Comprehensive survey of conserved RNA secondary structures in full-genome alignment of Hepatitis C virus.

Hepatitis C virus (HCV) is a plus-stranded RNA virus that often chronically infects liver hepatocytes and causes liver cirrhosis and cancer. These viruses replicate their genomes employing error-prone replicases. Thereby, they routinely generate a large 'cloud' of RNA genomes (quasispecies) which-by trial and error-comprehensively explore the sequence space available for functional RNA genomes that maintain the ability for efficient replication and immune escape. In this context, it is important to identify which RNA secondary structures in the sequence space of the HCV genome are conserved, likely due to functional requirements. Here, we provide the first genome-wide multiple sequence alignment (MSA) with the prediction of RNA secondary structures throughout all representative full-length HCV genomes. We selected 57 representative genomes by clustering all complete HCV genomes from the BV-BRC database based on k-mer distributions and dimension reduction and adding RefSeq sequences. We include annotations of previously recognized features for easy comparison to other studies. Our results indicate that mainly the core coding region, the C-terminal NS5A region, and the NS5B region contain secondary structure elements that are conserved beyond coding sequence requirements, indicating functionality on the RNA level. In contrast, the genome regions in between contain less highly conserved structures. The results provide a complete description of all conserved RNA secondary structures and make clear that functionally important RNA secondary structures are present in certain HCV genome regions but are largely absent from other regions. Full-genome alignments of all branches of Hepacivirus C are provided in the supplement.

On leveraging self-supervised learning for accurate HCV genotyping.

Hepatitis C virus (HCV) is a major global health concern, affecting millions of individuals worldwide. While existing literature predominantly focuses on disease classification using clinical data, there exists a critical research gap concerning HCV genotyping based on genomic sequences. Accurate HCV genotyping is essential for patient management and treatment decisions. While the neural models excel at capturing complex patterns, they still face challenges, such as data scarcity, that exist a lot in computational genomics. To overcome this challenges, this paper introduces an advanced deep learning approach for HCV genotyping based on the graphical representation of nucleotide sequences that outperforms classical approaches. Notably, it is effective for both partial and complete HCV genomes and addresses challenges associated with imbalanced datasets. In this work, ten HCV genotypes: 1a, 1b, 2a, 2b, 2c, 3a, 3b, 4, 5, and 6 were used in the analysis. This study utilizes Chaos Game Representation for 2D mapping of genomic sequences, employing self-supervised learning using convolutional autoencoder for deep feature extraction, resulting in an outstanding performance for HCV genotyping compared to various machine learning and deep learning models. This baseline provides a benchmark against which the performance of the proposed approach and other models can be evaluated. The experimental results showcase a remarkable classification accuracy of over 99%, outperforming traditional deep learning models. This performance demonstrates the capability of the proposed model to accurately identify HCV genotypes in both partial and complete sequences and in dealing with data scarcity for certain genotypes. The results of the proposed model are compared to NCBI genotyping tool.

Coordinated expansion of memory T follicular helper and B cells mediates spontaneous clearance of HCV reinfection.

Introduction: Follicular helper T cells are essential for helping in the maturation of B cells and the production of neutralizing antibodies (NAbs) during primary viral infections. However, their role during recall responses is unclear. Here, we used hepatitis C virus (HCV) reinfection in humans as a model to study the recall collaborative interaction between circulating CD4 T follicular helper cells (cTfh) and memory B cells (MBCs) leading to the generation of NAbs. Methods: We evaluated this interaction longitudinally in subjects who have spontaneously resolved primary HCV infection during a subsequent reinfection episode that resulted in either another spontaneous resolution (SR/SR, n = 14) or chronic infection (SR/CI, n = 8). Results: Both groups exhibited virus-specific memory T cells that expanded upon reinfection. However, early expansion of activated cTfh (CD4+CXCR5+PD-1+ICOS+FoxP3-) occurred in SR/SR only. The frequency of activated cTfh negatively correlated with time post-infection. Concomitantly, NAbs and HCV-specific MBCs (CD19+CD27+IgM-E2-Tet+) peaked during the early acute phase in SR/SR but not in SR/CI. Finally, the frequency of the activated cTfh1 (CXCR3+CCR6-) subset correlated with the neutralization breadth and potency of NAbs. Conclusion: These results underscore a key role for early activation of cTfh1 cells in helping antigen-specific B cells to produce NAbs that mediate the clearance of HCV reinfection.

Same-visit hepatitis C testing and treatment to accelerate cure among people who inject drugs (the QuickStart Study): a cluster randomised cross-over trial protocol.

INTRODUCTION: Despite universal access to government-funded direct-acting antivirals (DAAs) in 2016, the rate of hepatitis C treatment uptake in Australia has declined substantially. Most hepatitis C is related to injecting drug use; reducing the hepatitis C burden among people who inject drugs (PWID) is, therefore, paramount to reach hepatitis C elimination targets. Increasing DAA uptake by PWID is important for interrupting transmission and reducing incidence, as well as reducing morbidity and mortality and improving quality of life of PWID and meeting Australia's hepatitis C elimination targets. METHODS AND ANALYSIS: A cluster randomised cross-over trial will be conducted with three intervention arms and a control arm. Arm A will receive rapid hepatitis C virus (HCV) antibody testing; arm B will receive rapid HCV antibody and rapid RNA testing; arm C will receive rapid HCV antibody testing and same-day treatment initiation for HCV antibody-positive participants; the control arm will receive standard of care. The primary outcomes will be (a) the proportion of participants with HCV commencing treatment and (b) the proportion of participants with HCV achieving cure. Analyses will be conducted on an intention-to-treat basis with mixed-effects logistic regression models. ETHICS AND DISSEMINATION: The study has been approved by the Alfred Ethics Committee (number HREC/64731/Alfred-2020-217547). Each participant will provide written informed consent. Reportable adverse events will be reported to the reviewing ethics committee. The findings will be presented at scientific conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05016609. TRIAL PROGRESSION: The study commenced recruitment on 9 March 2022 and is expected to complete recruitment in December 2024.

Real-life experience of chronic hepatitis C treatment in Switzerland: a retrospective analysis.

BACKGROUND AND AIM: Direct-acting antivirals (DAAs) have revolutionised the management of chronic hepatitis C. We analysed the use of different generations of DAAs over time in Switzerland and investigated factors predictive of treatment failure. METHODS: This retrospective study was conducted within the framework of the Swiss Association for the Study of the Liver and the Swiss Hepatitis C Cohort Study; it included all patients with chronic hepatitis C treated with DAAs between January 2015 and December 2019 at eight Swiss referral centres. RESULTS: A total of 3088 patients were included; 57.3% were male, and the median age was 54 years. Liver cirrhosis was present in 23.9% of the cohort, 87.8% of whom were compensated. The overall sustained virological response (SVR) rate (defined as undetectable HCV RNA at week 12 after the first course of DAA-based treatment) was 96.2%, with an increase over time. The rate of treatment failure dropped from 8.3% in 2015 to 2.5% in 2019. Multivariable analysis revealed that female sex, the use of the latest generation of pangenotypic DAA regimens, Caucasian origin, and genotype (gt) 1 were associated with SVR, whereas the presence of active hepatocellular carcinoma (HCC), gt 3, and increasing liver stiffness were associated with treatment failure. Notably, the presence of active HCC during treatment increased the risk of DAA failure by a factor of almost thirteen. CONCLUSIONS: SVR rates increased over time, and the highest success rates were identified after the introduction of the latest generation of pangenotypic DAA regimens. Active HCC, gt 3 and increasing liver stiffness were associated with DAA failure.

Immunogenicity study of a Novel DNA-Based HCV vaccine candidate.

In this study, we aimed to evaluate the immunogenic profile of a chimeric DNA-based hepatitis C virus (HCV) vaccine candidate encoding the full-length viral core-E1-E2 (HCV-CE) fragment. The vaccine candidate was designed to uniformly express the HCV genotype 4 core-E1-E2 protein. The recombinant HCV-CE protein was bacterially expressed in C41 (DE3) cells, and then BALB/c mice were immunized with different combinations of DNA/DNA or DNA/protein prime/boost immunizations. The proper construction of our vaccine candidate was confirmed by specific amplification of the encoded fragments and basic local alignment search tool (BLAST) results of the nucleotide sequence, which revealed a high degree of similarity with several HCV serotypes/genotypes. The platform for bacterial expression was optimized to maximize the yield of the purified recombinant HCV-CE protein. The recombinant protein showed high specific antigenicity against the sera of HCV-infected patients according to the ELISA and western blot results. The predicted B- and T-cell epitopes showed high antigenic and interferon-γ (IFN-γ) induction potential, in addition to cross-genotype conservation and population coverage. The mice antisera further demonstrated a remarkable ability to capture 100% of the native viral antigens circulating in the sera of HCV patients, with no cross-reactivity detected in control sera. In conclusion, the proposed HCV vaccination strategy demonstrated promising potential regarding its safety, immunogenicity, and population coverage.

After the Storm: Persistent Molecular Alterations Following HCV Cure.

The development of direct-acting antivirals (DAAs) against hepatitis C virus (HCV) has revolutionized the management of this pathology, as their use allows viral elimination in a large majority of patients. Nonetheless, HCV remains a major public health problem due to the multiple challenges associated with its diagnosis, treatment availability and development of a prophylactic vaccine. Moreover, HCV-cured patients still present an increased risk of developing hepatic complications such as hepatocellular carcinoma. In the present review, we aim to summarize the impact that HCV infection has on a wide variety of peripheral and intrahepatic cell populations, the alterations that remain following DAA treatment and the potential molecular mechanisms implicated in their long-term persistence. Finally, we consider how recent developments in single-cell multiomics could refine our understanding of this disease in each specific intrahepatic cell population and drive the field to explore new directions for the development of chemo-preventive strategies.

Pharmacokinetics of Sofosbuvir/Velpatasvir and efficacy of an alternate-day treatment in hemodialysis patients with chronic hepatitis C infection.

Sofosbuvir/Velpatasvir (SOF/VEL) is a combination drug used for chronic hepatitis C (HCV) infection. However, limited information exists regarding the pharmacokinetics of SOF/VEL and its metabolites in hemodialysis patients. We conducted a prospective investigation of the pharmacokinetic parameters of SOF/VEL after a single dose of SOF/VEL (400/100 mg) on days with and without dialysis in 12 Thai hemodialysis patients with chronic HCV infection, who had been undergoing hemodialysis for a duration of 0.5-20 years. Blood samples were collected before dose (0) and 0.5, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0, and 12.0 h after dose. Dialysate samples were also collected before dose (0) and 1.0, 2.0, 3.0, and 4.0 h after dose. Plasma and dialysate samples were quantified for SOF and its metabolite, GS-331007, and VEL concentrations using a fully validated LCMS technique. In addition, a preliminary efficacy study was conducted using the proposed SOF/VEL dose reduction regimen in all patients. No differences in SOF/VEL PK parameters between on- and off-dialysis studies. On the contrary, GS-331007 exhibited a 30% reduction in the area under the plasma concentration-time curve from time 0 to 24 h (AUC0-24h) on dialysis days compared with non-dialysis days (AUC0-24h ratio 0.68 vs. 1.04, respectively). The dialysis clearance of SOF and GS-331007 was 9.35 (8.72-15.11) and 8.89 (8.52-14.07) mL/min, respectively. Subsequently, an alternate-day regimen of SOF/VEL (400/100 mg) was administered for 12 weeks, resulting in an undetectable plasma HCV viral load without side effects. Further clinical studies are warranted to validate the efficacy and safety of our proposed dose reduction regimen.

The Role of Genedrive in Point of Care Method For Hepatitis C Elimination in Hemodialysis Center.

BACKGROUND: Point of care is laboratory testing conducted close to the site of the patient. Point of care assessment is essential to detect and treat the hepatitis C virus in a single visit. The potential use of Genedrive extends to remote areas and key populations Therefore, there is a need for a simple, and cost-effective examination of methods, such as Genedrive. Genedrive is a rapid and low-cost diagnostic tool for the identification and treatment selection of infectious diseases. The World Health Organization targets to eliminate hepatitis by 2030, which decreases infections by 90%, and decreases deaths by 65%. Point of care could play a significant role in contributing to the elimination of hepatitis C. Chronic kidney disease (CKD) patients on hemodialysis are among the population at risk of hepatitis C due to nosocomial transmission. This study aimed to assess the role of Genedrive in measuring hepatitis C in chronic hepatitis C patients with chronic kidney disease on hemodialysis. METHODS: This study used a cross-sectional design. There were 64 CKD on Hd patients in Cipto Mangunkusumo Hospital tested by Genedrive. ROC analysis was conducted to assess significant hepatitis C among chronic kidney disease on hemodialysis. RESULTS: The calculated detection limit of Genedrive was 3.1x103 IU/mL. Genedrive HCV assay showed 90.6% sensitivity, 96.8% specificity, 92% negative predictive value, and 97% positive predictive value to detect HCV, 10.36 positive likelihood ratio, and 0.09 negative likelihood ratio. CONCLUSION: Genedrive could be a simple and reliable point of care method to detect hepatitis C with chronic kidney disease on hemodialysis.

Hepatitis C Virus (HCV) genotypes distribution in South-Eastern Tuscany: a ten-year retrospective study.

Italy is recognized as having the highest Hepatitis C virus (HCV) prevalence in Europe. The Tuscany region, where the prevalence of HCV infection is approximately 0.8%, implemented two programs for the control of chronic hepatitis C in Tuscany from 2018 to 2022. This retrospective study aims to investigate the incidence of HCV in a population screened in Southeastern Tuscany from 2013 to 2022. The study population included 246,137 patients from the provincial area of Arezzo and Grosseto, Tuscany, spanning from January 2013 to October 2022. Among the subjects included in the study, 3,190 (1.29%) tested positive for anti-HCV antibodies. Of this population, 2,119 patients (66.43%) also tested positive for HCV-RNA quantification, leading to their enrolment for subsequent viral genotyping. 1,106 patients had genotype (GT) 1 (52.2%), 484 had GT 3 (22.8%), 371 had GT 2 (17.5%), and 158 had GT 4 (7.5%). Our study underscores the prevalence of HCV GTs 1 and 3 as the most predominant GTs in the Southeast Tuscany region. We also observe a correlation between age, sex and HCV genotypic distribution.

Analysis of the susceptibility of refractory hepatitis C virus resistant to nonstructural 5A inhibitors.

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs.

Investigating the role of killer cell immunoglobulin-like receptors and human leukocyte antigen genetic variants in hepatitis C virus infection.

The genetic diversity of killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) genes influences the host's immune response to viral pathogens. This study aims to explore the impact of five single nucleotide polymorphisms (SNPs) in KIR3DL2 and HLA-A genes on hepatitis C virus (HCV) infection. A total of 2251 individuals were included in the case-control study. SNPs including KIR3DL2 rs11672983, rs3745902, rs1654644, and HLA-A rs3869062, rs12202296 were genotyped. By controlling various confounding factors using a modified logistic regression model, as well as incorporating stratified analysis, joint effects analysis, and multidimensional bioinformatics analysis, we analyzed the relationship between SNPs and HCV infection. The logistic regression analysis showed a correlation between KIR3DL2 rs11672983 AA, KIR3DL2 rs3745902 TT, and increased HCV susceptibility (p < 0.01). Stratified analysis indicated that KIR3DL2 rs1654644 and HLA-A rs3869062 also heightened HCV susceptibility in certain subgroups. A linear trend of rising HCV infection rates was observed when combining KIR3DL2 rs11672983 AA and KIR3DL2 rs3745902 TT (ptrend = 0.007). Bioinformatics analysis suggested these SNPs' regulatory potential and their role in altering messenger RNA secondary structure, implying their functional relevance in HCV susceptibility. Our findings indicate that KIR3DL2 rs11672983 AA and KIR3DL2 rs3745902 TT are significantly associated with increased susceptibility to HCV infection.

Prevalence of HIV, HBV and HCV among livestock merchants and slaughterhouse workers in Ibadan, Nigeria.

Background: Most studies on viral infections among livestock handlers have focused on occupational exposure from inadvertent contact with infected animals. Consequently, little emphasis is given to the effect of their lifestyle on the acquisition of other blood-borne viruses. Objectives: To determine the prevalence and assess risk factors for HIV, HBV and HCV infections among livestock handlers in Ibadan, Nigeria. Methods: Blood samples were collected from 265 livestock handlers between October 2016 to April 2017 in Ibadan. The samples were tested for the presence of antibodies to HIV and HCV; and surface antigen to HBV using ELISA. Structured questionnaire was administered to collect information on risk factors associated with the transmission of these viruses. Data analysis was carried out using Chi-square test and logistic regression to determine the association between risk factors and predictors of infection (p < 0.05). Results: Of 265 participants, 11 (4.2%), 29 (10.9%) and 13 (4.9%) individuals tested positive for HIV, HBV and HCV infections respectively. Two (0.8%) of the participants were coinfected with HIV and HBV while 1(0.4%) was coinfected with both HBV and HCV. Individuals who travelled frequently in the course of Livestock trades had a higher rate of HIV infection. Conclusions: A high Infection with HIV, HBV and HCV is common among the study participants. There is a need for continued surveillance and awareness creation on preventive measures against these viruses.

Hepatitis C virus-induced differential transcriptional traits in host cells after persistent infection elimination by direct-acting antivirals in cell culture.

Chronic hepatitis C virus infection (HCV) causes liver inflammation and fibrosis, leading to the development of severe liver disease, such as cirrhosis or hepatocellular carcinoma (HCC). Approval of direct-acting antiviral drug combinations has revolutionized chronic HCV therapy, with virus eradication in >98% of the treated patients. The efficacy of these treatments is such that it is formally possible for cured patients to carry formerly infected cells that display irreversible transcriptional alterations directly caused by chronic HCV Infection. Combining differential transcriptomes from two different persistent infection models, we observed a major reversion of infection-related transcripts after complete infection elimination. However, a small number of transcripts were abnormally expressed in formerly infected cells. Comparison of the results obtained in proliferating and growth-arrested cell culture models suggest that permanent transcriptional alterations may be established by several mechanisms. Interestingly, some of these alterations were also observed in the liver biopsies of virologically cured patients. Overall, our data suggest a direct and permanent impact of persistent HCV infection on the host cell transcriptome even after virus elimination, possibly contributing to the development of HCC.

A pilot project harnessing surveillance systems to support clinicians providing clinical care for people diagnosed with hepatitis C in Victoria, Australia, September 2021 to 31 March 2022.

BackgroundActive follow-up of chronic hepatitis C notifications to promote linkage to care is a promising strategy to support elimination.AimThis pilot study in Victoria, Australia, explored if the Department of Health could follow-up on hepatitis C cases through their diagnosing clinicians, to assess and support linkage to care and complete data missing from the notification.MethodsFor notifications received between 1 September 2021 and 31 March 2022 of unspecified hepatitis C cases (i.e. acquired > 24 months ago or of unknown duration), contact with diagnosing clinicians was attempted. Data were collected on risk exposures, clinical and demographic characteristics and follow-up care (i.e. HCV RNA test; referral or ascertainment of previous negative testing or treatment history). Reasons for unsuccessful doctor contact and gaps in care provision were investigated. Advice to clinicians on care and resources for clinical support were given on demand.ResultsOf 513 cases where information was sought, this was able to be obtained for 356 (69.4%). Reasons for unsuccessful contact included incomplete contact details or difficulties getting in touch across three attempts, particularly for hospital diagnoses. Among the 356 cases, 307 (86.2%) had received follow-up care. Patient-management resources were requested by 100 of 286 contacted diagnosing clinicians.ConclusionsMost doctors successfully contacted had provided follow-up care. Missing contact information and the time taken to reach clinicians significantly impeded the feasibility of the intervention. Enhancing system automation, such as integration of laboratory results, could improve completeness of notifications and support further linkage to care where needed.

Decrease in active hepatitis C infection among people who use drugs in Madrid, Spain, 2017 to 2023: a retrospective study.

BackgroundPeople who use drugs (PWUD) are a key target population to reduce the burden of hepatitis C virus (HCV) infection.AimTo assess risk factors and temporal trends of active HCV infection in PWUD in Madrid, Spain.MethodsWe conducted a retrospective study between 2017 and 2023, including 2,264 PWUD visiting a mobile screening unit. Data about epidemiology, substance use and sexual risk behaviour were obtained through a 92-item questionnaire. HCV was detected by antibody test, followed by RNA test. The primary outcome variable was active HCV infection prevalence, calculated considering all individuals who underwent RNA testing and analysed by logistic regression adjusted by the main risk factors.ResultsOf all participants, 685 tested positive for anti-HCV antibodies, and 605 underwent RNA testing; 314 had active HCV infection, and 218 initiated treatment. People who inject drugs (PWID) were identified as the main risk group. The active HCV infection rate showed a significant downward trend between 2017 and 2023 in the entire study population (23.4% to 6.0%), among PWID (41.0% to 15.0%) and PWUD without injecting drug use (7.0% to 1.3%) (p < 0.001 for all). These downward trends were confirmed by adjusted logistic regression for the entire study population (adjusted odds ratio (aOR): 0.78), PWID (aOR: 0.78), and PWUD non-IDU (aOR: 0.78).ConclusionsOur study demonstrates a significant reduction in active HCV infection prevalence among PWUD, particularly in PWID, which suggests that efforts in the prevention and treatment of HCV in Madrid, Spain, have had an impact on the control of HCV infection.

Hepatitis C Virus Infection and ABO Blood Group Assessment: Tertiary Hospital Study in India.

People with certain blood groups and Rh positive are more prone to infections transmitted by blood transfusion. The aim of this research was to survey the accompaniment of ABO Blood Group System and Rh type with infection to hepatitis C virus in India. This was a retrospective study in patients during October 2019-March2022 in India. The population of blood donors was tested for blood borne infections, including HCV. Logistic regression was used and collected data were analyzed using SPSS v.16.A total number of 901 people referred to the organization for donating blood during aforementioned years. Of these, 224 people had a history of hepatitis C disease, including 189 unmarried persons and the rest were married. 167 individuals were males and 57individuals were females. People who had viral diseases were comprised of 76 persons with negative Rh and 148positive persons with Rh.Future aims should include studies into blood groups and Rh types, according to the results of this study, in order to avoid the spread of blood-borne infections. Furthermore, further study is needed to establish the particular blood kinds that provide an elevated danger for classified donors.

Trends in hepatitis C virus seroprevalence and associated risk factors among msm in Pakistan: insights from a community-based study.

Pakistan bears a substantial burden of hepatitis C virus (HCV) infection, with the second-highest prevalence globally. This community-based cross-sectional study, conducted from January to December 2022 in Punjab, Pakistan, investigates the seroprevalence of HCV among the men who have sex with men (MSM) population. The study identifies demographic and behavioral risk factors associated with HCV infection within this population group. Among the 501 participants, the study found an HCV seroprevalence of 14.86%. The association between demographic characteristics and seroprevalence is assessed by calculating the percentage of positive cases, revealing notable associations with age, education level, and self-identified sexual orientation. Furthermore, the study identified several behavioral risk factors positively associated with HCV seroprevalence, including sharing personal items such as razors and toothbrushes, histories of surgery, blood transfusion, dental procedures, intravenous drug use, and therapeutic injection histories. These risk factors were identified through structured interviews, and the prevalence of HCV seropositivity among the exposed groups was calculated accordingly. Interestingly, a lower HCV positivity rate was observed among self-reported HIV-positive individuals, contradicting previous research. The findings underscore the need for comprehensive, targeted prevention strategies such as risk factor awareness campaigns and educational programs tailored for the MSM population in Pakistan. Further research is warranted to validate these findings and better understand the complex interplay of factors contributing to HCV seroprevalence in this high-risk population.

HCV 5-Methylcytosine Enhances Viral RNA Replication through Interaction with m5C Reader YBX1.

Hepatitis C virus (HCV) is a positive-stranded RNA virus that mainly causes chronic hepatitis, cirrhosis and hepatocellular carcinoma. Recently we confirmed m5C modifications within NS5A gene of HCV RNA genome. However, the roles of the m5C modification and its interaction with host proteins in regulating HCV's life cycle, remain unexplored. Here, we demonstrate that HCV infection enhances the expression of the host m5C reader YBX1 through the transcription factor MAX. YBX1 acts as an m5C reader, recognizing the m5C-modified NS5A C7525 site in the HCV RNA genome and significantly enhancing HCV RNA stability. This m5C-modification is also required for YBX1 colocalization with lipid droplets and HCV Core protein. Moreover, YBX1 facilitates HCV RNA replication, as well as viral assembly/budding. The tryptophan residue at position 65 (W65) of YBX1 is critical for these functions. Knockout of YBX1 or the application of YBX1 inhibitor SU056 suppresses HCV RNA replication and viral protein translation. To our knowledge, this is the first report demonstrating that the interaction between host m5C reader YBX1 and HCV RNA m5C methylation facilitates viral replication. Therefore, hepatic-YBX1 knockdown holds promise as a potential host-directed strategy for HCV therapy.