Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5′UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3′UTR by constructing an intragenotypic recombinant using 5′UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3′UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5′UTR-NS5A and NS5B-3′UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml−1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Diagnostic approach to elucidate the efficacy and side effects of direct-acting antivirals in HCV infected patients

Introduction: The conventional interferon therapy of hepatitis C virus has been substituted substantially with sofosbuvir and daclatasvir due to constraints in efficacy and tolerability. This study aimed diagnostically to monitor the effectiveness and side effects of direct-acting antivirals in the management of HCV infections. Methodology: This prospective study was conducted on HCV-infected patients treated with sofosbuvir and daclatasvir. Different serological, biochemical, hematological, and molecular techniques were used for the assessment of patients. Only treatment-naive patients aged ≥ 18 to 75 years received 12 weeks of treatment. The primary endpoint was a sustained virologic response with undetectable HCV RNA in the patients’ serum at the end of the treatment. Results: We identified 229 cases of confirmed HCV infections by PCR, 94.3% of which had genotype 3. The study population comprised 66% females and 34% males with a median age of 42.2 ± 10.6 SD. Ninety-three percent of the patients accomplished SVR at week 12. The combined therapy of SOF/DAC achieved the highest efficacy rate (92.6%) among the different HCV genotype 3 patients. A statistically significant relationship was observed between low baseline viral load (p < 0.001; 95% CI = 1.2-3.1) and HCV genotype 3 with minor side effects, including lethargy, headache, nausea, insomnia, diarrhea, and fever. Conclusions: HCV-infected patients can be treated well with an interferon-free SOF/DAC regimen, tolerated with generally mild adverse effects with a higher SVR.

Circulating microRNA-122 in HCV Cirrhotic Patients with High Frequency of Genotype 3

MicroRNA-122 (miR-122) is a liver abundant microRNA that is released upon liver injury. In the present study, we investigated the circulating miR-122 profiles in a Pakistani patients´ cohort with HCV chronic liver disease that was mainly based on HCV genotype 3 infections.From 222 patients with chronic HCV liver disease, classified as mild, moderate, or gross, serum samples were collected. Cell-free RNA was isolated and used for miR-122 quantification by qPCR. More than 60% of 222 patients were infected with HCV genotype 3. ALT values and HCV viral load showed no correlation with the HCV genotype. Circulating miR-122 levels were significantly upregulated in patients with cirrhosis. Notably, HCV patients with mild cirrhosis showed the most marked increase in serum miR-122 levels (p=0.0001). Furthermore, we proved a positive correlation (r=0.46) of miR-122 with the ALT values in patients with mild cirrhosis. Importantly, the increased miR-122 profiles of serum samples obtained from patients with mild, moderate, and gross cirrhosis did not depend on the HCV genotype. Importantly, our findings confirm that serum miR-122 levels are significantly upregulated in the HCV cirrhotic patients serving in particular as a biomarker for the non-advanced stages of cirrhosis, independently of the HCV genotype.

Efficacy and safety of sofosbuvir plus ribavirin in treatment-naive chronic hepatitis c genotype 3 patients of South Punjab, Pakistan

Background: To evaluate the efficacy and safety of sofosbuvir (SOF) plus ribavirin (RIB) in naive patients with chronic HCV genotype 3. The study design was open label, quasi experimental study. The study was conducted at Medical Outpatient Department of Medical Unit-1, Bahawal Victoria Hospital, affiliated with Quaid e Azam Medical College (QAMC), Bahawalpur, from April 2016 to June 2019.Methods: A total of 627 treatment-naive patients, aged above 18 years, with chronic Hepatitis C virus (HCV) genotype 3 infection were enrolled. SOF as 400 mg once a day plus weight-based RIB (1000 mg/day <75 kg and 1200 mg/day >75 kg) was given to all the study participants for 24 weeks. Qualitative polymerase chain reaction (PCR) for HCV ribonucleic acid (RNA) were done at 4 weeks to note the rapid virological response (RVR) whereas end of treatment response (ETR) was recorded at 24 weeks and sustained virological response (SVR) was noted 3 months after completion of treatment.Results: By 4th week, PCR of 524 (83.6%) patients was available, out of which, 492 (93.9%) had undetectable HCV RNA. By the end of treatment (24 weeks), PCR of 401 (64.0%) patients was available, out of which, 393 (98.0%) had undetectable HCV RNA. Data of 291 (46.4%) patients was available for SVR, 274 (94.1%) had undetectable HCV RNA. Weakness and fatigue turned out to be the commonest side effects, observed in 236 (37.6%) patients.Conclusions: Sofosbuvir was found to have good efficacy and safety in the local population of South Punjab having treatment-naïve chronic HCV genotype 3 infection.