Genotype 1 Research Articles

An RNA Interference/Adeno-Associated Virus Vector–Based Combinatorial Gene Therapy Approach Against Hepatitis E Virus

Hepatitis E virus (HEV) is a major public health problem with limited therapeutic options. Here, we engineered adeno‐associated viral vectors of serotype 6 (AAV6) to express short hairpin RNAs (shRNAs) against HEV transcripts with the prospect of down‐regulating HEV replication in vivo. We designed 20 different shRNAs, targeting the genome of the HEV genotype 3 (GT3) Kernow‐C1 p6 strain, for delivery upon AAV6 transduction. Using an original selectable HEV GT3 reporter replicon, we identified three shRNAs that efficiently down‐regulated HEV replication. We further confirmed their inhibitory potency with full‐length HEV infection. Seventy‐two hours following transduction, HEV replication in both systems decreased by up to 95%. The three most potent inhibitory shRNAs identified were directed against the methyltransferase domain, the junction region between the open reading frames (ORFs), and the 3´ end of ORF2. Targeting all three regions by multiplexing the shRNAs further enhanced their inhibitory potency over a prolonged period of up to 21 days following transduction. Conclusion: Combining RNA interference and AAV vector–based gene therapy has great potential for suppressing HEV replication. Our strategy to target the viral RNA with multiplexed shRNAs should help to counteract viral escape through mutations. Considering the widely documented safety of AAV vector–based gene therapies, our approach is, in principle, amenable to clinical translation.

The Impact of Steatosis on Chronic Hepatitis C Progression and Response to Antiviral Treatments.

Metabolic derangement is characteristic in patients with hepatitis C virus (HCV) infection. Aside from established liver injury, various extrahepatic metabolic disorders impact the natural history of the disease, clinical outcomes, and the efficacy of antiviral therapy. The presence of steatosis, recently redefined as metabolic-associated fatty liver disease (MAFLD), is a common feature in HCV-infected patients, induced by host and/or viral factors. Most chronic HCV-infected (CHC) patients have mild steatosis within the periportal region of the liver with an estimated prevalence of 40% to 86%. Indeed, this is higher than the 19% to 50% prevalence observed in patients with other chronic liver diseases such as chronic hepatitis B (CHB). The histological manifestations of HCV infection are frequently observed in genotype 3 (G-3), where relative to other genotypes, the prevalence and severity of steatosis is also increased. Steatosis may independently influence the treatment efficacy of either interferon-based or interferon-free antiviral regimens. This review aimed to provide updated evidence of the prevalence and risk factors behind HCV-associated steatosis, as well as explore the impact of steatosis on HCV-related outcomes.

Hepatitis C Resistance-Associated Substitutions Among People Who Inject Drugs Treated With Direct-Acting Antiviral-Containing Regimens.

BackgroundResistance-associated substitutions (RASs) to HCV direct-acting antivirals (DAAs) can contribute to virologic failure and limit retreatment options. People who inject drugs (PWID) are at highest risk for transmission of resistant virus. We report on RASs at baseline and after virologic failure in DAA-naive and protease inhibitor-experienced PWID.MethodsWe sequenced the NS3/4A, NS5A, and NS5B regions from 150 PWID with genotype 1 (GT1) viruses; 128 (85.3%) GT1a, 22 (14.7%) GT1b.ResultsAmong the 139 (92.7%) DAA-naive PWID, 85 of 139 (61.2%) had baseline RASs—67 of 139 (48.2%) in NS3 (predominantly Q80K/L); 25 of 139 (18.0%) in NS5A; and 8 of 139 (5.8%) in NS5B. Of the 11 protease inhibitor-experienced participants, 9 had baseline NS3 RASs (V36L N = 1, Q80K N = 9) and 4 had baseline NS5A RASs (M28V N = 2, H58P N = 1, A92T N = 1). Among the 11 participants who had posttreatment samples with detectable virus (7 treatment failures, 1 late relapse, 3 reinfections), 1 sofosbuvir/ledipasvir failure had a baseline H58P. Two sofosbuvir/ledipasvir-treated participants developed new NS5A mutations (Q30H, Y93H, L31M/V). Otherwise, no RASs were detected.ConclusionsOur results demonstrate RAS prevalence among DAA-naive PWID is comparable to that in the general population. Only 2 of 150 (1.3%) in our longitudinal cohort developed treatment-emergent RASs. Concern for transmission of resistant virus may therefore be minimal.

Sequence to structure analysis of the ORF4 protein from Hepatitis E virus.

Hepatitis E virus (HEV) is the main cause of acute hepatitis worldwide. HEV accounts for up to 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (G1) HEV. The contributing factors in adverse cases during pregnancy in women due to HEV infection is still debated. The mechanism underlying the pathogenesis of viral infection is attributed to different genomic component of HEV, i.e., open reading frames (ORFs): ORF1, ORF2, ORF3 and ORF4. Recently, ORF4 has been discovered in enhancing the replication of GI isolates of HEV through regulation of an IRES-like RNA element. However, its characterization through computational methodologies remains unexplored. In this novel study, we provide comprehensive overview of ORF4 protein's genetic and molecular characteristics through analyzing its sequence and different structural levels. A total of three different datasets (Human, Rat and Ferret) of ORF4 genomes were built and comparatively analyzed. Several non-synonymous mutations in conjunction with higher entropy values were observed in rat and ferret datasets, however, limited variation was observed in human ORF4 genomes. Higher transition to tranversion ratio was observed in the ORF4 genomes. Studies have reported the association of intrinsic disordered proteins (IDP) with drug discovery due to its role in several signaling and regulatory processes through protein-protein interactions (PPIs). As PPIs are potent drug target sources, thus the ORF4 protein was explored by analyzing its polypeptide structure in order to shed light on its intrinsic disorder. Pressures that lead towards preponderance of disordered-promoting amino acid residues shaped the evolution of ORF4. The intrinsic disorder propensity analysis revealed ORF4 protein (Human) as a highly disordered protein (IDP). Predominance of coils and lack of secondary structure further substantiated our findings suggesting its involvement in binding to ligand molecules. Thus, ORF4 contributes to cellular signaling processes through protein-protein interactions, as IDPs are targets for regulation to accelerate the process of drug designing strategies against HEV infections.

Rubella epidemiology in the Central African Republic, 2015-2016 and molecular characterization of virus strains from 2008-2016

Objectives: Rubella cases in the Central African Republic (CAF) are currently identified during measles surveillance. This study aimed to investigate rubella epidemiology between 2015 and 2016 and to provide baseline genotype data for monitoring future rubella control efforts.Methods: 831 measles IgM negative or equivocal sera from 2015/2016 were tested for rubella IgM antibodies and 350 rubella IgM positive sera collected between 2008 and 2016 were selected for PCR and sequencing.Results: 411 of the 831 sera (49.5%) were rubella IgM positive and most cases (n=391, 95.1%) occurred between January and April. Most patients were between 5 and 9 years old (50.2%) and more than half of the rubella cases (56.7%) originated from the capital Bangui. Genotype information was obtained for 37 of the 350 selected rubella IgM-positive specimens, with the majority of the patients originating from Bangui (n=24, 64.9%) and sequences covering all years except 2009. Phylogenetic analysis identified genotypes 1E (n=12), 1G (n=5) and 2B (n=20), with 2B being detected from 2014 onwards.Conclusions: Our study confirmed the important role of rubella as a rash and fever disease in CAF and provided comprehensive data on rubella epidemiology and the first information on rubella genotypes in the country.

Hepatitis C virus genotype 4: A poorly characterized endemic genotype.

Globally, 13% of all hepatitis C virus (HCV) infections are caused by genotype 4 (GT4), which consists of 17 subtypes with various levels of susceptibility to anti-HCV therapy. This genotype is endemic in the Middle East and Africa and has considerably spread to Europe lately. The molecular features of HCV-GT4 infection, as well as its appropriate therapeutics, are poorly characterized as it has not been the subject of widespread basic research. As such, in this review, we aim to gather the current state of knowledge of this genotype with a particular emphasis on its heterogeneity, sequence signatures, resistance-associated substitutions, and available in vivo and in vitro models used for its study. We urge developing more cell-culture models based on different GT4 subtypes to better understand the virology and therapeutic response of this particular genotype. This review may raise more awareness about this genotype and trigger more basic research work to develop its research tools. This will be critical to design better therapeutics and help to provide adequate guidelines forphysicians working with HCV-GT4 patients.

Linkage of resistance-associated substitutions in GT1 sofosbuvir + NS5A inhibitor failures treated with glecaprevir/pibrentasvir

Retreatment with glecaprevir/pibrentasvir (G/P) resulted in a rate of sustained virologic response 12 weeks after treatment completion (SVR12) of >90% in HCV genotype 1 (GT1) patients who previously failed a regimen of sofosbuvir plus an NS5A inhibitor (NS5Ai). This study investigated the prevalence and impact of baseline NS3 and NS5A resistance-associated substitutions (RASs) on the efficacy of G/P in prior GT1 sofosbuvir+NS5Ai failures and the persistence of treatment-emergent RASs. Longitudinal samples from 177 patients enrolled in a phase IIIb, randomized pragmatic clinical trial were analyzed. Patients without cirrhosis were randomized to 12 or 16 weeks of G/P, and patients with compensated cirrhosis were randomized to G/P and ribavirin for 12 weeks or G/P for 16 weeks. Linkage of RAS was identified using Primer-ID next-generation sequencing at a 15% cut-off. Of 177 patients, 169 (95.5%) were PI-naïve. All 33 GT1b-infected patients achieved SVR12. In GT1a-infected patients, baseline NS5A RASs were prevalent (74.5%, 105/141) but NS3 RASs were uncommon. Baseline NS3 RASs had no impact on G/P efficacy and patients with baseline NS5A RASs showed a numerically but not statistically significantly lower SVR12 rate compared to those without NS5A RASs (89% vs. 97%). SVR12 was achieved in 34 of 35 (97%) patients without NS5A baseline substitution, and 53 of 57 (93%), 35 of 40 (88%), 5 of 8 (63%) with single, double-linked, and triple-linked NS5A substitutions, respectively. Among 13 patients with virologic failure, 4 acquired treatment-emergent NS3 RASs and 10 acquired NS5A RASs. Baseline NS5A RASs were highly prevalent. The presence of an increasing number of linked NS5A RASs in GT1a showed a trend in decreasing SVR12 rates, although no specific NS5A RASs or their linkage pattern were associated with lower SVR12 rates. Direct-acting antivirals have revolutionized the treatment of chronic hepatitis C infection, but treatment failure occurs in some patients. Retreatment of patients who previously failed a regimen consisting of sofosbuvir and an NS5A inhibitor with a regimen of glecaprevir and pibrentasvir (G/P) is >90% effective. Herein, we analyzed samples from these patients and showed that retreatment efficacy with G/P is lower in patients with double- or triple-linked NS5A resistance mutations than in patients with single or no NS5A resistance mutations. NCT03092375.

Transmission Dynamics of the Rubella Virus Circulating in China During 2010-2019: 2 Lineage Switches Between Genotypes 1E and 2B.

To provide a better understanding of the progress on rubella control and elimination in China, a genetic analysis was conducted to examine the transmission pattern of the endemic rubella virus in China during 2010-2019. A total of 4895 strains were obtained from 29 of the 31 provinces in mainland China during 2010-2019. The genotyping regions of the strains were amplified, determined, and assembled. Genotyping analysis and lineage division were performed by comparisons with the World Health Organization reference strains and reported lineage reference strains, respectively. Further phylogenetic analyses were performed to compare the genetic relationship. During 2010-2019, the domestic lineage 1E-L1 and multiple imported lineages of rubella viruses including 2B-L1, 1E-L2, and 2B-L2c were identified. Further analysis of the circulation trend of the different lineages indicated that 2 switches occurred among the lineages. The first shift was from lineage 1E-L1 to 2B-L1, which occurred around 2015-2016, followed by the lowest rubella incidence in 2017. The second shift was from lineage 2B-L1 to 1E-L2 and 2B-L2c, which occurred around 2018-2019, coinciding with rubella resurgence and the subsequent nationwide epidemic during 2018-2019. Insufficient genomic information worldwide made it impossible to trace the origin of the imported viruses. China was moving toward rubella elimination, as evidenced by the fact that previous endemic lineages were not detected. However, rubella reemerged in 2018 2019 due to the newly imported rubella viruses. Therefore, to realize the rubella elimination goal, joint efforts are required for all countries worldwide.

Genotyping and sequence characterization of the NSP4 gene of human group A rotavirus strains in Northern Iran.

Rotavirus is known to be responsible for remarkable numbers of severe diarrheal episodes and even death in infants and young children. In this study, we aimed to survey genetic diversity and variation analysis of viroporin, which is encoded by the rotavirus NSP4 segment. Thirty-five rotavirus-positive specimens were obtained, and RNA extraction and polymerase chain reaction amplification were performed. After the sequencing process, four specimens were excluded, and the final 31 samples remained for genetic diversity and variation analysis. The predominant single G/P combination was G1P[8] (~78%), followed by G2P[8] (~13%), and equal percentages (3%) of G2P[4], G3P[8], and G-non-typeable-P[8]. Further analyses revealed that variations could be found in the three regions of NSP4, including VP4 binding site (aa 112-146), double-layered particle binding site (aa 161-175), and finally, in the predicted amphipathic alpha-helix. Phylogenic tree analysis demonstrated that the mentioned samples clustered with genotype E1 and E2 reference sequences. As previously reported in the literature, in this study, it was revealed that no apparent correlation exists in the deduced amino acid sequences corresponding to this region between the rotaviruses collected from patients with and without diarrhea.

Real-World Experience with Coformulated Ledipasvir and Sofosbuvir for HIV-Positive Patients with HCV Genotype 2 Infection: A Multicenter, Retrospective Study.

IntroductionWhile coformulated ledipasvir (90 mg)/sofosbuvir (400 mg) (LDV/SOF) is approved for the treatment of hepatitis C virus (HCV) genotype 2 (GT2) infection in Taiwan, Japan, and New Zealand, data regarding its use for HIV (Human Immunodeficiency Virus)-positive patients infected with HCV GT2 are sparse. We aimed to assess the effectiveness and tolerability of LDV/SOF for HIV-positive patients with HCV GT2 coinfection.MethodsFrom January 2019 to July 2020, consecutive HIV-positive Taiwanese patients infected with HCV GT2 who received LDV/SOF were retrospectively included for analysis. The effectiveness was determined by sustained virologic response 12 weeks off-therapy (SVR12).ResultsOf the 114 patients (mean age, 38.6 years) initiating LDV/SOF during the study period, 0.9% had liver cirrhosis and 4.4% were HCV treatment-experienced. All patients had estimated glomerular filtration rate (eGFR) > 30 ml/min/1.73 m2 and were receiving antiretroviral therapy with 98.2% having CD4 counts ≥ 200 cells/mm3 and 93.9% plasma HIV RNA load < 50 copies/ml. Antiretrovirals prescribed included tenofovir alafenamide/emtricitabine in 42.1%, tenofovir disoproxil fumarate (TDF)/emtricitabine 18.4%, other nucleoside reverse transcriptase inhibitors (NRTIs) 39.5%, non-NRTIs 12.3%, protease inhibitors 13.2%, and integrase inhibitors 74.6%. All patients had undetectable plasma HCV RNA load at the end of treatment, and 96.5% achieved SVR12 in intention-to-treat analysis. The on-treatment eGFR decline was more pronounced in those receiving TDF-containing antiretroviral therapy (mean change, − 8.33 ml/min/1.73 m2), which was reversible after discontinuation of LDV/SOF. None of the patients interrupted LDV/SOF during the 12-week treatment course.ConclusionSimilar to the response observed among HIV-negative patients, LDV/SOF is effective for HIV-positive patients coinfected with HCV GT2.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40121-021-00424-8.

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

This study aimed to investigate the role of resistance-associated substitutions (RASs) to direct-acting-antivirals (DAAs) in HCV genotype 3 (GT3). 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. 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). 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.

Anti-HEV IgG Avidity Testing: Utility for Diagnosing Acute and Resolved Genotype 3 Infections.

European Association of the Study of the Liver (EASL) guidelines specify HEV RNA, as well as anti-HEV IgG and IgM as positive markers for acute HEV infection. HEV RNA assay sensitivity limitations may lead to false negative test results in patients with low levels of viremia. Moreover, anti-HEV IgM positivity is not a reliable indicator for distinguishing between acute and resolved infections given the ability of this antibody to persist several months after a resolved infection. Our study aims were to assess HEV IgG avidity for diagnosing acute and resolved infections, regardless of the anti-HEV IgM serostatus, and examine assay reliability when evaluating different genotype 3 (GT3) HEV subtypes. Patient serum samples (n = 104) were tested for HEV IgG avidity by utilizing the DIA.PRO kit on a DSX automated instrument. Among patients identified with acute HEV infections, 32 were infected with GT3: GT3c (n = 5), GT3e (n = 8), 3f (n = 17) and GT3-unsubtyped (n = 2). Avidity sensitivity was 91.2% and specificity was 100%. For patients with long-lasting anti-HEV IgM persistence, an Avidity Index >70% was observed. Thus, the DIA.PRO avidity assay may be utilized to distinguish between recently acquired and resolved HEV GT3 infections. However, for equivocal results (Avidity Index > 40–70%), HEV RNA molecular testing will be required to confirm a recent infection.

Противовирусная терапия хронического гепатита С: многолетний опыт реальной клинической практики

Direct-acting antivirals (DAAs) have significantly changed the treatment of chronic hepatitis C (CHC) and the prognosis for patients since their introduction into clinical practice in 2014–2015. Objective. Study of the evolution and efficacy of antiviral therapy (AVT) for CHC based on the results of providing medical care to patients in Moscow in 2002–2020. Patients and methods. The study was conducted in the Center for the Treatment of Chronic Viral Hepatitis “Infectious Clinical Hospital No 1 of the Moscow City Health Department”. For quantitative variables, the mean and standard deviation were used, while for categorical variables, frequency and proportion were counted (%). Results. 69,745 patients were diagnosed with CHC among all the patients who were examined in the Center. The pooled SVR rate of various therapy regimens was as follows in the HCV genotype 1 (GT1) cohort: IFN + ribavirin – 41.7%; DAAs + PegIFN + ribavirin – 77.4%; IFN-free regimens – 94.0%. Age older than 40 years, diabetes mellitus (DM), history of AVT failure (retreatment), and liver cirrhosis (LC) adversely affected the achievement of SVR. In the patients with HCV GT2 and GT3, the IFN + ribavirin combination allowed SVR achievement in 68.5% of patients, while IFN-free regimens were effective in 95.2% of patients. The predictive factors for achieving SVR were similar to those in the GT1 subgroup, except that the effect of age and DM did not reach statistical significance. Conclusions. The efficacy of DAAs in real clinical practice significantly exceeds the efficacy of IFN-based regimens in patients with various HCV infection genotypes. Achievement of SVR as a result of AVT reduces the likelihood of LC and HCC development. Key words: viral hepatitis, chronic hepatitis, antiviral therapy, peginterferon, interferon-free, dual therapy, triple therapy

Ravidasvir hydrochloride for genotype 1 hepatitis C treatment.

Effective oral combination regimens have been approved for treatment of hepatitis C virus (HCV) infection and demonstrated high cure rates in different HCV genotypes. These direct-acting agents target a variety of HCV proteins, including HCV-NS5A (nonstructural protein 5A). Ravidasvir hydrochloride, a potent pan-genotypic HCV-NS5A inhibitor approved in Egypt for treatment of HCV genotype 4 (G4), demonstrated impressive efficacy, safety profiles and a high barrier to resistance in multiple clinical trials when used as a key component in combination with other direct-acting agents in treating patients with HCV-G1.

Sustained virological response in patients with HCV treated with daclatasvir plus sofosbuvir, with or without ribavirin: a large, field-practice study.

BackgroundThe once-daily oral combination of daclatasvir (DCV) and sofosbuvir (SOF), with or without ribavirin (RBV), is effective and well tolerated in patients with hepatitis C virus (HCV). However, further field-practice studies are necessary to investigate the effectiveness and safety of the DCV+SOF combination in diverse subpopulations of patients with HCV, including those who are more challenging to treat such as patients with a genotype 3 (G3) infection. The aim of this retrospective, multicenter, field-practice study was to investigate the therapeutic efficacy and safety of the oral combination of DCV and SOF, with or without RBV (DCV+SOF±RBV), in a large unselected cohort of patients with chronic HCV infection (CHC).Patients and methodsConsecutive patients received DCV+SOF±RBV for 12 or 24 weeks. The efficacy endpoint was sustained virological response at 12 weeks after the end of treatment (SVR12). Safety factors were also considered.ResultsA total of 620 patients were included in this study; the predominant genotype was G3 (55.3%). Of the total sample, 248 (40%) patients were treated with DCV+SOF+RBV and 372 (60%) did not receive RBV. The majority of patients assessed at week 12 (98%, 596/608) achieved SVR12. Among G3 patients, 98.8% (335/339) achieved SVR12. The most common adverse event was elevated bilirubin (30.6%), recorded in 4.9% of cases as a grade 3–4 adverse event.ConclusionThis study shows the high pan-genotypic effectiveness and safety of the DCV+SOF±RBV combination in a large, unselected sample of CHC patients with G1–4, including a wide proportion of G3 CHC patients.

Efficacy and safety of sofosbuvir/velpatasvir in a real-world chronic hepatitis C genotype 3 cohort.

Real-world data on sofosbuvir/velpatasvir with and without ribavirin (SOF/VEL±RBV), particularly among patients with genotype 3 (GT3) decompensated cirrhosis, prior treatment, coinfection, and hepatocellular carcinoma (HCC), are scarce. We aimed to assess the efficacy and safety of SOF/VEL±RBV in a real-world setting that included both community and incarcerated GT3 hepatitis C virus (HCV) patients. We included all GT3 HCV patients treated with SOF/VEL±RBV in our institution. The primary outcome measure was the overall sustained virological response 12weeks after treatment (SVR12), reported in both intention-to-treat (ITT) and per-protocol analyses. The secondary outcome measures were SVR12 stratified by the presence of decompensated cirrhosis, prior treatment, HCC, and HIV/hepatitis C virus coinfection and the occurrence rate of serious adverse events requiring treatment cessation or hospitalization. A total of 779 HCV patients were treated with 12weeks of SOF/VEL±RBV, of which 85% were treated during incarceration. Among the 530 GT3 HCV patients, 31% had liver cirrhosis, and 6% were treatment-experienced. The overall SVR12 for GT3 was 98.7% (95% confidence interval: 97.3%, 99.5%) and 99.2% (95% confidence interval: 98.1%, 99.8%) in ITT and per-protocol analyses, respectively. High SVR12 was also seen in ITT analysis among GT3 HCV patients with decompensated cirrhosis (88%), prior treatment (100%), HCC (100%), and HIV/hepatitis B virus coinfection (100%). Apart from one patient who developed myositis, no other serious adverse events were observed. The SOF/VEL±RBV is a safe and efficacious treatment option for GT3 HCV patients in a real-world setting. SOF/VEL with RBV may be considered for decompensated GT3 HCV patients.

The A150V Polymorphism of Genotype 3 Hepatitis C Virus Polymerase Inhibits Interferon Alfa by Suppressing Protein Kinase R Activation.

Background & AimsDespite recent advances in antiviral therapy for hepatitis C virus (HCV), a proportion of patients with genotype 3 (G3) HCV infection do not respond to current all oral treatment regimens. Genomic analyses have identified key polymorphisms correlating with increased resistance to direct-acting antivirals. We previously reported that amino the acid polymorphism, A150V, in the polymerase (NS5B) of G3 HCV reduces response to sofosbuvir. We now demonstrate that this polymorphism alters the response to interferon alpha.MethodsQuantitative polymerase chain reaction, immunofluorescence, luciferase activity assay, immunoblotting, and flow cytometry were used to study the antiviral effect of interferon (IFN) on DBN G3 HCV-infected cells and G3 HCV replicons.ResultsWe show the presence of the A150V polymorphism markedly reduces the response to IFN alpha (IC50 of S52_WT = 1.162 IU/mL and IC50 of S52_A150V = 14.45 IU/mL, 12.4-fold difference). The induction of IFN-stimulated genes in A150V replicon cells is unaffected, but nuclear localization of active protein kinase R (PKR) is reduced. Blockade of PKR activity reduced the antiviral effect of IFN on wild-type replicons, whereas augmented PKR activation promoted the antiviral effect of IFN on A150V replicons. Furthermore, we show that impaired activation of PKR in A150V replicon cells diminishes cellular apoptosis.ConclusionsThese results demonstrate that polymorphisms reducing response rates to direct-acting antivirals may function beyond conferring drug resistance by modulating the intrinsic cellular antiviral response.

Combination treatment with sofosbuvir and ribavirin for patients diagnosed with hepatitis C genotype 2: A real-world, single-center study

Background and study aimsThe demand for treatments for viral hepatitis using direct antiviral agents (DAAs) has increased; however, few real-world clinical studies are available. The objective of this study was to evaluate the efficacy and safety of sofosbuvir combined with ribavirin for patients with chronic hepatitis C (CHC) genotype 2 (GT2). Patients and methodsA total of 106 consecutive CHC GT2 patients treated with sofosbuvir plus ribavirin between May 2016 and August 2018 (median age: 52.5 years, male: 51 [48.1%], treatment-naïve patients: 98 [92.5%]) were analyzed. The primary endpoint was sustained virologic response at 12 weeks (SVR12). The secondary endpoint was the occurrence of side effects during treatment. ResultsOf a total of 106 patients with CHC GT2, 103 were genotype 2a (97.2%), and 3 were 2b (2.8%). SVR12 was confirmed in 105 of 106 patients (99.1%). The one patient with treatment failure had combined liver cirrhosis and hepatocellular carcinoma. Twenty-five patients had liver cirrhosis in addition to hepatitis C virus (HCV) (Child-Turcotte-Pugh (CTP)-A, n = 24; C, n = 1), and SVR12 was confirmed in 24 of these patients (96.0%). The mean HCV RNA titer was 2,629,159 IU/ml. Reductions in haemoglobin levels occurred in 23 patients during treatment (3.0 mg/dL, mean), and consequently, ribavirin dose reduction was required (365.2 mg, mean). ConclusionSofosbuvir plus ribavirin was highly effective for the treatment of patients with CHC GT2 and had no serious, treatment-related adverse effects.

Real-world effectiveness of direct-acting antiviral agents for chronic hepatitis C patients with genotype-2 infection after completed treatment.

Chronic hepatitis C (CHC) is a major cause of cirrhosis, hepatocellular carcinoma (HCC), and mortality. Eliminating hepatitis C virus (HCV) can greatly improve long-term outcomes. Several direct-acting antiviral agents (DAAs), including sofosbuvir (SOF) plus different NS5A inhibitors, as well as non-SOF-based DAAs, including glecaprevir/pibrentasvir (GLE/PIB), have been approved for treating CHC genotype-2 (GT-2) patients in Taiwan. However, there is limited real-world effectiveness data regarding these different regimens. Thus, we aimed to evaluate the real-world efficacy in CHC GT-2 patients who underwent these DAA regimens. We retrospectively enrolled CHC GT-2 patients who were treated with SOF-based DAAs or GLE/PIB at a single medical center. A total of 704 enrolled patients were treated with either SOF + ribavirin (RBV), SOF/daclatasvir (DCV) ± RBV, SOF/ledipasvir (LDV) ± RBV, SOF/velpatasvir (VEL) ± RBV, or with GLE/PIB. The overall sustained virological response (SVR) rate was 97.9%. The SVR rate was significantly lower in the SOF + RBV group (95.6%) than in the non-SOF + RBV (98.9%) group, especially compared to the SOF/DCV (100%) and GLE/PIB groups (99.5%). Among patients treated with SOF + RBV, cirrhotic patients had significantly lower SVR rates than noncirrhotic patients (89.4% vs 98.2%). Multivariate analysis showed that patients with a younger age, hepatitis B virus coinfection, baseline cirrhosis, or those who received SOF + RBV were less likely to achieve SVR. In conclusion, for CHC GT-2 patients, SOF in combination with DCV, LDV, or VEL, as well as GLE/PIB, achieved similar high efficacies, regardless of cirrhosis, treatment experience, or chronic kidney disease status. Therefore, the use of DAA therapy to eradicate HCV should not be delayed in these populations.

Clinical Characteristics of Acute Hepatitis E and Their Correlation with HEV Genotype 3 Subtypes in Italy.

Genotype 3 (GT3) is responsible for most European autochthonous hepatitis E virus (HEV) infections. This study analyzed circulating genotypes and GT3 subtypes in the Lazio region, Italy, between 2011 and 2019, as well as their pathogenic characteristics. Of the 64 evaluable HEV GT3 patient-derived sequences, identified subtypes included GT3f (n = 36), GT3e (n = 15), GT3c (n = 9), GT3a (n = 1) and three unsubtyped GT3 sequences. GT3c strains were similar to Dutch sequences (96.8–98.1% identity), GT3e strains showed high similarity (96.8%) with a United Kingdom sequence, while the most related sequences to GT3f Italian strains were isolated in France, Belgium and Japan. One sequence was closely related to another Italian strain isolated in raw sewage in 2016. The liver functioning test median values for 56 evaluable GT3 patients were: alanine aminotransferase (ALT), 461 (range 52–4835 U/L); aspartate aminotransferase (AST), 659 (range 64–6588 U/L); and total bilirubin, 3.49 (range 0.4–33 mg/dL). The median HEV RNA viral load for 26 evaluable GT3 patients was 42,240 IU/mL (range 5680–895,490 IU/mL). Of the 37 GT3 patients with available clinical information, no correlation was observed between HEV clinical manifestations and GT3 subtype. HEV symptoms were comparable among GT3c/e/f patients across most analyzed categories except for epigastric pain, which occurred more frequently in patients with HEV GT3e (75%) than in patients with GT3c (50%) or GT3f (19%) (p = 0.01). Additionally, patients with HEV GT3c exhibited significantly higher median international normalized ratio (INR) than patients with GT3e and GT3f (p = 0.033). The severity of GT3 acute hepatitis E was not linked to HEV RNA viral load or to the GT3 subtype.