Resistance-associated Substitutions Research Articles

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.

Hepatitis C virus treatment failure: Clinical utility for testing resistance-associated substitutions.

The hepatitis C virus has a high mutation capacity that leads to the emergence of resistance-associated substitutions (RAS). However, the consequence of resistance selection during new direct-acting antiviral drug (DAA) treatment is not necessarily the therapeutic failure. In fact, DAA treatment has shown a high rate (> 95%) of sustained virological response even when high baseline RAS prevalence has been reported. In the context of RAS emergence and high rates of sustained viral response, the clinical relevance of variants harboring RAS is still controversial. Therefore, in order to summarize the data available in international guidelines, we have reviewed the clinical utility of testing RAS in the era of new pangenotypic DAA drugs.

Prevalence of resistance-associated substitutions and phylogenetic analysis of hepatitis C virus infection in Russia

ObjectivesDue to limited hepatitis C virus (HCV) sequence availability from patients in Russia, the relationship between subtypes and baseline resistance-associated substitutions (RAS) to direct antiretroviral treatment outcome is not fully understood. MethodsDeep sequencing of HCV NS3, NS5A, and NS5B sequences was performed on plasma HCV samples from 412 direct-acting antiviral (DAA)-naïve patients from Russia. Phylogenetic analysis was performed to investigate sequence similarities between HCV strains from Russia, Asia, Europe, and North America. Pretreatment HCV RAS was assessed with a 15% cutoff. ResultsHCV genotype GT1b and GT3a sequences in Russia were related to strains in Europe and Asia. The prevalence of GT1a and GT2a was low in Russia. In GT1b, the prevalence of NS5A Y93H was lower in Russia (6%) compared with Asia (15%). The prevalence of NS5B L159F was similar between Russia and Europe (26–39%). GT3a RAS prevalence was similar between Russia and Asia, Europe, and North America. The 2k/1b recombinant strain in Russia was related to strains from Europe. A higher prevalence of the NS5A RAS L31M (10%) was observed in 2k/1b sequences compared to GT1b (1–6%). ConclusionsThe prevalence of RASs and the phylogenetic analysis showed similarities in HCV strains between Russia, Europe, and North America. This information may be useful for HCV regimens in Russia.

Genetic Subtypes and Natural Resistance Mutations in HCV Genotype 4 Infected Saudi Arabian Patients.

This study aimed to characterize the HCV genetic subtypes variability and the presence of natural occurring resistance-associated substitutions (RASs) in Saudi Arabia patients. A total of 17 GT patients were analyzed. Sequence analysis of NS3, NS5A, and NS5B regions was performed by direct sequencing, and phylogenetic analyses were used to determine genetic subtypes, RAS, and polymorphisms. Nine patients were infected by GT 4a, two with GT 4o and three with GT 4d. Two patients were infected with apparent recombinant virus (4a/4o/4a in NS3/NS5A/NS5B), and one patient was infected with a previously unknown, unclassifiable, virus of GT 4. Natural RASs were found in six patients (35%), including three infected by GT 4a, two by GT 4a/GT 4o/GT 4a, and one patient infected by an unknown, unclassifiable, virus of GT 4. In particular, NS3-RAS V170I was demonstrated in three patients, while NS5A-RASs (L28M, L30R, L28M + M31L) were detected in the remaining three patients. All patients were treated with sofosbuvir plus daclatasvir; three patients were lost to follow-up, whereas 14 patients completed the treatment. A sustained virological response (SVR) was obtained in all but one patient carrying NS3-RAS V170I who later relapsed. GT 4a is the most common subtype in this small cohort of Saudi Arabia patients infected with hepatitis C infection. Natural RASs were observed in about one-third of patients, but only one of them showed a treatment failure.

Characterization of a Novel Hepatitis C Subtype, 6xj, and Its Consequences for Direct-Acting Antiviral Treatment in Yunnan, China.

ABSTRACTHepatitis C virus (HCV) has a high rate of genetic variability, with eight genotypes and 91 subtypes. The genetic diversity of HCV genotype 6 (HCV-6) is the highest with 31 subtypes, and this genotype is prevalent in Southeast Asia. In this study, we investigated 160 individuals with chronic hepatitis C in Yunnan Province, China. Using reverse transcription (RT)-PCR and Sanger sequencing, 147 cases were successfully amplified and genotyped as 3b (4.9%), 3a (19.73%), 6n (12.24%), 1b (7.48%), 2a (6.12%), 6a (2.04%), 1a (0.68%), 6v (0.68%), and 6xa (0.68%), with eight sequences remaining unclassified. Subsequently, the eight nearly full-length genomes were successfully amplified and analyzed. The eight complete coding sequences formed a phylogenetic group that was distinct from the previously assigned HCV-6 subtypes and clustered with two previously unnamed HCV-6 sequences. Furthermore, Simplot analysis showed no recombination and the p-distance was more than 15% in comparison to the 6a to 6xi subtypes. Taken together, we identified a new HCV-6 subtype, 6xj, which originated approximately in 1775 according to Bayesian analyses. Moreover, all eight individuals received follow-up assessments at 44 weeks from the beginning of their 12-week treatments of sofosbuvir/velpatasvir (after-treatment week 32). One case relapsed at after-treatment week 32. Next-generation sequencing (NGS) was conducted and showed that the treatment failure case had two suspected antiviral resistance mutations, NS5A V28M (a change of V to M at position 28) and NS5B A442V, compared with the baseline. Overall, this newly identified 6xj subtype further confirmed the high diversity of the HCV-6 genotype. The newly identified resistance-associated amino acid substitutions may help inform future clinical treatments.IMPORTANCE This study investigated the genetic diversity of hepatitis C virus (HCV), particularly in relation to genotype 6, which is prevalent in Yunnan, China, and is often difficult to treat successfully. We identified a new HCV-6 subtype, 6xj, which is an ancient strain. Moreover, all eight individuals with the novel subtype received follow-up assessments at 44 weeks from the beginning of their treatments. One case relapsed after 8 months of withdrawal. NGS was conducted and showed that the isolate from the treatment failure case had two suspected antiviral resistance mutations, NS5A V28M and NS5B A442V, compared with the baseline. Overall, this newly identified 6xj subtype further confirmed the high diversity of the HCV-6 genotype. The newly identified resistance-associated amino acid substitutions may help inform future clinical treatments. We believe that our study makes a significant contribution to the literature based on the results described above.

Prevalence of Naturally Occurring Resistance Associated Substitutions in NS3/4AProtease Inhibitors in Iranian HCV/HIV Infected Patients.

Hepatitis C virus (HCV) acts in the host as a complicated mixture of related variants with the potency to genetically escape host immune responses. Direct acting antivirals (DAAs) have been approved for HCV treatment with shorter duration, better cure rates and lower side effects. However, naturally occurring resistance associated substitutions (RASs) create some obstacles to this antiviral therapy success. In this study, we aimed at the determination of the naturally occurring NS3/4A RASs in HCV/human immunodeficiency virus (HIV)infected patients. A total of 120 DAA-naïve HCV-HIV co-infected patients were included. HCV NS3/4Agenome region was amplified with PCR and mutation analysis was performed by Sanger sequencing technique. The amino acid sequence diversity of the region was analyzed using geno2pheno HCV. Phylogenetic analysis showed that 73 cases were infected by 3a and 47 subjects by subtype1a. The overall RASs among studied subjects were observed in 6 (5%) individuals from 120 studied cases who were infected with HCV 1a. V36M/L, Q80L, S122G/L, R155T/G, A156S, D168Y/N and S174A/N/T mutations were detected in this study. Although the prevalence of RASs was totally low in this study, the presence of several cases of double and triple mutants among this population suggests prior evaluation of protease inhibitors related mutations before initiation of standard treatment and also an investigation on a large population could be of high value.

Validation of a Genotype-Independent Hepatitis C Virus Near-Whole Genome Sequencing Assay.

Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1–6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving >100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load >5 log10 IU/mL. This genotype-independent sequencing method resulted in >99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.

Compartmentalization of Resistance-Associated Substitutions in HIV/HCV-Infected Patients: Possible Correlation with Infecting HCV Genotype

Resistance-associated substitutions (RASs) may exist prior to treatment and contribute to the failure of treatment with direct-acting antivirals (DAAs). As the major site of HCV replication, naturally occurring variants with RASs may segregate into the liver. In the present study, we performed viral population sequencing to retrospectively investigate the NS3 and NS5A RAS profiles in 34 HIV/HCV coinfected patients naïve to anti-HCV treatment who underwent diagnostic liver biopsy between 2000 and 2006 and had liver and plasma samples available. Sixteen were infected by HCV genotype (GT) 1a, 11 by GT3a, and 7 by GT4d. The analysis of the NS3 domain in GT1a showed a difference in strain between the liver and plasma in three cases, with a preponderance of specific RASs in the liver compartment. In GT4d samples, 6/7 coupled liver and plasma samples were concordant with no RASs. Sequence analysis of the NS5A domain showed the presence of RASs in the livers of 2/16 patients harboring GT1a but not in the corresponding plasma. In GT4d, NS5A RASs were detected in 7/7 liver tissues and 5/7 plasma samples. NS3 domain and NS5A domain were found to be conserved in plasma and livers of patients infected with GT3a. Thus, RASs within GT1a and GT4d more likely segregate into the liver and may explain the emergence of resistant strains during DAA treatment.

Clinical evaluation of a Hepatitis C Virus whole-genome sequencing pipeline for genotyping and resistance testing

ObjectivesWe sought to evaluate clinically a hepatitis C virus (HCV) whole-genome, next-generation sequencing (NGS) pipeline that is agnostic to viral genotype. MethodsPerformance of the NGS pipeline was assessed through comparison of results with Sanger sequencing (SS) of partial HCV genomes. ResultsThere was 98.7% (376/381) concordance for viral subtype between SS and NGS. The positive and negative per cent agreements for determination of resistance-associated substitutions were 97.8% (95% CI 92.5–99.4%) and 99.9% (95% CI 99.5–100.0%), respectively. The NGS pipeline was also able to detect novel subtypes, mixtures, recombinants, transiently occurring resistance mutations and distinguish re-infection with the same subtype from relapse. DiscussionParticular scenarios where NGS may be used include settings without universal access to pan-genotypic antiviral regimens, those infected with a ‘rare’ subtype or who have been failed by first-line therapy, and in cases of suspected re-infection.

Inferior cure rate in pilot study of 4-week glecaprevir/pibrentasvir treatment with or without ribavirin of chronic hepatitis C.

Shortening the treatment duration for chronic hepatitis C may increase feasibility and reduce the cost of cure. The aims of this study were to compare 4weeks of glecaprevir/pibrentasvir (GLE/PIB) treatment with and without ribavirin for patients with chronic hepatitis C and favourable baseline characteristics and to monitor the development of resistance-associated substitutions (RAS) and re-treatment outcomes if treatment failed. We performed an open-label single-centre randomized controlled trial, in which patients with chronic hepatitis C were randomized 1:1 to GLE/PIB±ribavirin, stratified by genotype 3. The main inclusion criteria were treatment-naive patients, aged 18-49 with all genotypes accepted, and absence of liver fibrosis, determined by liver stiffness measurement less than 8kPa. Viral genome sequences were determined by deep sequencing at baseline and at the time of relapse. A total of 32 patients started treatment. Sustained virological response at week 12 (SVR12) was 59% (10/17) for GLE/PIB without ribavirin and 73% (11/15) for GLE/PIB with ribavirin. Drug target-specific NS5A RAS were detected at baseline for 45% (5/11) of patients with treatment failure and for 14% (3/21) of patients who achieved SVR12. Ten failure patients were retreated 12weeks with sofosbuvir-based regimens; all have been cured. In this pilot study of 4-week treatment with GLE/PIB with and without ribavirin, we found that baseline RAS were more frequent in patients with virological failure. Development of RAS did occur after short treatment but did not result in retreatment failure with a different regimen. EudraCT no: 2017-005179-21.

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.

High Cure Rates for Hepatitis C Virus Genotype 6 in Advanced Liver Fibrosis With 12 Weeks Sofosbuvir and Daclatasvir: The Vietnam SEARCH Study.

BackgroundGenotype 6 is the most genetically diverse lineage of hepatitis C virus, and it predominates in Vietnam. It can be treated with sofosbuvir with daclatasvir (SOF/DCV), the least expensive treatment combination globally. In regional guidelines, longer treatment durations of SOF/DCV (24 weeks) are recommended for cirrhotic individuals, compared with other pangenotypic regimens (12 weeks), based on sparse data. Early on-treatment virological response may offer means of reducing length and cost of therapy in patients with liver fibrosis.MethodsIn this prospective trial in Vietnam, genotype 6-infected adults with advanced liver fibrosis or compensated cirrhosis were treated with SOF/DCV. Day 14 viral load was used to guide duration of therapy: participants with viral load <500 IU/mL at day 14 were treated with 12 weeks of SOF/DCV and those ≥500 IU/mL received 24 weeks. Primary endpoint was sustained virological response (SVR).ResultsOf 41 individuals with advanced fibrosis or compensated cirrhosis who commenced treatment, 51% had genotype 6a and 34% had 6e. The remainder had 6h, 6k, 6l, or 6o. One hundred percent had viral load <500 IU/mL by day 14, meaning that all received 12 weeks of SOF/DCV. One hundred percent achieved SVR12 despite a high frequency of putative NS5A inhibitor resistance-associated substitutions at baseline.ConclusionsPrescribing 12 weeks of SOF/DCV results in excellent cure rates in this population. These data support the removal of costly genotyping in countries where genotype 3 prevalence is <5%, in keeping with World Health Organization guidelines. NS5A resistance-associated mutations in isolation do not affect efficacy of SOF/DCV therapy. Wider evaluation of response-guided therapy is warranted.

Real world SOF/VEL/VOX retreatment outcomes and viral resistance analysis for HCV patients with prior failure to DAA therapy.

Sustained viral response (SVR) rates for direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) infection routinely exceed 95%. However, a small number of patients require retreatment. Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is a potent DAA combination primarily used for the retreatment of patients who failed by DAA therapies. Here we evaluate retreatment outcomes and the effects of resistance-associated substitutions (RAS) in a real-world cohort, including a large number of genotype (GT)3 infected patients. 144 patients from the UK were retreated with SOF/VEL/VOX following virologic failure with first-line DAA treatment regimens. Full-length HCV genome sequencing was performed prior to retreatment with SOF/VEL/VOX. HCV subtypes were assigned and RAS relevant to each genotype were identified. GT1a and GT3a each made up 38% (GT1a n=55, GT3a n=54) of the cohort. 40% (n=58) of patients had liver cirrhosis of whom 7% (n=4) were decompensated, 10% (n=14) had hepatocellular carcinoma (HCC) and 8% (n=12) had received a liver transplant prior to retreatment. The overall retreatment SVR12 rate was 90% (129/144). On univariate analysis, GT3 infection (50/62; SVR=81%, p=.009), cirrhosis (47/58; SVR=81%, p=.01) and prior treatment with SOF/VEL (12/17; SVR=71%, p=.02) or SOF+DCV (14/19; SVR=74%, p=.012) were significantly associated with retreatment failure, but existence of pre-retreatment RAS was not when viral genotype was taken into account. Retreatment with SOF/VEL/VOX is very successful for non-GT3-infected patients. However, for GT3-infected patients, particularly those with cirrhosis and failed by initial SOF/VEL treatment, SVR rates were significantly lower and alternative retreatment regimens should be considered.

HCV resistance-associated substitutions following direct-acting antiviral therapy failure - Real-life data from Poland.

This study analysed the NS3 and NS5A mutation frequencies, persistence and drug susceptibility in a cohort of real-life patients, with failed hepatitis C virus (HCV) therapy following directly acting antiviral (DAA) treatment. NS3/NS5A Sanger sequences from 105 patients infected with HCV genotype (G) 1a (6,5.7%), G1b (94,89.5%), G3a (4,3.8%), and G4 (1,1.0%) post DAA treatment failure were analysed. NS3 and NS5A resistance-associated substitutions (RASs) were identified using the geno2pheno algorithm and associated with clinical variables. Time trends were examined using logistic regression. NS5A RAS were found in 87.9% of sequences derived from patients exposed to this class of agents, whereas NS3 RAS was found in 59.1% of HCV protease-exposed subjects. The frequency of the NS3 RAS increased with fibrosis stage, from 40.0% among F0/F1 individuals to 81.8% among patients with liver cirrhosis (F4, p=0.094). NS5A mutation frequencies were 7.6% for 28A/V/M, 10.6% for 30K/Q/R, 42.4% for 31I/F/M/V, and 75.8% for 93H. For NS3, the most common RASs were 56F-23.7%, 168A/E/I/Y/T/V-14.0%, and 117H-5.4%. Susceptibility to glecaprevir/pibrentasvir, velpatasvir/voxlaprevir, and elbasvir/grazoprevir was retained in 92.9%, 43.4%, and, 25.3% of patients, respectively. The frequency of NS3 RAS decreased with time elapsed from failure to sampling (p=0.034 for trend). NS5A RAS frequency remained stable over the 24-months. Following DAA treatment failure, NS5A and NS3 RASs were common with increasing frequency among patients with advanced liver disease. In most cases, despite the presence of RASs, susceptibility to DAA combinations with higher genetic barrier was retained.

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.

The combination of the NS5A and cyclophilin inhibitors results in an additive anti-HCV inhibition in humanized mice without development of resistance.

We and others previously reported that the direct-acting agents (DAA) NS5A inhibitors (NS5Ai) and the host-targeting agents cyclophilin inhibitors (CypIs) inhibit HCV replication in vitro. In this study, we investigated whether the combination of NS5Ai and CypI offers a potent anti-HCV effect in vivo. A single administration of NS5Ai or CypI alone to HCV-infected humanized-mice inhibits HCV replication. The combination of NS5Ai with CypI suppresses HCV (GT1a, GT2a, GT3a and GT4a) replication in an additive manner. NS5Ai/CypI combinations provide a statistically more profound anti-HCV inhibition for GT2a and GT3a than GT1a and GT4a, leading to a fastest and deepest inhibition of GT2a and GT3a replications. Combining CypI with NS5Ai prevents the viral rebound normally observed in mice treated with NS5Ai alone. Results were confirmed in mice implanted with human hepatocytes from different donors. Therefore, the combination of NS5Ai with CypI may serve as a regimen for the treatment of HCV patients with specific genotypes and disorder conditions, which diminish sustain viral response levels to DAA, such as GT3a infection, cirrhosis, and DAA resistance associated with the selection of resistance-associated substitutions present at baseline or are acquired during treatment.

Direct-acting Antiviral Regimens for Patients with Chronic Infection of Hepatitis C Virus Genotype 3 in China

Hepatitis C virus (HCV) genotype (GT)3 infection is associated with a more rapid hepatic disease progression than the other genotypes. Hence, early HCV clearance slows down the disease progression and is important for improving prognosis in GT3-infected patients. Nevertheless, compared with other genotypes, GT3 is difficult-to-treat with direct-acting antivirals, especially in the presence of cirrhosis. Current guidelines recommend several regimens which have been proven to be effective in GT3-infected patients from the Western world (North America, Europe, and Oceania). In China, GT3 infection comprises 8.7–11.7% of the 10 million patients infected with HCV and has strikingly different characteristics from that in Western countries. Unlike the Western countries, where GT3a is the predominant subtype, GT3a and 3b each affect roughly half of Chinese GT3-infected patients, with 94–96% of the GT3b-infected patients carrying A30K+L31M double NS5A resistance-associated substitutions. Phase 3 clinical trials including GT3b-infected patients have suggested that GT3b infection is difficult to cure, making the regimen choice for GT3b-infected patients an urgent clinical gap to be filled. This review includes discussions on the epidemiology of HCV GT3 in China, recommendations from guidelines, and clinical data from both Western countries and China. The aim is to provide knowledge that will elucidate the challenges in treating Chinese GT3-infected patients and propose potential solutions and future research directions.

High frequency of fungicide resistance-associated mutations in the wheat yellow rust pathogen Puccinia striiformis f. sp. tritici.

Reliance on fungicides to manage disease creates selection pressure for the evolution of resistance in fungal and oomycete pathogens. Rust fungi (Pucciniales) are major pathogens of cereals and other crops and have been classified as low-risk for developing resistance to fungicides; no case of field failure of fungicides in a cereal rust disease has yet been recorded. Recently, the Asian soybean rust pathogen, Phakopsora pachyrhizi evolved resistance to several fungicide classes, prompting us to screen a large sample of the globally widespread wheat yellow rust pathogen, Puccinia striiformis f. sp. tritici (Pst), for mutations associated with fungicide resistance. We evaluated 363 Pst isolates from Europe, the USA, Ethiopia, Chile, China and New Zealand for mutations in the target genes of demethylase inhibitor (DMI; Cyp51) and succinate dehydrogenase inhibitor (SDHI; SdhB, SdhC and SdhD) fungicides. A high proportion of Pst isolates carrying a Y134F DMI resistance-associated substitution in the Cyp51 gene was found among those from China and New Zealand. A set of geographically diverse Pst isolates was also found to display a substitution in SdhC (I85V) that is homologous to that reported recently in P. pachyrhizi and linked to SDHI resistance. The identification of resistance-associated alleles confirms that cereal rusts are not immune to fungicide resistance and that selection for resistance evolution is operating at high levels in certain locations. It highlights the need to adopt fungicide resistance management practices and to monitor cereal rust species for development of resistance. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

WITHDRAWN: Sofosbuvir resistance-associated substitutions in the palm domain of HCV-NS5B RNA dependent RNA polymerase; study of two Sofosbuvir non-responders

In current study we performed sequencing of palm domain of HCV-NS5B gene, its ancestral analysis along with amino acids substitution analysis. These analysis were done to find the molecular basis of the viral resistance against Sofosbuvir drug. Blood samples from individuals with chronic Hepatitis C infection that were resistant to Sofosbuvir were collected. The samples were processed for their molecular characterization that included RNA extraction, Complementary DNA (cDNA) synthesize, nested PCR, gel elution, Sequencing, ancestral and 3D structure analysis. Evolutionary analysis revealed that current study sequences (QAU-01, QAU-02) clustered with a previously studied sequence, KY971494.1. Moreover, we reports multiple novel amino acid substitutions in the palm domain of NS5B gene such as Ile116Val, Asn117Gly, Glu246Ala, Val252Ala, Glu258Gln, Cys262Leu, Ser269Arg, Ala272Thr, Ile293Leu, Lys304Arg, Asn307Gly, Ala338Val and Arg345Gly in our query sequence (QAU-01). At 246 and 269 position in (QAU-02), no substitution was observed. We have noticed that the current sequences are relatively emerging and could have been originated from aforementioned sequence recently. Based on the current results, we suggests that these substitutions could be associated with structural or functional impairment of protein and could also be may be considered as resistance associated substitutions (RAS) to Sofosbuvir drug.

Novel drug resistance-associated substitutions against pibrentasvir emerged in genotype 1b hepatitis C virus-infected human hepatocyte transplanted mice

Combination therapy with glecaprevir and pibrentasvir (PIB) has high efficacy for patients with hepatitis C virus (HCV) infection except among those who experienced NS5A-P32 deletion (del) mutation during prior DAA treatment failure. However, some patients fail to achieve SVR through combination treatment even in the absence of NS5A-P32del. We analyzed emergence of NS5A resistance-associated substitutions (RASs) against PIB using HCV-infected mice. Male human hepatocyte transplanted mice were infected with genotype 1b wild-type HCV. Mice were treated with PIB, resulting in a transient decrease in serum HCV RNA levels but followed by relapse during the treatment. Direct sequence analysis showed emergences of various mutations in the NS5A region, including L31V/P32del, L31F/P32del/Y93H, NS5A-P29del/Y85C, and NS5A-F37Y. PIB was less effective in mice with NS5A-F37Y mutations compared to mice with wild-type HCV. NS5A-F37Y showed 5.4-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. The present in vivo and in vitro studies identified NS5A-F37Y as a novel RAS against PIB and showed the possibility of emergence of various NS5A RASs including P29del, P32del and F37Y following PIB treatment. These mutations might emerge and lead to failure to respond to DAA therapies including PIB-based regimens in chronic hepatitis C patients.