Two mutations in the ORF1 of genotype 1 hepatitis E virus enhance virus replication and may associate with fulminant hepatic failure.

Hepatitis E virus (HEV) infection in pregnant women has a high incidence of developing fulminant hepatic failure (FHF) with significant mortality. Multiple amino acid changes in genotype 1 HEV (HEV-1) are reportedly linked to FHF clinical cases, but experimental confirmation of the roles of these changes in FHF is lacking. By utilizing the HEV-1 indicator replicon and infectious clone, we generated 11 HEV-1 single mutants, each with an individual mutation, and investigated the effect of these mutations on HEV replication and infection in human liver cells. We demonstrated that most of the mutations actually impaired HEV-1 replication efficiency compared with the wild type (WT), likely due to altered physicochemical properties and structural conformations. However, two mutations, A317T and V1120I, significantly increased HEV-1 replication. Notably, these two mutations simultaneously occurred in 100% of 21 HEV-1 variants from patients with FHF in Bangladesh. We further created an HEV-1 A317T/V1120I double mutant and found that it greatly enhanced HEV replication, which may explain the rapid viral replication and severe disease. Furthermore, we tested the effect of these FHF-associated mutations on genotype 3 HEV (HEV-3) replication and found that all the mutants had a reduced level of replication ability and infectivity, which is not unexpected due to distinct infection patterns between HEV-1 and HEV-3. Additionally, we demonstrated that these FHF-associated mutations do not appear to alter their sensitivity to ribavirin (RBV), suggesting that ribavirin remains a viable option for antiviral therapy for patients with FHF. The results have important implications for understanding the mechanism of HEV-1-associated FHF.

Full-length sequence analysis of hepatitis C virus genotype 3b strains and development of an in vivo infectious 3b cDNA clone.

IMPORTANCE: HCV genotype 3b is a difficult-to-treat subtype, associated with accelerated progression of liver disease and resistance to antivirals. Moreover, its prevalence has significantly increased among persons who inject drugs posing a serious risk of transmission in the general population. Thus, more genetic information and antiviral testing systems are required to develop novel therapeutic options for this genotype 3 subtype. We determined the complete genomic sequence and complexity of three genotype 3b isolates, which will be beneficial to study its biology and evolution. Furthermore, we developed a full-length in vivo infectious cDNA clone of genotype 3b and showed its robustness and genetic stability in human-liver chimeric mice. This is, to our knowledge the first reported infectious cDNA clone of HCV genotype 3b and will provide a valuable tool to evaluate antivirals and neutralizing antibodies in vivo, as well as in the development of infectious cell culture systems required for further research.

Performance of HCV core antigen and PCR testing in a predominantly genotype 3 population.

Hepatitis C core antigen (HCVcAg) is becoming increasingly recognized as an alternative to molecular testing for the confirmation of chronic hepatitis C. However, there are limited data on the performance of this assay in a genotype 3 (GT3) predominant country like Pakistan. We conducted a study to evaluate the diagnostic performance of HCVcAg against the HCV polymerase chain reaction (PCR) molecular test. HCV antibody-positive patients requiring confirmatory testing were recruited from August to October 2018 at the Pakistan Kidney and Liver Institute and Research Center (PKLI&RC), Lahore, Pakistan. Patients with previously known diagnoses or treatment histories were excluded. The Abbott HCV Ag assay was used for HCVcAg testing. Results ≥3.00 fmol/L were considered positive for HCVcAg. The Abbott RealTime HCV assay was used for PCR testing with a lower detection limit of ≥12 IU/mL. We computed the sensitivity, specificity and correlation of HCVcAg against HCV PCR. A total of 394 patients were recruited. The median age of the patients was 42 years. Most participants were females (51.5%, n = 203), 30.7% (n = 121) had HTN, 10.4% DM (n = 41) and 5% had APRI ≥2. The overall sensitivity was 98.0% and the specificity was 98.6%. The lowest detection limit of cAg was an HCV RNA value of 4657 IU/mL. The levels of cAg were highly correlated with those of HCV RNA by Spearman's rank correlation test (r = 0.935, p < .001). HCVcAg represents a suitable alternative with high sensitivity and specificity compared with HCV PCR in the GT3-predominant population and can be incorporated into algorithms to improve linkage to care.

Efficacy, safety, and quality of life profile of Genotype-3 Chronic Hepatitis-C Pakistani patients receiving ledipasvir plus sofosbuvir treatment.

Objective: This study aimed to assess the overall treatment response of Genotype-3 Chronic HCV Pakistani Patients with or without cirrhosis to Ledipasvir plus Sofosbuvir combination. Method: In this observational study, HCV Genotype-3 patients were enrolled from Liver Center, DHQ Hospital, Faisalabad and divided into two groups, i.e., non-cirrhotic and compensated cirrhotic patients. The study spanned for a period of 24 months (November 2019 - November 2021) from the first enrollment to the last follow up. Non-cirrhotic patients received Ledipasvir/Sofosbuvir (LDV/SOF) 90/400mg for 12 weeks and cirrhotic patients received LDV/SOF with Ribavirin (RBV) for 12 weeks and without RBV for 24 weeks. The treatment efficacy in terms of sustained virological response (SVR12) was monitored 12 weeks post-treatment. The safety profile, and health-related quality of life (HRQoL) were monitored from baseline to follow-up visits. Results: Two hundred and ninety out of 309 (93.85%) non-cirrhotic and 31 out of 33 (93.94%) compensated cirrhotic patients achieved SVR-12. The safety profile of the non-cirrhotic and compensated cirrhotic patients was comparable throughout the study duration. Fatigue was the most commonly reported adverse event (AE) in non-cirrhotic and compensated cirrhotic patients, followed by headache, nausea, and fever. The HRQoL improved from baseline to follow-up visits among patients of both groups. Conclusion: It is concluded that LDV and SOF combination regimen is safe and effective for treating Genotype-3 HCV patients without cirrhosis/compensated cirrhosis, and also improves the patient's HRQoL.

Modulation of SOCS3 Levels via STAT3 and Estrogen-ERαp66 Signaling during Hepatitis E Virus Replication in Hepatocellular Carcinoma Cells.

Hepatitis E virus (HEV) infection usually results in a self-limiting acute disease; however, in infected pregnant women, it is associated with increased mortality and fulminant hepatic failure. Estrogen is known to be elevated during pregnancy, and estrogen signaling via classical estrogen receptor-ERα is known to regulate hepatocyte function and host innate immune response, including the STAT3 pathway. In this study, we investigated whether the estrogen classical signaling pathway via ERαp66 has any effect on STAT3 activation during HEV replication and HEV-induced IFN response. We first demonstrated that Huh7-S10-3 liver cells expressed the nonfunctional estrogen receptor ERαp36 isoform and lack the functional ERαp66 isoform. We further showed persistent phosphorylated-STAT3 levels in genotype 3 human HEV (Kernow P6 strain) RNA-transfected cells at later time points. In Huh7-S10-3 cells, estrogen at first-to-third trimester concentration (7.3 to 73 nM) did not significantly affect HEV replication; however, blocking of STAT3 activation led to a decrease in the HEV ORF2 protein level. Our mechanistic study revealed that STAT3 differentially regulates SOCS3 and type-III interferon (IFN) levels during HEV replication and the presence of estrogen-ERαp66 signaling stabilizes SOCS3 levels in vitro. We also demonstrate that HEV infection in pregnant and nonpregnant rabbits led to a significant increase in IFN response as measured by increased levels of IFN-stimulated-gene-15 (ISG15) mRNA levels irrespective of pregnancy status. Collectively, the results indicate that estrogen signaling and STAT3 regulate SOCS3 and IFN responses in vitro during HEV replication. The results have important implications for understanding HEV replication and HEV-induced innate immune response in pregnant women. IMPORTANCE Hepatitis E is usually a self-resolving acute disease; however, in pregnant women, HEV infection is associated with high mortality and fulminant hepatic failure. During pregnancy, estrogen levels are elevated, and in the liver, the estrogen receptor ERα is predominant and estrogen signaling is known to regulate hepatocyte metabolism and leptin-induced STAT3 levels. Viruses can module host innate immune response via STAT3. Therefore, in this study, we investigated whether STAT3 and estrogen-classical signaling via the ERαp66 pathway modulate HEV replication and HEV-induced innate immune response. We demonstrated that estrogen signaling did not affect HEV replication in human liver cells, but blocking of STAT3 activation reduced HEV capsid protein levels in human liver cells. We also showed that inhibition of STAT3 activation reduced SOCS3 levels, while the presence of the estrogen-ERαp66 signaling pathway stabilized SOCS3 levels. The results from this study will aid our understanding of the mechanism of HEV pathogenesis and immune response during pregnancy.

Dynamic risk assessment for hepatocellular carcinoma in patients with chronic hepatitis C.

Chronic hepatitis C (HCV) is a primary cause of hepatocellular carcinoma (HCC). Although antiviral treatment reduces risk of HCC, few studies quantify the impact of treatment on long-term risk in the era of direct-acting antivirals (DAA). Using data from the Chronic Hepatitis Cohort Study, we evaluated the impact of treatment type (DAA, interferon-based [IFN], or none) and outcome (sustained virological response [SVR] or treatment failure [TF]) on risk of HCC. We then developed and validated a predictive risk model. 17186 HCV patients were followed until HCC, death or last follow-up. We used extended landmark modelling, with time-varying covariates and propensity score justification and generalized estimating equations with a link function for discrete time-to-event data. Death was considered a competing risk. We observed 586 HCC cases across 104,000 interval-years of follow-up. SVR from DAA or IFN-based treatment reduced risk of HCC (aHR 0.13, 95% CI 0.08-0.20; and aHR 0.45, 95% CI 0.31-0.65); DAA SVR reduced risk more than IFN SVR (aHR 0.29, 95% CI 0.17-0.48). Independent of treatment, cirrhosis was the strongest risk factor for HCC (aHR 3.94, 95% CI 3.17-4.89 vs. no cirrhosis). Other risk factors included male sex, White race and genotype 3. Our six-variable predictive model had 'excellent' accuracy (AUROC 0.94) in independent validation. Our novel landmark interval-based model identified HCC risk factors across antiviral treatment status and interactions with cirrhosis. This model demonstrated excellent predictive accuracy in a large, racially diverse cohort of patients and could be adapted for 'real world' HCC monitoring.

Identification and characterization of Sofosbuvir-resistant mutations of hepatitis C virus genotype 3a replicon.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Among its 8 genotypes (GT), GT3 has a relatively lower sustained virological response to highly effective direct-acting antiviral agents (DAA). Sofosbuvir (SOF), an anti-NS5B polymerase inhibitor, is a core component of many anti-HCV DAA cocktail regimens, and its resistant mutations are rare in clinics because these mutations usually severely impair the NS5B polymerase activity, including a mutation S282T in NS5B, the most frequently reported SOF-resistant mutation. In this study, we selected SOF-resistant variants of a previously developed GT3 subgenomic replicon (PR87A7). Two mutations were identified in the viral genome of SOF-resistant PR87A7 variants, Q606R in nontargeted NS3 and S282T in targeted N5SB. We demonstrated that Q606R could rescue the replication defect of S282T in PR87A7, and the resulting double mutant confers the SOF resistance. Finally, we showed that NS3-606R could not compensate for the replication defect of S282T in other GTs. In conclusion, we identified a novel GT3-specific combination of two mutations that confers SOF resistance. Our result calls for attention to potential mutations that may arise in nontargeted viral proteins during the SOF-based DAA treatment of chronic HCV.

Sero-reactivity to three distinct regions within the hepatitis C virus alternative reading frame protein (ARFP/core+1) in patients with chronic HCV genotype-3 infection.

Hepatitis C virus (HCV) infection affects more than 71 million people worldwide. The disease slowly progresses to chronic, long-term liver injury which leads to hepatocellular carcinoma (HCC) in 5 % of infections. The alternative reading frame protein (ARFP/core+1) is encoded by a sequence overlapping the HCV core gene in the +1 reading frame. Its role in hepatitis C pathogenesis and the viral life cycle is unclear, although some observers have related its production to disease progression and the development of HCC. The aim of this study was to determine whether ARFP is immunogenic in patients with chronic HCV genotype 3 infection and to assess whether sero-reactivity is associated with disease progression, particularly to HCC. Immunogenic epitopes within the protein were predicted by a bioinformatics tool, and three -20 aa length-peptides (ARFP-P1, ARFP-P2 and ARFP-P3) were synthesized and used in an avidin-biotin ARFP/core+1 peptide ELISA. Serum samples from 50 patients with chronic HCV genotype 3 infection, 50 genotype-1 patients, 50 HBV patients and 110 healthy controls were tested. Sero-reactivity to the ARFP peptides was also tested and compared in 114 chronic HCV genotype-3 patients subdivided on the basis of disease severity into non-cirrhotic, cirrhotic and HCC groups. Chronic HCV genotype-3 patients showed noticeable rates of reactivity to ARFP and core peptides. Seropositivity rates were 58% for ARFP-P1, 47 % for ARFP-P2, 5.9 % for ARFP-P3 and 100 % for C22 peptides. There was no significant difference between these seroreactivities between HCV genotype-3 patients with HCC, and HCV genotype-3 patients with and without liver cirrhosis. Patients with chronic HCV genotype-3 infection frequently produce antibodies against ARFP/core+1 protein. ARFP peptide reactivity was not associated with disease severity in patients with HCV genotype-3. These results support the conclusion that ARFP/core+1 is produced during HCV infection, but they do not confirm that antibodies to ARFP can indicate HCV disease progression.

Efficacy and safety of direct-acting antiviral therapies and baseline predictors for treatment outcomes in hepatitis C patients: A multicenter, real-world study in Guangdong, China.

The data on direct-acting antivirals (DAAs) in chronic hepatitis C (CHC) patients in southern China with multiple genotypes circulating are limited. This study aims to evaluate the efficacy and safety of DAA regimens among CHC patients in Guangdong, China. A total of 220 patients receiving a variety of DAA were enrolled. The primary outcome was sustained virologic response (SVR) at 12 weeks. Resistance associated substitutions (RASs) were evaluated by deep sequencing. The overall SVR rate was 96.4%, and was 97.7% for genotype 1, 100% for genotype 2, 91.9% for genotype 3, 95.7% for genotype 6, and 100% for untyped. The overall incidence of adverse events (AEs) was 8.2% (18/220) and all the AEs were mild. Nonstructural proteins 5A RAS, 30K/31M, and Y93H were most prevalent at baseline and the end of treatment in non-SVR patients, respectively. Logistics regression showed that elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at baseline were specifically associated with non-SVR in patients with genotype 3 and 6 infections (p = 0.029 and p = 0.017) but not genotype 1 infection (p = 0.746 and p = 0.971), and baseline AST was the best predictor for SVR in genotypes 3 and 6 patients (area under curve = 0.890). Our studies demonstrated all DAA regimens achieved ideal SVR and were well tolerated. NS5A RAS were prevalent in non-SVR patients. Elevated ALT and AST as baseline predictors for non-SVR in genotypes 3 and 6 infections warrant further research in a larger cohort.

Partial nonstructural 3 region analysis of hepatitis C virus genotype 3a.

BACKGROUND: The hepatitis C virus (HCV) is a major cause of illness around the world. HCV genotype 3a is the most prevalent genotype in Thailand. Direct-acting antiviral (DAA) drugs are available for treatment, and these drugs target the NS3, NS5A, and NS5b proteins of HCV. However, HCV variants that are resistant to NS3 protease inhibitors have been found during treatment. This resistance can be naturally occurring or in response to treatment. The purpose of this study is to analyze the codon positions of the main mutation of the partial NS3 gene region of HCV genotype 3a. METHODS: In order to detect mutations and confirm the genotype of HCV genotype 3a, the nucleotide sequencing and amino acid portion of NS3 were analyzed. RESULTS: Twenty-six samples were successfully sequenced and clustered within two sub-clades defined as 3a-1 and 3a-2. Through amino acid mutation analysis, the variations were detected at codon positions 122 (3.8%), 132 (84.6%), 168 (100%), 170 (92.3%), 174 (100%), and 175 (100%). CONCLUSIONS: In conclusion, mutations at positions 168, 170, 174, and 175 of the NS3 region are common within the HCV genotype 3a. This information should be useful in the development of effective anti-viral drugs that can successfully treat HCV infection.

Hepatitis E virus genotype 3 microbiological surveillance by the Spanish Reference Laboratory: geographic distribution and phylogenetic analysis of subtypes from 2009 to 2019.

BackgroundHepatitis E virus genotype 3 (HEV-3) is widely distributed throughout Europe, with incidence of infections increasing in many countries. Belgium, Bulgaria, France, Germany, Italy, the Netherlands and the United Kingdom have reported the distribution of HEV-3 subtypes in cohorts of patients with hepatic disease.AimTo describe the distribution of the HEV-3 subtypes in Spain at national and autonomous community (AC) levels between 2009 and 2019. The study was also extended to Andorra.MethodsOf 5,197 samples received by the National Reference Laboratory during the study, 409 were HEV-RNA-positive. Among these, 294 (71.9%) were further typed based on an ORF2 sequence fragment, or, for a subset of 74, based on the full-coding genome sequence.ResultsHEV-3 was detected in 291 samples. The dominant subtype in Spain was HEV-3f (88.3%; 257/291), which occurred in all ACs, with no change in detection level over time. Within this subtype, three subclusters were characterised: HEV-3f-B, HEV-3f-A1 and HEV-3f-A2. The second most common HEV subtype was the recently described HEV-3m (7%; 21/291), with two subclusters identified: HEV-3m-A, which has been known since 2010, and HEV-3m-B, since 2014. The third most encountered subtype was HEV-3c (4.1%; 12/291), with a frequency not increasing over time, unlike observations in some European countries.ConclusionThe importance of the surveillance of HEV-3 subtype and subcluster circulation is yet to be assessed. This surveillance together with the comprehensive epidemiological characterisation of clinical cases, could support the identification of sources of transmission and the establishment of control measures nationally and internationally.

Efficacy and Safety of Sofosbuvir/Velpatasvir/Voxilaprevir for Hepatitis C Virus (HCV) NS5A-Inhibitor Experienced Patients With Difficult to Cure Characteristics.

BACKGROUND: In clinical trials, hepatitis C virus (HCV) salvage treatment with sofosbuvir/velpatasvir/voxilaprevir (SOF/VEL/VOX) achieved an SVR12 rate of >95% in NS5A-experienced participants. Lower SVR12 rates have been reported in real-world studies, particularly for genotype (GT)3 infection and cirrhosis. We determined the efficacy and safety of SOF/VEL/VOX in a large real-world cohort. METHODS: We assessed the efficacy of salvage SOF/VEL/VOX for HCV infection in NS5A-inhibitor experienced participants with cirrhosis and portal hypertension, prior liver transplantation (LT) or severe extra-hepatic manifestations. SOF/VEL/VOX was available via an early access program. The primary outcome was SVR12. Secondary outcome was frequency of adverse events (AE). FINDINGS: Ninety-seven participants were included. Median age was 58, 82% were male, 78% had cirrhosis, most with portal hypertension (61%, n = 46/76), and 18% had prior-LT. Of the cirrhotic participants, 96% were Child-Turcotte-Pugh class A, and 4% were class B. Of the 72% with GT3, 76% were also cirrhotic. By intention-to-treat analysis, SVR12 rate was 85% (n = 82/97). Per protocol, the SVR12 rate was 90%, including 91% in GT1 (GT1a n = 18/18, GT1b n = 2/4), 89% in GT3 (n = 59/66) and 100% in GT6 (n = 3/3). SVR12 in participants with GT3 and cirrhosis was 90%. No predictors of non-SVR12 were identified. There were 4 serious AEs including 1 death and 3 hepatic decompensation events. NS5A resistance-associated substitutions detected at baseline did not affect SVR12. CONCLUSIONS: This real-world study confirms high efficacy of SOF/VEL/VOX for the treatment of difficult-to-cure NS5A-inhibitor experienced patients, including those with GT3 and cirrhosis. Treatment was well tolerated in most; however, serious AEs can occur in those with advanced liver disease.

A novel substitution in NS5A enhances the resistance of hepatitis C virus genotype 3 to daclatasvir.

Hepatitis C virus (HCV) genotype 3 presents a high level of both baseline and acquired resistance to direct-acting antivirals (DAAs), particularly those targeting the NS5A protein. To understand this resistance we studied a cohort of Brazilian patients treated with the NS5A DAA, daclatasvir and the nucleoside analogue, sofosbuvir. We observed a novel substitution at NS5A amino acid residue 98 [serine to glycine (S98G)] in patients who relapsed post-treatment. The effect of this substitution on both replication fitness and resistance to DAAs was evaluated using two genotype 3 subgenomic replicons. S98G had a modest effect on replication, but in combination with the previously characterized resistance-associated substitution (RAS), Y93H, resulted in a significant increase in daclatasvir resistance. This result suggests that combinations of substitutions may drive a high level of DAA resistance and provide some clues to the mechanism of action of the NS5A-targeting DAAs.

Effect of baseline resistance-associated substitutions on the efficiency of glecaprevir/pibrentasvir in chronic hepatitis C subjects: A meta-analysis.

The effect of baseline resistance-associated substitutions on the sustained virologic response at 12 weeks in chronic hepatitis C subjects has drawn considerable attention. However, it has been reported that the relationship between such substitutions and sustained virologic response at 12 weeks in chronic hepatitis C subjects is variable in different treatments. This meta-analysis was performed to evaluate this relationship in subjects treated with glecaprevir/pibrentasvir. A systematic literature search up to May 2020 was done, and 17 studies were identified with 6501 chronic hepatitis C subjects. They were reporting relationships between baseline resistance-associated substitutions and sustained virologic response at 12 weeks in chronic hepatitis C subjects treated with glecaprevir/pibrentasvir. The odds ratio (OR) with 95% confidence intervals (CIs) was calculated to evaluate the prognostic role of baseline resistance-associated substitutions on the sustained virologic response at 12 weeks in chronic hepatitis C subjects treated with glecaprevir/pibrentasvir using the dichotomous method with a random or fixed-effect model. Lower sustained virologic response at 12 weeks post-treatment in chronic hepatitis C subjects was significantly related to baseline resistance-associated substitutions in overall genotypes (OR, 0.03; 95% CI, 0.15-0.61, P < .001), baseline NS5a resistance-associated substitutions in genotype-1 (OR, 0.16; 95% CI, 0.04-0.57, P = .005), baseline resistance-associated substitutions in genotype-3 (OR, 0.14; 95% CI, 0.05-0.38, P < .001), and baseline NS5a resistance-associated substitutions in genotype-3 (OR, 0.21; 95% CI, 0.09-0.49, P < .001). Sustained virologic response at 12 weeks in chronic hepatitis C subjects was not significantly related to the baseline NS5a resistance-associated substitutions (OR, 0.61; 95% CI, 0.17-2.22, P = .45), and baseline resistance-associated substitutions in genotype-1 (OR, 0.35; 95% CI, 0.12-1.088, P = .07). In conclusion, the impact of baseline resistance-associated substitutions on the sustained virologic response at 12 weeks in chronic hepatitis C subjects treated with glecaprevir/pibrentasvir may have a great prognostic effect, especially in genotype-3 as a tool to improve treatment prediction. Chronic hepatitis C subjects with baseline resistance-associated substitutions may have an independent risk relationship with poor treatment outcomes. This relationship forces us to recommend testing prior to treatment selection to avoid any possible treatment failure.

Resistance analysis and treatment outcomes in hepatitis C virus genotype 3-infected patients within the Italian network VIRONET-C.

AIM: This study aimed to investigate the role of resistance-associated substitutions (RASs) to direct-acting-antivirals (DAAs) in HCV genotype 3 (GT3). METHODS: Within the Italian VIRONET-C network, a total of 539 GT3-infected patients (417 DAA-naïve and 135 DAA-failures, of them, 13 at both baseline and failure) were analysed. Sanger sequencing of NS3/NS5A/NS5B was performed following home-made protocols. RESULTS: The majority of patients were male (79.4%), 91.4% were injection drug users, 49.3% were cirrhotic and 13.9% were HIV co-infected. Phylogenetic analysis classified sequences as GT3a-b-g-h (98%-0.4%-0.2%-1.2%) respectively. Overall, 135 patients failed a DAA regimen: sofosbuvir (SOF)/daclatasvir (DCV) or velpatasvir (VEL)±ribavirin (RBV) (N = 91/15) and glecaprevir (G)/pibrentasvir (P) (N = 9). Moreover, 14.8% of patients were treated with suboptimal regimens for GT3: 3D ± RBV (Paritaprevir/r + Ombitasvir+Dasabuvir, N = 15), SOF + Simeprevir (SIM) (N = 1) or SOF/Ledipasvir (LDV) ± RBV (N = 4). RAS prevalence was 15.8% in DAA-naïve patients. At failure, 81.5% patients showed at least one RAS: 11/25 (44.0%) in NS3, 109/135 (80.7%) in NS5A, 7/111 (6.3%) in NS5B SOF-failures. In NS5A-failures, Y93H RAS was the most prevalent (68.5% vs 5.1% DAA-naïve, P < .001) followed by A30K (12.7% vs 2.8% in DAA-naïve, P < .001). Analysing baseline samples, a higher prevalence of NS5A-RASs was observed before treatment in DAA-failures (5/13, 38.5%) vs DAA-naïves (61/393, 15.5%, P = .04). Regarding 228 DAA-naïve patients with an available outcome, 93.9% achieved a SVR. Interestingly, patients with baseline Y93H and/or A30K had SVR rate of 72.2% vs 95.7% for patients without NS5A-RASs (P = .002). CONCLUSIONS: In this real-life GT3 cohort, the majority of failures harboured resistant variants carrying NS5A-RASs, the most frequent being Y93H. The presence of natural NS5A-RASs before treatment was associated with failure. Further analyses are needed to confirm this observation, particularly for the new current regimens.

Real-World Effectiveness of Direct-Acting Antiviral Regimens against Hepatitis C Virus (HCV) Genotype 3 Infection: A Systematic Review and Meta-Analysis.

Patients with hepatitis C virus (HCV) genotype 3 (GT3) infection are resistant to direct-acting antiviral (DAA) treatments. This study aimed to analyze the effectiveness of sofosbuvir (SOF)+daclatasvir (DCV) ± ribavirin (RBV); SOF+velpatasvir (VEL)±RBV; SOF+VEL+voxilaprevir (VOX); and glecaprevir (GLE)+pibrentasvir (PIB) in the treatment of HCV GT3-infected patients in real-world studies. Articles were identified by searching the PubMed, EMBASE, and Cochrane Library databases from January 1, 2016 to September 10, 2019. The meta-analysis was conducted to determine the sustained virologic response (SVR) rate, using R 3.6.2 software. Thirty-four studies, conducted on a total of 7328 patients from 22 countries, met the inclusion criteria. The pooled SVR rate after 12/24 weeks of treatment was 92.07% (95% CI: 90.39-93.61%) for the evaluated regimens. Also, the SVR rate was 91.17% (95% CI: 89.23-92.94%) in patients treated with SOF+DCV±RBV; 95.08% (95% CI: 90.88-98.13%) in patients treated with SOF+VEL±RBV; 84.97% (95% CI: 73.32-93.91%) in patients treated with SOF+VEL+VOX; and 98.54% (95% CI: 96.40-99.82%) in patients treated with GLE+PIB. The pooled SVR rate of the four regimens was 95.24% (95% CI: 93.50-96.75%) in non-cirrhotic patients and 89.39% (95% CI: 86.07-92.33%) in cirrhotic patients. The pooled SVR rate was 94.41% (95% CI: 92.02-96.42%) in treatment-naive patients and 87.98% (95% CI: 84.31-91.25%) in treatment-experienced patients. The SVR rate of GLE+PIB was higher than other regimens. SOF+VEL+VOX can be used as a treatment regimen following DAA treatment failure.

[Twelve-week of sofosbuvir/velpatasvir therapeutic regimen for chronic hepatitis C patients in northwest region of China: a real-world multicenter clinical study].

Objective: To study the real-world outcome of China FDA-approved Sofosbuvir (SOF)/Velpatasvir (VEL) in Northwest China. Methods: In this multicenter, prospective, real-world cohort study, we recruited patients from 10 sites from Northwest China, who were chronically infected with HCV GTs 1-6 from 06/2018 to 09/2019. Patients received SOF (400mg)/VEL (100mg) for 12 weeks, and with ribavirin 900-1200 mg for GT3 cirrhosis and for any genotype decompensated cirrhosis. The primary endpoint was sustained virological response at 12-weeks post-treatment (SVR12) and safety. The secondary endpoint was the change of liver function after the achievement of SVR12. Results: Totally, 143 patients were enrolled in the study, four patients were lost to follow-up and one died during the follow-up, 138 patients were included in per-protocol analysis. Of the 138 patients, the mean age 53 years, 53.6% male, 94.2% Han nationality, 53.6% liver cirrhosis, 10.1% HBsAg(+), 6.5% renal dysfunction, 5.1% treatment-experienced, and 16.7% patients received ribavirin treatment. The genotype distribution was as follows: 35.5% GT1, 42.8% GT2, 15.9% GT3, and 5.8% un-typed. The SVR12 rate was 96.5% (138/143, 95%CI: 93.5%-99.6%) for intention-to-treat analysis, and in per-protocol analysis, all 138 patients obtained SVR12 (100%). Compared with baseline, the serum total bilirubin, ALT and AFP levels decreased (all P < 0.05), as well as increased ALB and platelet count (all P < 0.001) at post-treatment 12-weeks. Overall adverse events (AEs) rate is 29.0%, and the most common AEs were anemia (14.5%) and fatigue (8.0%). Severe side effects (edema and fatigue) occurred in 2 patients, one of whom needed a short-term interruption of treatment due to fatigue. Conclusion: In this real-world cohort study, 12-week SOF/VEL regimen with or without ribavirin achieved high SVR12 rates (96.5%-100% overall) with excellent safety profile among patients with HCV GT1/2/3 infection including patients with GT3 and cirrhosis, and led to improvement of liver function.

Insights into the unique characteristics of hepatitis C virus genotype 3 revealed by development of a robust sub-genomic DBN3a replicon.

Hepatitis C virus (HCV) is an important human pathogen causing 400 000 chronic liver disease-related deaths annually. Until recently, the majority of laboratory-based investigations into the biology of HCV have focused on the genotype 2 isolate, JFH-1, involving replicons and infectious cell culture systems. However, genotype 2 is one of eight major genotypes of HCV and there is great sequence variation among these genotypes (>30 % nucleotide divergence). In this regard, genotype 3 is the second most common genotype and accounts for 30 % of global HCV cases. Further, genotype 3 is associated with both high levels of inherent resistance to direct-acting antiviral (DAA) therapy, and a more rapid progression to chronic liver diseases. Neither of these two attributes are fully understood, thus robust genotype 3 culture systems to unravel viral replication are required. Here we describe the generation of robust genotype 3 sub-genomic replicons (SGRs) based on the adapted HCV NS3-NS5B replicase from the DBN3a cell culture infectious clone. Such infectious cell culture-adaptive mutations could potentially promote the development of robust SGRs for other HCV strains and genotypes. The novel genotype 3 SGRs have been used both transiently and to establish stable SGR-harbouring cell lines. We show that these resources can be used to investigate aspects of genotype 3 biology, including NS5A function and DAA resistance. They will be useful tools for these studies, circumventing the need to work under the biosafety level 3 (BSL3) containment required in many countries.

Eight weeks of sofosbuvir/velpatasvir for genotype 3 hepatitis C in previously untreated patients with significant (F2/3) fibrosis.

Twelve weeks sofosbuvir/velpatasvir (SOF/VEL) is a highly effective pan-genotypic regimen for hepatitis C. Phase 2 data suggest 8 weeks of treatment may be sufficient for previously untreated noncirrhotic patients with genotype 3 (GT3) infection. To maximize the number of patients potentially cured within a fixed treatment budget, we elected to treat such patients locally eligible for treatment (F2/3), with 8 weeks of SOF/VEL. By local protocol, treatment-naive patients with F2 (LSM > 6.9kPa < 9.5kPa) or F3 fibrosis (≥9.5kPa < 12.5kPa) were eligible for 8-week SOF/VEL treatment. Patients commencing treatment before 1 Oct 2017 were identified from the Scottish HCV database. Baseline and treatment outcome data obtained. Ninety patients were included for analysis (72 (80%) male, mean age 45 (IQR ± 8.4), 28 (31.1%) F3 fibrosis). Opioid agonist therapy (OAT) was prescribed in 82 (91.1%) patients. Of 49 patients attending Glasgow city Alcohol and Drug Services, 27 (55.1%) had evidence of recent drug use (< 3 months) including 8 (16.3%) with self-reported intravenous drug use. On an intention-to-treat basis, SVR rates were 86/90 (95.6%, 95% CI 89.0-98.8). Excluding those who prematurely discontinued treatment (n = 4), died prior to SVR testing (n = 1) or whom experienced reinfection (n = 1), per-protocol SVR rate was 84/84 (100%, 95% CI 95.7-100.0). In conclusion, eight-week SOF/VEL is highly effective in treatment-naive GT3 patients with significant fibrosis. This offers a non-protease inhibitor-based 8-week regimen which may be useful for complex drug interactions or where time-limited opportunity for treatment. In limited resource settings, reduction in drug acquisition costs may help achieve progress towards the goal of HCV elimination.

Direct-acting antiviral agents in the treatment of chronic hepatitis C-Real-life experience from clinical practices in Pakistan.

This study aims to evaluate the clinical effectiveness in terms of sustained virological response (SVR), predictors of SVR and safety of available second-generation generic direct-acting antivirals in Pakistani chronic hepatitis C patients. This is a retrospective study conducted in multiple centers of Pakistan from January 2015 to January 2019. The samples include patients infected with chronic hepatitis C virus, regardless of virus genotype, cirrhosis, or prior treatment. A total of 993 patients were included in the present study, with the majority receiving sofosbuvir with daclatasvir (95%), sofosbuvir with daclatasvir and ribavirin (4%), and sofosbuvir with ribavirin (1%). There were 96% cases of chronic hepatitis, 3% cases compensated cirrhosis, and 1% cases of decompensated cirrhosis. Genotype 3 (99.6%) was the most common genotype. Overall SVR after 12 weeks was 98% for all treatment regimens. High SVR12 was observed with sofosbuvir in combination with daclatasvir (98.5%), then sofosbuvir in combination with daclatasvir and ribavirin (90.2%) and sofosbuvir in combination with ribavirin (75%). SVR rates were high in chronic hepatitis C patients (98.2%) as compared with cirrhotic patients (92.1%) and it was high in treatment-naive (98.8%) then interferon experienced patients (90.1%). In multivariate binary logistic regression analysis, patients' education status, treatment strategy, viral load, and alanine aminotransferase had a statistically significant association with SVR at 12 weeks. No major adverse events occurred which required treatment discontinuation. Generic oral direct acting antiviralss (sofosbuvir with daclatasvir) achieved higher SVR12 rates and were well tolerated in this large real-world cohort of genotype 3 infected patients.