Identification of a Novel Hepatitis C Virus Genotype From Punjab, India: Expanding Classification of Hepatitis C Virus Into 8 Genotypes

Abstract

Background

Hepatitis C virus (HCV) exhibits great genetic diversity and is classified into 7 genotypes (GTs), with varied geographic prevalence. Until the recent development of pangenotypic direct-acting antiviral regimens, the determination of HCV GT was necessary to inform optimal treatment.

Methods

Plasma samples with unresolved GT using standard commercial genotyping methods were subjected to HCV full-genome sequencing, and phylogenetic analysis was performed to assign GT.

Results

Four patients, previously classified as GT5 by LiPA or Abbott RealTime polymerase chain reaction assays, were identified as infected with a novel HCV GT. This novel HCV GT, GT8, is genetically distinct from previously identified HCV GT1–7 with >30% nucleotide sequence divergence to the established HCV subtypes. All 4 patients were originally from Punjab, India, but now reside in Canada and are epidemiologically unlinked. Despite presence of baseline resistance-associated substitutions within the GT8 virus of all 4 patients (NS3: V36L, Q80K/R; NS5A: Q30S, Y93S), all patients achieved a sustained virologic response; 2 treated with sofosbuvir/velpatasvir/voxilaprevir for 8 weeks, 1 with sofosbuvir/ledipasvir plus ribavirin for 24 weeks and 1 with sofosbuvir plus daclatasvir for 12 weeks.

Conclusions

The discovery of a novel HCV GT8 confirms the circulation of this newly identified lineage in the human population.

genetic diversity, genotype classification, HCV full-genome sequencing

Hepatitis C virus (HCV) is a major worldwide pathogen belonging to the family of Flaviviridae in the genus Hepacivirus. It is a significant cause of chronic liver disease and is estimated to infect 71 million people globally [1]. In industrialized countries, transmission occurs mainly between people who inject drugs; other mechanisms of HCV transmission include perinatal transmission, invasive medical and dental procedures, and rarely via sexual intercourse.

The HCV genome consists of a single strand of positive-sense ribonucleic acid (RNA) encoding a single polyprotein [2]. Seven major genotypes (GTs) have been recognized to date, the complete genomes of which differ from each other by at least 30% at the nucleotide level [3–7]. Genotypes 1–4 and 6 contain multiple subtypes that typically differ by 15%–25% and display high genetic variability, whereas GT5 has 1 subtype identified [4]. In 2006, a novel HCV GT was identified in a patient originating from the Democratic Republic of Congo, which was later classified as HCV GT7a with subsequent identification of GT7b [4, 5, 8, 9]. Genotype 1 is the most prevalent globally (46%) and predominates in Europe, North America, and Australia followed by GT3 (30%) primarily distributed in South Asia, particularly the Indian subcontinent [10, 11]. Genotypes 2, 4, and 6 are responsible for approximately 23% of cases, whereas GT5 and GT7 comprise <1% [12]. The GT of HCV and resistance-associated substitutions (RASs) occur naturally in different HCV subtypes and can affect treatment outcome depending on the selected HCV treatment regimen [13, 14].

The estimated prevalence of HCV infection in India is approximately 0.5%–2.0%, with GT3 being most common [15, 16]. Despite the low prevalence of HCV, India with its large population accounts for a significant proportion of the global HCV burden with approximately 12–18 million people infected [17]. Significant variability in prevalence has been described across Indian geographical regions [18, 19]. The State of Punjab in northwest India has among the highest rates of HCV infection in India, estimated to be between 3.2% and 5.2%, predominantly GT3 [20]. In one epidemiological survey conducted in the region, Ludhiana, Moga, and Sangrur districts represented 30%, 18%, and 12% of the cases, respectively [21].

METHODS

Patient Samples

The clinical study POLARIS-2 (NCT02607800) was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice guidelines, and local regulatory requirements. All patients provided written informed consent.

Laboratory Assessments

Hepatitis C virus RNA was determined at a central laboratory using COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test, version 2.0 (Roche Molecular Diagnostics, Pleasanton, CA) with a lower limit of quantitation of 15 IU/mL. Hepatitis C virus GT was determined using VERSANT HCV Genotype 2.0 assay (LiPA) or by Abbott RealTime HCV Genotype II assay (Abbott, Des Plaines, IL).

Hepatitis C Virus Genome Sequencing and Resistance-Associated Substitutions Definitions

Hepatitis C virus RNA was isolated from 200 µL plasma using the QIAamp MinElute Virus spin kit (QIAGEN, Hilden, Germany), and full HCV genome sequencing was performed at DDL Diagnostic Laboratory (Rijswijk, The Netherlands) as previously described [22]. In brief, RNA was reverse transcribed and amplified using the Ovation RNA-Seq V2 system (NuGEN, San Carlos, CA). Double-stranded deoxyribonucleic acid was generated and amplified using single-primer isothermal linear amplification, according to the manufacturer’s protocol, with minor modifications. Amplified products were fragmented using the Covaris system (Covaris, Inc., Woburn, MA), and paired-end libraries were created for each sample using Ovation Ultralow DR Multiplex Systems (NuGEN), following the manufacturer’s instructions. Libraries were subjected to Illumina MiSeq deep sequencing. In cases of low sequencing coverage, target gene amplification was used to refine sequence. Internally developed software (Gilead Sciences) was used to process and align sequencing data. Consensus sequences were submitted to GenBank (accession numbers: MH590698–MH590701). The RASs are defined as substitutions that confer reduced susceptibility to any approved direct-acting antiviral (DAA) inhibitor with >2.5-fold change compared GT1a reference (HCV1a H77 NC AF009606; NS3 RASs: V36A/G/I/L/M/T, Q41R/H/K, F43L/S/V, T54A/C/G/S, V55A/I, Y56H, Q80K/L/R, S122D/N/R, R155any, A156any, D168any, and I170A/T/V; NS5A RASs: K24A/E/G/N/R, M28A/G/T/V, Q30any, L31I/F/M/V, P32L, S38F, H58D/L/N, A92K/P/T, and Y93any; NS5B NI RASs: L159F, E237G, S282any, L320F, and V321A). Patient material will be made available upon request for further analysis of 3’-untranslated region (UTR) region.

Phylogenetic Analyses and Sequence Homology Calculations

Full-genome consensus sequences were aligned to HCV GT1-7 reference set downloaded from ITCV website containing complete genomes of all 67 HCV subtypes described by Smith et al [4] including GT7b and subtypes not yet assigned a subtype (https://talk.ictvonline.org/ictv_wikis/flaviviridae/w/sg_flavi/56/hcv-classification). Maximum likelihood phylogenetic trees were inferred using GARLi (version 2.0) software, which optimizes the substitution model iteratively [23]. Default parameters were used. The confidence of the branches was assessed by approximate likelihood-ratio test [24] using PhyML 3.0 [25]. The phylogenetic trees were visualized using FigTree (version 1.3.1).

Sequence homology between HCV sequences were calculated by basic local alignment search tool, BLASTN 2.6.1 [26]. The following reference sequences were used: GT1a H77 NC AF009606 [27], GT1b Con1 AJ238799 [28], GT2a JFH1 AB047639 [29], GT2b JQ745651 [30], GT3a S52 GU814263 [31], GT4a D43 GU814265 [31], GT5a SA13 AF064490 [32], GT6a EUHK2 Y12083 [33], and GT7a EF108306 [5]. In addition, the full-genome sequences of GT8 virus were compared across the genome to GT1 to GT7 by SimPlot v.3.5.1 using the GT8 virus as query. Analysis was performed on sequence window of 200 base pairs (bp), incremented by 20 bp in successive fragments across the genome, using default settings (Window: 200 bp; Step: 20 bp; GapStrip: On, Kimura [2-parameter]; T/t: 2.0).

Recombination Analysis

The generated full-genome sequences were analyzed by BootScan in SimPlot v.3.5.1 using default settings (Step: 20 bp; GapString: On, Reps: 100, Kimura [2-parameter]; T/t: 2.0, Neighbor-Joining). Analysis was performed at the nucleotide level of GT1 to GT7 with GT8 sequences as query to identify possible recombination breakpoints.

Phenotypic Analysis

Phenotypic analysis was performed on GT8 clinical isolates to investigate in vitro susceptibility of GT8 to sofosbuvir, velpatasvir, and voxilaprevir as previously described [34]. In brief, patient specimen-derived HCV NS3, NS5A, and NS5B coding sequence was inserted in chimeric replicon vector (NS3 – JFH-1 GT2a backbone [35], NS5A and NS5B – Con1 GT1b backbone [28]). The RNA was transcribed from the vector in vitro and transfected by electroporation into “cured” Huh-7 cell lines. The “cured” cells were generated by prolonged treatment of Huh-7 stably transfected replicon cells to obtain cells free of HCV RNA, which are more permissive to transfection, as previously described [36]. Transfected cells were cultured in the presence of a range in concentration of different inhibitors and luciferase activity, measured 3 days posttransfection, and was used to derive half maximal effective concentration (EC₅₀). The EC₅₀ is the compound concentration at which a 50% reduction in the level of Renilla reporter activity was observed when compared with control samples with dimethyl sulfoxide. Dose-response curves and EC₅₀ values were generated using GraphPad Prism software package (GraphPad Software, La Jolla, CA) by nonlinear regression analysis, as previously described [37].

Role of the Funding Source

The funder of the study had a role in study design, data collection, data analysis, and data interpretation and approved the content. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

RESULTS

We report the finding of a novel HCV GT identified in 4 patients originating from Punjab, India, which confirms the circulation of this newly identified lineage in the human population.

Two patients in Canada, originating from Punjab, India, and previously identified to be infected with GT5 by commercial assays were enrolled in the POLARIS-2 study [38]. Initial sequencing using GT5 primers for amplification failed. Therefore, full HCV genome sequencing was performed, and a maximum likelihood phylogenetic analysis revealed a novel HCV GT [39]. Full HCV genome sequencing was performed on 2 additional patients in Canada also originating from Punjab and previously classified as HCV GT5 by commercial assays (Table 1). The full HCV genome sequencing approach generated complete 5’-UTR sequence in 3 of 4 patients (94.7%–100% of total nucleotide length) and complete sequence of the HCV open reading frame in 2 of 4 patients (99.3%–100% of total nucleotide length). Limited sequence of the 3’-UTR was obtained due to technical difficulty in amplifying the poly U region (≤58% of total nucleotide length) (Supplementary Table 1).

Table 1.

Characteristics of Patients with GT8 HCV Infection

Patient	HCV VL (IU/mL)	Country (Origin)	Race	Age	Sex	GT by Abbott or LiPA	GT by Phylo Analyses	Resistance-Associated Substitutionsa			Treatment	SVR12
								NS3 RASs	NS5A RASs	NS5B RASs
1	20100000	CAN (Kalala village, Barnala District, Punjab, India)	Asian	28	Male	GT5	GT8	V36L Q80K	Q30S Y93S	None	SOF/VEL/VOX 8 wks	Yes
2	8710000	CAN (Rampura village, Sangrur District, Punjab State, India)	Asian	31	Male	GT5	GT8	V36L Q80R	Q30S Y93S	None	SOF/VEL/VOX 8 wks	Yes
3	4735001	CAN (Ludhiana City, Ludhiana District, Punjab, India)	Asian	40	Male	GT5	GT8	V36L Q80R	Q30S Y93S	None	SOF + DCV 12 wks	Yes
4	4200000	CAN (Raikot City, Ludhiana District, Punjab State, India)	Asian	66	Female	GT5	GT8	V36L Q80K	Q30S Y93S	None	LDV/SOF + RBV 24 wks	Yes

Abbreviations: CAN, Canada; DAA, direct-acting antiviral; DCV, daclatasvir; GT, genotype; HCV, hepatitis C virus; LVD, ledipasvir; RAS, resistance-associated substitution; RBV, ribavirin; SOF/VEL/VOX, sofosbuvir/velpatasvir/voxilaprevir; SVR, sustained virologic response; VL, viral load.

^aRASs are defined as substitutions that confer reduced susceptibility to any approved DAA inhibitor with >2.5-fold change compared with GT1a reference (HCV1a H77 NC AF009606).

The generated sequences were aligned to HCV GT1–7 reference sequences (67 subtypes) obtained from the ICTV website. The majority of the reference sequence contained full-length open reading frames; however, some reference sequences were missing 5’-UTR and the end of NS5B. To allocate for the differences in genome coverage of sequences in the alignment, the 5’-UTR regions were removed and the 3’ ends were truncated. The final alignment covered 99% of the open reading frame and had 90 amino acids absent in the end of NS5B. Maximum likelihood phylogenetic analysis of the aligned sequences showed that these 4 patients were infected with a novel HCV GT, GT8, clearly distinct from previously described HCV GTs (Figure 1), and absent of recombination break points using SimPlot analysis (Supplementary Figure 1A).

Figure 1.

Phylogenetic tree of hepatitis C virus (HCV) genotype (GT) 1 through 8. The maximum likelihood tree includes the novel GT, GT8, from the 4 patients from Punjab district in India, colored in red. Alignment contained a total of 170 sequences of nearly complete coding region of HCV (99% of open reading frame; 8841 nucleotides), including Core to mid NS5B due to ambiguous nucleotides in end of NS5B for 4d reference (4d_FJ462437, position 9109 in 1a H77 coordinates). Subtype information for the 67 subtypes described by Smith et al [4] are indicated in the tree with corresponding letter as well as GT7b [8] and GT8a as the subtype of GT8. Unassigned subtypes (n = 25) are included in the tree without letter code.

Homology calculations of GT8 to other HCV GTs showed an average of 67%–71% sequence identity of GT8 to GT1–7 across the HCV NS3, NS5A, and NS5B genes, which are the protein targets for DAAs (Table 2). In addition, analysis of the complete genome of GT8 in comparison to GT1–7 showed that 5’-UTR region and core are conserved among GTs. Moreover, high diversity was observed in E1 and E2 and the beginning of NS2 with <50% identity of GT8 to other GTs in specific regions (Supplementary Figure 1B). The 4 GT8 viruses were closely related to each other, with 89%–90% homology between sequences, suggesting that they belong to the same subtype, GT8a (Table 2, Supplementary Figure 2).

Table 2.

Sequence Homology of GT8 to Other HCV Genotypes^a

	NS3					NS5A					NS5Bb					Complete Genomec
GT8 Patient	1	2	3	4	Aver	1	2	3	4	Aver	1	2	3	4	Aver	1	2	3	4	Aver
GT1a	73	72	73	73	71	67	68	68	67	67	72	72	73	73	71	70	70	70	70	69
GT1b	71	71	72	72		69	70	69	68		71	70	72	71		70	70	70	70
GT2a	69	70	70	71		66	68	67	66		70	69	70	71		68	68	68	68
GT2b	70	70	71	70		69	70	68	64		71	70	71	70		67	67	68	67
GT3a	70	70	70	70		65	66	65	66		72	72	72	72		68	68	68	69
GT4a	72	72	72	72		68	66	66	67		72	74	73	72		70	70	70	70
GT5a	73	73	73	73		69	70	70	70		71	70	72	71		70	71	70	70
GT6a	71	71	72	72		66	67	67	66		72	72	72	72		69	69	69	69
GT7a	68	68	69	72		65	65	65	66		69	68	69	69		66	66	71	66
GT8 Patient 1	-	-	-	-	91	-	-	-	-	89	-	-	-	-	94	-	-	-	-	91
GT8 Patient 2	90	-	-	-		89	-	-	-		93	-	-	-		90	-	-	-
GT8 Patient 3	91	91	-	-		88	90	-	-		94	93	-	-		90	91	-	-
GT8 Patient 4	91	90	92	-		89	89	90	-		94	93	94	-		90	91	92	-

Abbreviations: Aver, average; GT, genotype; HCV, hepatitis C virus. Bold text emphasizes column/row headers.

^aSequence homology between HCV nucleotide sequences were calculated by basic local alignment search tool, BLASTN 2.6.1.

^bNS5B nucleotide alignment contained 1692 nucleotides of total 1779 nucleotides of NS5B due to removal of ambiguous nucleotides in end of NS5B.

^cAlignment containing 9198 nucleotides (core to NS5B) with ambiguous bases removed in end of NS5B.

For GT8 sequences, amino acid positions in NS3, NS5A, and NS5B associated with resistance in other GTs were investigated. Amino acids associated with resistance to GT1a were observed at specific positions in GT8. Within NS3, all 4 patients harbored V36L with 2 harboring Q80K and the other 2 harboring Q80R. Within NS5A, all 4 patients harbored Q30S and Y93S. No amino acids associated with resistance were observed in NS5B (Table 1, Supplementary Figure 1). All other amino acid substitutions compared with other GTs are shown in the alignment (Supplementary Figure 2). Phenotypic analysis was performed as previously described [34] on clinical isolates to investigate in vitro susceptibility of GT8 to sofosbuvir, velpatasvir, and voxilaprevir (Table 3). The clinical isolates from patients 2 and 3 containing NS3 V36L and Q80R, respectively, demonstrated no change in susceptibility to voxilaprevir. The isolates from all 4 patients containing NS5A RASs Q30S and Y93S demonstrated no change in susceptibility to velpatasvir; however, they demonstrated high resistance to ledipasvir (Table 3). The NS5B clinical isolates did not replicate. Taken together, the GT8 NS3 and NS5A clinical isolates harboring amino acids associated with resistance to other GTs showed no evidence of resistance to voxilaprevir or velpatasvir, and both GT8 patients treated with sofosbuvir/velpatasvir/voxilaprevir for 8-weeks achieved sustained virologic response (SVR). Despite presence of NS5A amino acids conferring high-fold resistance in GT1a (Q30S, Y93S) to ledipasvir (Table 3), patient 4 achieved SVR after treatment with sofosbuvir/ledipasvir plus ribavirin for 24 weeks.

Table 3.

Phenotypic Analysis of Clinical Isolates

	EC50 Fold Change vs Wild-Type
	VOX	VEL	LDV
GT1b WT EC50(nM)	2.9	0.002	0.002
	GT8 Clinical Patient Isolates
NS3
V36L, Q80Ra	0.59 ± 0.2	NA	NA
NS5A
Q30S, Y93Sb	NA	1.6 ± 1.5	2819 ± 604

Abbreviations: EC₅₀, half maximal effective concentration; GT, genotype; LVD, ledipasvir; NA, not applicable; VEL, velpatasvir; VOX, voxilaprevir; WT, wild type.

^aAverage EC₅₀ values from clinical isolates of patient 2 and patient 3. Virus isolate from the other 2 patients did not replicate.

^bAverage EC₅₀ values from clinical isolates of all 4 GT8 patients.

Patient 1 is a 27-year-old man born in 1987 in Kalala village (30.50°N 75.52°E), Barnala district, Punjab, India (Figure 2). He had no history of injection drug use, surgery, or having received blood products. He moved from India to Alberta, Canada in 2014 and was diagnosed with HCV infection in 2015. He was classified as having GT5 by the Abbott RealTime HCV Geno-II assay. Transient elastography revealed minimal fibrosis (5.1 kPa). He underwent treatment with sofosbuvir/velpatasvir/voxilaprevir for 8-weeks in the POLARIS-2 clinical trial and achieved a SVR (Table 1).

Figure 2.

Punjabi districts from which patients with hepatitis C virus genotype 8 originated. Two patients were from the Ludhiana district, 1 from the Barnala district and 1 from the Sangrur district. Insert, Punjab State in red.

Patient 2 is a 31-year-old man born in 1986 in Uppali village (30.24°N 75.85°E), Sangrur district, Punjab, India (Figure 2). He was raised and lived 136 kilometers away in Rampura village until 2010. He denied a history of surgery, blood transfusion, or injection drug use but underwent routine dental care and sutures for a foot laceration. He immigrated to Ontario, Canada, in 2010 with no permanent residence in any other country. Chronic HCV infection was detected in 2014. Transient elastography revealed minimal fibrosis (5.1 kPa). He was classified as having GT5 by the Abbott RealTime HCV Geno-II assay. He underwent treatment with sofosbuvir/velpatasvir/voxilapravir for 8-weeks in the POLARIS-2 clinical trial and achieved a SVR (Table 1).

Patient 3 is a 40-year-old man born in 1976 in Ludhiana city (30.90°N 75.86°E), Ludhiana district, Punjab, India (Figure 2). He received multiple medical injections and dental treatments in India but denied injection drug use or receipt of blood products. He immigrated to Alberta, Canada in 2009 and was diagnosed with chronic HCV in January 2017. He was classified as having GT5 by the Abbott RealTime HCV Geno-II assay. Transient elastography revealed minimal fibrosis (liver stiffness 4.6 kPa). In 2017, he went to visit family in India where he was treated with a 12-week course of sofosbuvir plus daclatasvir; after return, SVR was documented in Canada. (Table 1)

Patient 4 is a 66-year-old woman born in 1951 in Raikot city (30.65°N 75.6°E.), Ludhiana district, Punjab, India (Figure 2). She underwent appendectomy in Ludhiana District Hospital. She never traveled to Rampura village, which is 65 kilometers away; however, she has visited friends and relatives often in Kalala village, which is 22 kilometers from Raikot. She immigrated to Ontario, Canada, in 2006 with no permanent residence in any other country. She was classified in 2007 as having GT5 by the VERSANT HCV Genotype 2.0 assay (LiPA). In January 2015, she was found to have Child-Pugh B cirrhosis. Genotyping, using the Abbott RealTime HCV Geno-II assay, again revealed GT5. In February 2016, the patient went to India for evaluation at Ludhiana District Hospital and began a 6-month treatment course with sofosbuvir/ledipasvir and ribavirin. She returned to Canada and was admitted to hospital for hepatic decompensation with encephalopathy, refractory ascites requiring paracentesis, and secondary bacterial peritonitis but did not disclose that she was taking antiviral treatment. She was re-evaluated during her fourth month of treatment and found to have an undetectable viral load. She was again lost to care until January 2017, at which time an SVR was documented with clinical improvement (Table 1).

DISCUSSION

The discovery of a novel HCV GT8 confirms the endemic nature of HCV in the Indian subcontinent, particularly in the Punjab State, which has one of the highest prevalence rates in the country and has important implications for the genetic and epidemiological characterization of the HCV epidemic worldwide. The recent development of highly effective, safe, pangenotypic DAAs has transformed HCV treatment.

Before the development of pangenotypic DAA treatments, knowledge of the HCV GT was required to inform the optimal treatment regimen, a limitation that complicated HCV treatment at the individual and population levels and represented a significant barrier to worldwide elimination goals. The current focus is on global elimination invoking the simplest and most comprehensive identification and treatment strategies, particularly in resource-limited countries where access to detailed HCV molecular characterization and wide choice of DAA regimens may not be available. Broad uptake of pangenotypic regimens will serve to mitigate the risks of GT misidentification using current commercially available assays, which target the 5’-UTR and are designed to capture GT1–6.

Commercial tests use proprietary probes, and without the precise sequence they utilize the exact nature of the explanation for the cross-reactivity, resulting in GT8 initially being identified as GT5 cannot be provided. Although all 4 patients with GT8 strains in our report were identified initially as GT5 by a commercial assay, it is difficult to comment on how many other patients may have also been similarly affected (the prevalence of GT5 in the Punjab state has not been reported, but it is likely to be very low). Of note, it is important to recognize that the identification of patients with GT8 as GT5 only suggests that GT8 is similar to GT5 in the 5’-UTR probe binding site; across the near entire HCV sequence, GT8 is as diverse from GT5 as it is from all the other known GTs.

CONCLUSIONS

Because GTs, in addition to being vital for the characterization of the epidemiology and evolution of HCV, are also important in determining the treatment regimen, the inaccurate identification of infecting GTs may place patients at risk of treatment failure, if non-pangenotypic regimens are used. Further research into the discovery and characterization of additional cohorts of patients with HCV GT8 infection, and yet unidentified GTs, will enhance the confidence in pangenotypic treatments as well as help to better understand the role of HCV GT in a highly mobile global population.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Presented in part: American Association for the Study of Liver Disease, October 2017, Washington DC; 27th Annual Conference of the Asian Pacific Association of the Study of the Liver, March 2018, New Delhi.

Notes

Acknowledgments.  We thank Professor Peter Simmonds (University of Oxford) for important discussions about hepatitis C virus (HCV) diversity.

Potential conflicts of interest.  S. M. B. has received research and study agreement grants from Merck, AbbVie, and Gilead Sciences. He is on the advisory boards for Merck, AbbVie, and Gilead Sciences and has consultancy agreements with Merck and Gilead Sciences. C. H., B. P., R. H. H., L. M. S., D. M. B., M. G. S., J. G. M., H. M., and E. S. are employees and stock holders of Gilead Sciences. S. D. S. has received funding for HCV clinical trials from AbbVie, BMS, Gilead Sciences, and Merck and honoraria from BMS, Gilead Sciences, and Merck. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Author notes

S. M. B. and S. D. S. contributed equally to this work.