comparison of full-length genomes Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 amplify these new fragments, we used the products of the The full sequence (9182nt) deposited in the GenBank external RT-PCRs obtained previously on the FastStart High database with accession number “MH921830” includes Fidelity PCR System, dNTPack (#04738292001 Sigma, the almost complete coding region and a partial sequence Roche), adapting the thermocycler programs to the charac- from 5ʹ UTR. teristics of each different primer pair. Again all fragments Phylogenetic analysis of the complete new genome were checked and purified on 1.5% agarose gel, and were with the confirmed subtypes of all 8 genotypes showed deep-sequenced using the MiSeq Reagent Kit v3 (600 that our sequence clustered with G1 (Figure 1). cycle) (#MS-102–3003, Illumina) according to Furthermore, the new genome did not cluster with any of a previously described method.16 Briefly, each nested-PCR the confirmed G1 subtypes when compared to the con- purified product was pooled and normalized to 4nM, and firmed subtype reference sequences (Figure 2). appropriate volumes of each pool were added to the final For personal use only. The heatmap on the matrix of p-distances further library, which was quantified by LightCycler480 (Kapa demonstrated that our sequence did not cluster with any Library Quantification Kit, KapaBiosystems, Roche, confirmed subtype or unassigned isolate (Figure 3). The Pleasanton, CA, USA). The last dilution of the library was shortest genetic p-distance measured (0.2447) was to a still prepared and mixed with an internal DNA control (PhiX unassigned isolate “KY348757”,11 whereas the shortest control V3, Illumina, San Diego, CA) before sequencing on p-distance to a confirmed subtype was 0.2495 with sub- the MiSeq platform using the MiSeq Reagent Kit V3 type 1k (Table S2). Therefore, our sequence could not be (Illumina, San Diego, CA). Data management includes classified into any of the existing G1 subtypes and could a bioinformatics haplotype-centric procedure to exclude not be grouped with any other unassigned isolate. full reads that did not meet minimum quality requirements.16 The final steps included demultiplexing by specific primers to obtain a fasta file by region. Reads were Recombination: sliding window analysis then collapsed into haplotypes with corresponding frequen- Sliding window analysis was conducted to exclude the cies. Haplotypes were aligned with the reference sequence, possibility of viral recombination between HCV genotype and haplotypes containing more than two indeterminations, 1 subtypes (Figure 4). The p-distance of our sequence to three gaps, or 99 differences were also discarded. Accepted the nearest G1 subtype at any given position was higher indeterminations and gaps were repaired as per the contents than the minimum p-distance between all subtypes. of the dominant haplotype. The yield of this process was above 90% in all cases. Then, reads were translated to Resistance-associated substitutions (RAS) amino acids, and the intersection between forward and To characterize the viral genome, RAS were analyzed reverse haplotypes with abundances not below 0.2% was for the NS3, NS5A and NS5B proteins by deep sequen- performed. The yield of this process has been found in the cing using the Illumina MiSeq sequencing platform, to range of 45%–60%. Based on the controls, all amino acid identify resistances to various DAAs between our iso- variants by site or haplotype at 1% or above are reported. late and the wild-type replicons 1a and 1b, used as The global yield is 15%–30% of raw reads. references (Table 1). RAS S122T, D168E and I170V were found in NS3 in almost all genomes reported after Results NGS, and the substitutions Y93F/L and R30Q in NS5A. HCV subtyping of a short fragment of the NS5B gene using Interestingly, variability was found in the Y93 residue of a high-resolution method based on deep sequencing NS5A, in which 72% of viral genomes were carrying (HRCS) revealed that the all reads belonged to the same amino acid 93L (leucine) and 28% were carrying 93F subtype and that this subtype was included in the genotype (phenylalanine). The amino acid composition of our submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 949 DovePress Powered by TCPDF (www.tcpdf.org) von Massow et al Dovepress Neighbor- joining tree on p-distances 100 6t EU246939 RF2014 100 6t EF632071 RF2014 6p EF424626 RF2014 6o EF424627 RF2014 6q EF424625 RF2014 100 100 6u EU246940 RF2014 6e DQ314805 RF2014 100 6d D84263 RF2014 6c EF424629 RF2014 100 6r EU408328 RF2014 6f DQ835760 RF2014 6s EU408329 RF2014 100 6g D63822 RF2014 100 Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 6w EU643834 RF2014 75 6w DQ278892 RF2014 100 6h D84265 RF2014 100 6j DQ835769 RF2014 100 6i DQ835770 RF2014 71 100 6m DQ835767 RF2014 100 6n DQ835768 RF2014 100 6n DQ278894 RF2014 100 6k D84264 RF2014 6l EF424628 RF2014 100 6v EU798760 RF2014 100 76 100 6xa6v EU158186 RF2014 EU408332 RF2014 6xa EU408330 RF2014 100 6b D84262 RF2014 100 6a Y12083 RF2014 6a AY859526 RF2014 100 5a Y13184 RF2014 5a AF064490 RF2014 100 1c AY051292 RF2014 100 1a M67463 RF2014 100 1a M62321 RF2014 100 1I KC248197 RF2014 83 1I KC248193 RF2014 100 1g NA RF2014 1e KC248194 RF2014 69 100 Px Our isolate 1h KC248199 RF2014 100 1h KC248198 RF2014 100 100 1b M58335 RF2014 For personal use only. 1b D90208 RF2014 100 4r FJ462439 RF2014 57 4g FJ462432 RF2014 100 4k FJ462438 RF2014 4k EU392173 RF2014 100100 100 4b FJ462435 RF2014 4w FJ025856 RF2014 4w FJ025855 RF2014 100 4t FJ839869 RF2014 4p FJ462431 RF2014 100 4f EU392175 RF2014 100 4f EF589161 RF2014 100 4c FJ462436 RF2014 4a Y11604 RF2014 100 3 4n FJ462441 RF2014 100 4I FJ839870 RF2014 100 4q FJ462434 RF2014 100 4v JX227959 RF2014 4v HQ537009 RF2014 4o FJ462440 RF2014 4m FJ462433 RF2014 100 4d FJ462437 RF2014 4d DQ418786 RF2014 3h JF35121 RF2014 100 100 3k JF735122 RF2014 100 3k D63821 RF2014 100 3a D28917 RF2014 100 3a D17763 RF2014 100 3g JX227954 RF2014 74 100 3g JF735123 RF2014 79 3b D49374 RF2014 100 3i JX227955 RF2014 3i FJ407092 RF2014 7a EF108306 RF2014 100 100 2b D10988 RF2014 2b AB030907 RF2014 100 100 2a D00944 RF2014 100 2a AB047639 RF2014 2e JF735120 RF2014 100 2j JE735113 RF2014 71 2j HM777358 RF2014 100 2m JX227967 RF2014 98 2m JF735111 RF2014 100 2k AB031663 RF2014 100 2q FN666429 RF2014 2q FN666428 RF2014 2r JF735115 RF2014 2i DQ155561 RF2014 2c D50409 RF2014 2d JF735114 RF2014 0.25 0.2 0.15 0.1 0.05 0 Figure 1 Phylogenetic tree of the entire sequences, including reference from accepted genotypes2 (shown in different colors) and our sequence (Px). Trees were generated using neighbor-joining based on the p-distances. Only those bootstrap-values greater than 60 are shown. isolate in the main RAS positions in the NS3 protein (Table 1). Later, we discuss the scope of each mutation was 80Q, 122T, 155R, 156A, 168E and 170V; and in and the combination of them in the same genome. No the NS5A protein 24K, 28L, 30Q, 58P, 62Q and 93L/F RAS were detected in the NS5B protein. submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 950 DovePress Powered by TCPDF (www.tcpdf.org) Dovepress von Massow et al Neighbor- joining tree on p-distances G1m.KJ439782 100 G1m.KJ439778 G1 .KJ439780 G1 .KJ439779 100 100 G1c.D14853 Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 G1c.AY051292 G1c.HQ537007 G1e.KJ439769 100 G1e.KC248194 100 G1g.KJ439770 100 G1g.AM910652 G1.KC248195 100 G1.KJ439776 100 G1I.KC248197 100 G1I.KC248193 Px Our isolate For personal use only. 76 G1h.KC248199 100 G1h.KC248198 97 G1 .AJ851228 100 95 G1 .KY348757 G1d .KJ439768 100 G1I.KJ439772 77 G1b.M58335 100 G1b.D90208 G1a.M67463 100 G1a.M62321 76 G1n.KJ439775 100 G1n.KJ439781 G1J.KJ439773 94 100 G1k.KJ439774 G1 .KJ439777 0.15 0.1 0.05 0 Figure 2 Phylogenetic tree of entire genomes from all 13 subtypes of genotype 1, and including 8 unassigned isolates (green) and our Px sequence (orange). The tree was generated using neighbor-joining based on p-distances. Only those bootstrap-values greater than 60 are shown. Discussion a short region of the NS5B gene, confirming that the isolate This report describes and characterizes the full-length gen- could be member of a new subtype. ome of a putative new HCV subtype identified during routine The complete genome sequence was then obtained HCV classification of a serum sample from an Equatorial after PCR and Sanger sequencing and uploaded to Guinea patient. Commercial methods were unable to geno- GenBank (MH921830). Through phylogenetic analysis, type the viral isolate and it was then subjected to high- genetic p-distance calculations by pairs, and heat- resolution HCV subtyping, a methodology based on NGS10 mapping, we found that our sequence (Px) had a genetic adapted to the MiSeq platform, and phylogenetic analysis of distance greater that 15% to the closest confirmed submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 951 DovePress Powered by TCPDF (www.tcpdf.org) von Massow et al Dovepress p–distances G1_.HQ537007 0.28726 G1_.KC248195 G1_.KJ439776 G1I.KC248193 0.25853 G1I.KC248197 G1g.AM910652 G1g.KJ439770 0.22980 G1e.KC248194 Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 G1e.KJ439769 Our isolate Px G1h.KC248198 0.20108 G1h.KC248199 G1_.AJ851228 G1_.KY348757 0.17235 G1d.KJ439768 G1i.KJ439772 G1b.D90208 0.14363 G1b.M58335 G1_.KJ439780 G1m.KJ439778 0.11490 G1m.KJ439782 G1n.KJ439781 G1n.KJ439775 G1a.M62321 0.08617 For personal use only. G1a.M67463 G1j.KJ439773 G1_.KJ439777 0.05745 G1k.KJ439774 G1_.KJ439779 G1c.AY051292 0.02872 G1c.D14853 G1m.KJ439778 G1m.KJ439782 G1b.D90208 G1c.AY051292 G1_.KJ439776 G1e.KJ439769 G1_.AJ851228 G1_.KJ439780 G1n.KJ439781 G1n.KJ439775 G1_.KJ439779 G1b.M58335 G1a.M62321 G1a.M67463 G1_.HQ537007 G1g.AM910652 G1_.KC248195 G1e.KC248194 G1h.KC248198 G1h.KC248199 G1c.D14853 G1i.KJ439772 G1j.KJ439773 G1I.KC248193 G1k.KJ439774 G1_.KY348757 Px G1g.KJ439770 G1d.KJ439768 G1_.KJ439777 G1l.KC248197 0 Our isolate Figure 3 Heatmap visualizing the p-distances between all G1 reference sequences and the new isolate (Px). The scale on the right indicates the p-distance according to color, with red representing a short and blue a long p-distance. The clusters shown in light colors indicate the grouping of subtypes based on their similarity. The new isolate did not cluster with any subtype. reference sequence, which belonged to G1k (Table S2). exactly report the frequency at which each mutation was Interestingly, the closest sequence was a subtype- present in the viral population, and determine whether two unassigned sequence (KY348757), also isolated from an mutations were combined in the same viral genome, parti- Equatorial Guinean patient. This suggests that G1 has cularly minor mutations. Interestingly, all genomes iso- a long-lasting presence in that country, which has facili- lated from this new isolate were carrying the NS3 tated divergence of this genotype into new, unclassified mutations S122T and D168E.17 S122T has been found to subtypes. Additionally, sliding window analysis provided be a polymorphism of genotypes 4 and 6, but not genotype evidence that the sequence did not result from recombina- 1. S122T and D168E have been associated with resistance tion of different G1 subtypes. However, it will remain as to antivirals, especially in subtypes 1a and 1b, and D168E an unclassified lineage until a related sequence is discov- additionally in subtypes 2a, 2b, 4a and 6,7,18 suggesting ered, in keeping with the recommendations for a unified that this subtype maybe basically resistant to NS3 nomenclature system.2 inhibitors.7,8,9,18 NS3-D168E is especially relevant, as in Deep sequencing analysis was used to trace the RAS subtypes 1a and 1b, it can make the virus resistant to profile of the targeted NS3, NS5A, and NS5B proteins, to almost all NS3 inhibitors, including vaniprevir (used for identify with maximum reliability minor mutations, genotype 1 in many countries) and voxilaprevir (NS3 submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 952 DovePress Powered by TCPDF (www.tcpdf.org) Dovepress von Massow et al 0.5 0.4 Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 0.3 p-dist 0.2 0.1 Px min p- dist. intersubtype min p- dist. intersubtype average p- dist. 0.0 intersubtype max p- dist. For personal use only. 0 2,000 4,000 6,000 8,000 Nucleotide position (from 1 to 8,830) Figure 4 Visual representation of the sliding window analysis (500-bp, by 10-bp steps). The minimum p-distance between the new isolate (Px) and any reference sequence of accepted subtypes for each 500-bp window is shown in black. The green dotted line represents the minimum p-distance between the closest sequences belonging to the various subtypes. The blue dotted line indicates the average p-distance of all accepted G1 subtypes for each window. The red dotted line represents the maximum p-distance between the farthest sequences belonging to the accepted subtypes for each window. Table 1 Mean fold change in resistance compared with wild type, found in G1a and G1b (in brackets)7,8,9,18 Amino acids at the RAS positions: NS3: V36, F43, T54, V55, Y56, Q80, S122T, I132, R155, A156, A158, A166, D168E, I170V, L175 NS5A: Q(1b)24K, L28, R(1b)30Q, L31, P32, S38, H58P, E62Q, A92, Y93L/F NS5B: L159, E237, S282, L314, C316, S368, M414, Y448 RAS Frequency % HCV protein ASV GLE GZR PTV SMV MK-7009 VOXI S122T 100.0 NS3 non-R 20 non-R D168E 100.0 78 non-R 16; 3 14; 4 26–49; 15–90 12–57; 21–40 non-R I170V 100.0 non-R 8 non-R DCV EBR LDV OMV PIB VEL Q (1b)24K 100.0 NS5A non-R <100 R (1b) 30Q 100.0 n.d 3 n.d non-R Y93L 71.9 3006 non-R <100 Y93F 28.1 10 <100 27 non-R Abbreviations: ASV, Asunaprevir; GLE, Lecaprevir; GZR, Grazoprevir; PTV, Paritaprevir; SMV, Simeprevir; MK-7009, Vaniprevir; VOXI, Voxilaprevir; DCV, Daclatasvir; EBR, Elbasvir; LDV, Ledipasvir, OMV, Ombitasvir; PIB, Pibretasvir; VEL, Velpatasvir; n.d., described as resistant, but with no data; non-R, non-resistant. inhibitor present in the pan-genotypic triple salvage treat- (Y93F/L). Y93F is reported to confer resistance in G1a to ment with velpatasvir and sofosbuvir).19 The new subtype ledipasvir and ombitasvir, and Y93L to ombitasvir and carries the amino acid 170V which is considered wild type velpatasvir in cell cultures as well as in treatment-failing for many subtypes, but we cannot exclude it as a RAS patients.18 Lysine (K) and glutamine (Q) in residues 24 because it has been described for subtype 1a.18 Variability and 30, respectively, have been found to be wild type in was found in the Y93 residue of NS5A in our new subtype G1a17 However, in genotype 1b, Q24K has been reported submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 953 DovePress Powered by TCPDF (www.tcpdf.org) von Massow et al Dovepress to confer resistance to daclatasvir and elbasvir, and R30Q “Plan Estratégico Nacional contra la Hepatitis C”. This to velpatasvir.18 Cell culture studies are needed to deter- study was funded by Instituto de Salud Carlos III grants, mine whether these NS5A mutations (93F/L and 30Q) and PI15/00856, PI15/00829 and PI16/00337, and cofinanced some of the NS3 mutations (170V) can be considered RAS by CIBERehd (Consorcio Centro de Investigación en Red for the new subtype. The level of resistance to an inhibitor de Enfermedades Hepaticas y Digestivas), which is funded depends on several conditions: the genetic barrier to resis- by Instituto de Salud Carlos III and Centro para el tance of the HCV region and the drug, type of RAS, Desarrollo Tecnológico Industrial-CDTI from the Spanish Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 freguency at which the RAS is present in the viral isolate, Ministry of Economy and Business, grant number, IDI- and presence of RAS combinations in the same viral 20151125. particle, all of which can significantly increase resistance The authors thank Celine Cavallo for English language to some DAA treatments. In the study of combinations of support and helpful editing suggestions. RAS residues in the same genome, none of the combina- tions found could be associated with a fold-change Disclosure increase in resistance to the inhibitors.18 The funder “Roche Diagnostics S.L.” provided support in Taking all this information together, the RAS report the form of salaries for one author (Josep Gregori), but the suggests that our naïve patient should be treated with a company did not have any additional role in the study combination of an NS5B inhibitor such as sofosbuvir, design, data collection and analysis, decision to publish an NS3 inhibitor such as glecaprevir or voxilaprevir, or preparation of the manuscript. The other authors report For personal use only. and/or the NS5A inhibitor pibrentasvir. As any treat- no conflicts of interest in this work. ment should include a combination of two or even three inhibitors with or without ribavirin, our patient References could be treated combining glecaprevir+pibrentasvir 1. United Nations-General Assembly. Global hepatitis report 2017. (Maviret)+RBV or using voxilaprevir+velpatasvir WHO; 2017. Available from: http://www.who.int/hepatitis/publica +sofosbuvir+RBV. In the latter option, velpatasvir tions/global-hepatitis-report2017/en/.2017/p1-83ed. 2. Smith D. HCV classification. A web resource to manage the classif- (which is included in the Vosevi commercial treatment) cation and genotype and subtype assignements of hepatits virus; will probably not work, but the combination of voxila- 2018. Available from: https://talk.ictvonline.org/ictv_wikis/flaviviri dae/w/sg_flavi/56/hcv-classification. previr plus sofosbuvir with ribavirin should be effec- 3. Smith DB, Bukh J, Kuiken C, et al. Expanded classification of tive. Inclusion of ribavirin is recommended, if possible, hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria due to the presence of several baseline RAS. and assignment web resource. Hepatology. 2014;59:318–327. doi:10.1002/hep.26744 In summary, high HCV variability among genotype 4. Martell M, Esteban JI, Quer J, et al. Hepatitis C virus (HCV) 1 subtypes has been observed in Equatorial Guinean circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol. patients. In general, HCV Isolates that cannot be 1992;66:3225–3229. assigned by means of commercially available techni- 5. Vermehren J, Sarrazin C. The role of resistance in HCV treatment. Best ques should be analyzed using a high-throughput tech- Pract Res Clin Gastroenterol. 2012;26:487–503. doi:10.1016/j. bpg.2012.09.011 nology, with the special recommendation to use NGS 6. Eltahla AA, Leung P, Pirozyan MR, et al. Dynamic evolution of and phylogenetic analysis for the NS5B region, as hepatitis C virus resistance-associated substitutions in the absence of antiviral treatment. Sci Rep. 2017;7:41719. doi:10.1038/srep41719 mixed infections and minority mutants can be evi- 7. EASL. EASL recommendations on treatment of hepatitis C 2018. denced with these techniques, Further characterization J Hepatol. 2018;69:461–511. doi:10.1016/j.jhep.2018.03.026 of the whole genome is interesting for epidemiological 8. Lontok E, Harrington P, Howe A, et al. Hepatitis C virus drug resistance-associated substitutions: state of the art summary. reasons and study of RAS mutations using a high- Hepatology. 2015;62:1623–1632. doi:10.1002/hep.27934 throughput technique is useful to identify whether the 9. Sarrazin C. The importance of resistance to direct antiviral drugs in HCV infection in clinical practice. J Hepatol. 2016;64:486–504. new subtype is naturally harboring resistance- doi:10.1016/j.jhep.2015.09.011 associated mutations, which will be of help to prescribe 10. Quer J, Gregori J, Rodriguez-Frias F, et al. High-resolution hepatitis the most effective antiviral treatment. C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods. J Clin Microbiol. 2015;53:219–226. doi:10.1128/JCM.02093-14 Acknowledgments 11. Ordeig L, Garcia-Cehic D, Gregori J, et al. New hepatitis C virus genotype 1 subtype naturally harbouring resistance-associated muta- This study was supported by the Spanish Ministry of tions to NS5A inhibitors. J Gen Virol. 2018;99:97–102. doi:10.1099/ Health, Consumption and Social Welfare grant name jgv.0.000996 submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 954 DovePress Powered by TCPDF (www.tcpdf.org) Dovepress von Massow et al 12. Rodriguez-Frias F, Nieto-Aponte L, Gregori J, et al. High HCV 17. Palanisamy N, Kalaghatgi P, Akaberi D, et al. Worldwide prevalence subtype heterogeneity in a chronically infected general population of baseline resistance-associated polymorphisms and resistance muta- revealed by high-resolution hepatitis C virus subtyping. tions in HCV against current direct-acting antivirals. Antivir Ther. Clin Microbiol Infect. 2017;23:775e1–775e6. doi:10.1016/j. 2018;23:485–493. doi:10.3851/IMP3237 cmi.2017.02.007 18. Sorbo MC, Cento V, Di Maio V, et al. Hepatitis C virus drug 13. Kuiken C, Combet C, Bukh J, et al. A comprehensive system for resistance associated substitutions and their clinical relevance: consistent numbering of HCV sequences, proteins and epitopes. update 2018. Drug Resist Updat. 2018;37:17–39. doi:10.1016/j. Hepatology. 2006;44:1355–1361. doi:10.1002/hep.21377 drup.2018.01.004 14. R Core Team. R: A language and environment for statistical comput- 19. Lawitz E, Yang JC, Stamm LM, et al. Characterization of HCV ing. Vienna, Austria: R Foundation for Statistical Computing; 2016. resistance from a 3-day monotherapy study of voxilaprevir, a novel Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 81.60.156.107 on 04-May-2021 15. Edgar RC. MUSCLE: a multiple sequence alignment method with pangenotypic NS3/4A protease inhibitor. Antivir Ther. reduced time and space complexity. BMC Bioinformatics. 2018;23:325–334. doi:10.3851/IMP3202 2004;5:113. doi:10.1186/1471-2105-5-113 16. Perales C, Chen Q, Soria ME, et al. Baseline hepatitis C virus resistance-associated substitutions present at frequencies lower than 15% may be clinically significant. Infect Drug Res. 2018;11:2207–2210. For personal use only. Infection and Drug Resistance Dovepress Publish your work in this journal Infection and Drug Resistance is an international, peer-reviewed open- antibiotic resistance and the mechanisms of resistance development and access journal that focuses on the optimal treatment of infection diffusion in both hospitals and the community. The manuscript manage- (bacterial, fungal and viral) and the development and institution of ment system is completely online and includes a very quick and fair peer- preventive strategies to minimize the development and spread of resis- review system, which is all easy to use. Visit http://www.dovepress.com/ tance. The journal is specifically concerned with the epidemiology of testimonials.php to read real quotes from published authors. Submit your manuscript here: https://www.dovepress.com/infection-and-drug-resistance-journal submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 955 DovePress Powered by TCPDF (www.tcpdf.org)