Inhibition of hepatitis C virus replication by single-stranded RNA structural mimics

AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were transfected with expression vectors generating HCV mimics and controls.Cellular HCV RNA and protein levels were quantified by real-time polymerase chain reaction and Western blotting,respectively.To evaluate possible antisense effects,complementary RNAs spanning a mimic were prepared.RESULTS: In the BB7 genotype 1b replicon system,mimics of the polymerase (NS-5B),X and BA regions inhibited replication by more than 90%,50%,and 60%,respectively.In the JFH-1 genotype 2 infection system,mimics that were only 74% and 46% identical in sequence relative to the corresponding region in JFH-1 inhibited HCV replication by 91.5% and 91.2%,respectively,as effectively as a mimic with complete identity to HCV genotype 2a.The inhibitory effects were confirmed by NS3 protein levels.Antisense RNA molecules spanning the 74% identical mimic had no significant effects.CONCLUSION: HCV RNA structural mimics can inhibit HCV RNA replication in replicon and infectious HCV systems and do so independent of close sequence identity with the target.

Diffusion-weighted magnetic resonance imaging and micro-RNA in the diagnosis of hepatic fibrosis in chronic hepatitis C virus

BACKGROUND Diffusion-weighted magnetic resonance imaging has shown promise in the detection and quantification of hepatic fibrosis. In addition, the liver has numerous endogenous micro-RNAs(miRs) that play important roles in the regulation of biological processes such as cell proliferation and hepatic fibrosis.AIM To assess diffusion-weighted magnetic resonance imaging and miRs in diagnosing and staging hepatic fibrosis in patients with chronic hepatitis C.METHODS This prospective study included 208 patients and 82 age-and sex-matched controls who underwent diffusion-weighted magnetic resonance imaging of the abdomen, miR profiling, and liver biopsy. Pathological scoring was classified according to the METAVIR scoring system. The apparent diffusion coefficient (ADC) and miR were calculated and correlated with pathological scoring.RESULTS The ADC value decreased significantly with the progression of fibrosis, from controls(F0) to patients with early fibrosis(F1 and F2) to those with late fibrosis(F3 and F4)(median 1.92, 1.53, and 1.25 × 10^(-3) mm^2/s, respectively)(P = 0.001).The cut-off ADC value used to differentiate patients from controls was 1.83 × 10^(-3) mm^2/s with an area under the curve(AUC) of 0.992. Combining ADC and miR-200 b revealed the highest AUC(0.995) for differentiating patients from controls with an accuracy of 96.9%. The cut-off ADC used to differentiate early fibrosis from late fibrosis was 1.54 × 10^(-3) mm^2/s with an AUC of 0.866. The combination of ADC and miR-200 b revealed the best AUC(0.925) for differentiating early fibrosis from late fibrosis with an accuracy of 80.2%. The ADC correlated with miR-200 b(r =-0.61, P = 0.001), miR-21(r =-0.62, P = 0.001), and miR-29(r = 0.52,P = 0.001).CONCLUSION Combining ADC and miRs offers an alternative surrogate non-invasive diagnostic tool for diagnosing and staging hepatic fibrosis in patients with chronic hepatitis C.

MicroRNA-mediated interactions between host andhepatitis C virus

Micro RNAs(mi RNAs) are small noncoding RNAs. More than 2500 mature mi RNAs are detected in plants, animals and several types of viruses. Hepatitis C virus(HCV), which is a positive-sense, singlestranded RNA virus, does not encode viral mi RNA. However, HCV infection alters the expression of host mi RNAs, either in cell culture or in patients with liver disease progression, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma. In turn, host mi RNAs regulate HCV life cycle through directly binding to HCV RNAs or indirectly targeting cellular m RNAs. Increasing evidence demonstrates that mi RNAs are one of the centered factors in the interaction network between virus and host. The competitive viral and host RNA hypothesis proposes a latent cross-regulation pattern between host m RNAs and HCV RNAs. High loads of HCV RNA sequester and de-repress host mi RNAs from their normal host targets and thus disturb host gene expression, indicating a means of adaptation for HCV to establish a persistent infection. Some special mi RNAs are closely correlated with liver-specific disease progression and the changed levels of mi RNAs are even higher sensitivity and specificity than those of traditional proteins. Therefore, some of them can serve as novel diagnostic/prognostic biomarkers in HCVinfected patients with liver diseases. They are also attractive therapeutic targets for development of new anti-HCV agents.

Micro RNA expression in hepatitis C virus-related malignancies: A brief review

Not only is chronic hepatitis C virus(HCV) infection a major public health problem,but also it can cause hepatocellular carcinoma and,more rarely,nonHodgkin's lymphoma.These characteristics mean that HCV is the only virus infecting humans that is able to cause two different cancers.The fine pathogenetic and molecular mechanisms by which HCV induces these two malignancies are not completely clear.In the last decade,it has been shown that microRNAs(miRNAs),a class of 21-23-nucleotide molecules modulating posttranscriptional gene expression,make an important contribution to the pathogenesis of several cancers and are also considered highly promising biomarkers.Here,we briefly describe the current knowledge about microRNAs' involvement in HCV-related molecular oncogenesis.We decided to focus our attention on studies fully conducted on ex vivo samples with this specific etiology,and on cultured cell lines partially or completely expressing the HCV genome.Some of the results reported in this review are controversial,possibly because of methodological issues,differences in sampling size and features,and ethnicity of patients.What is certain is that miRNAs play a remarkable role in regulating gene expression during oncogenetic processes and in viral infection.A clear understanding of their effects is fundamental to elucidating the mechanisms underlying virus-induced malignancies.

In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype

Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against NS3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods: Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin(G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration(G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results: RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested(50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24(80%, P=0.013) and 48 h(75%, P=0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70%(P<0.05) viral RNA reduction as compared to NS3-is33, which showed a 64%(P<0.05) decrease in viral copy number. siRNA synergism(NS3-is33 + NS3-is44) decreased viral load by 84%(P<0.05) as compared to individual inhibition by each siRNA(i.e., 64%–70%(P<0.05) in serum-inoculated cells. Synthetic siRNAs mixture(NS5Bis88 + NS3-is33) targeting different region of HCV genome(NS5B and NS3) also decreased HCV viral load by 85%(P< 0.05) as compared to siRNA inhibitory effects alone(70% and 64% respectively, P<0.05). Conclusions: siRNAs directed against NS3 gene significantly decreased m RNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.

Inhibition of Hepatitis C Virus Genotype 1a Non-Structural Proteins by Small Interference RNA in Human Hepatoma Cell Lines

Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the development of novel direct-acting antivirals, but such therapeutic options are still expensive and beyond the financial range of the most infected individuals in developing or even in resource replete countries. It demands an urgent need to search novel and improved alternate treatment strategies to treat the infection. The present study was aimed to develop an in vitro stable cell culture system, persistently expressing HCV genotype 1a non-structural genes and to characterize the inhibitory effects of synthetic siRNAs (short interference RNA) directed against the most conserved regions of nonstructural genes in an in vitro cell culture model. The continuous expression of nonstructural genes for more than 30 days post transfection was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis in stable human hepatoma cell line (Huh-7). The gene expression studies revealed significantly reduced gene expression of HCV nonstructural genes (i.e., NS2, NS4A and NS5A) both at mRNA and protein levels when treated against genome specific synthetic siRNAs in stable cell lines (51%, 47% and 54% respectively, p < 0.05). Similarly, a vivid decrease in HCV viral titer was exhibited by synthetic siRNAs in an in vitro viral replicate cell culture model (58%, 48% and 50%, respectively, p < 0.05) determined by quantitative Real-Time PCR (qPCR). Our data indicate that siRNA mediated gene silencing may be considered a promising alternate treatment strategy against HCV in combination with other effective therapeutic regimens in future.

Characteristics of short double stranded RNA against hepatitis C virus: a literature-based analysis

Objective： To describe the characteristics of short interfering double stranded RNA （dsRNA） against hepatitis C virus （HCV） and to fred out the determining factors in design for desirable inhibitory efficacy. Methods： The data were collected and analyzed by retrieval of 229 published short dsRNAs designed for degradation ofHCV RNA. Results： Statistical analyses showed that the most frequently involved short dsRNAs were directing against 5＇NTR/core and genotype lb, accounting for 64.2% and 69.9%, respectively. Inhibitory efficacy varied with the structural characteristics of short dsRNAs, of which the most potential were those directed against HCV core region with inhibitory efficacy of 70.2%. Moreover, the mean inhibitory efficacy of short dsRNAs with GC contents from 30% to 52% was higher than that of those with GC contents out of this range. Conclusion： Based on this pooled data in a relatively large sample, the present results provided clues to design for short dsRNAs with more potent inhibitory efficacy.

Structure-function relationship in viral RNA genomes: The case of hepatitis C virus

The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus(HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5'-untranslatable regions(5'UTRs) and 3'UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5' terminus of the viral genome and regulated by distal functional RNA domains placed at the 3' end. Subsequent RNA replication strongly depends on the 3'UTR folding and is also influenced by the 5' end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNARNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.

A prospective study of vertical transmission of hepatitis C virus

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Hepatitis C virus: Virology, diagnosis and management ofantiviral therapy

Hepatitis C virus (HCV) infects approximately 170 million individuals worldwide. Prevention of HCV infection complications is based on antiviral therapy with the combination of pegylated interferon alfa and ribavirin. The use of serological and virological tests has become essential in the management of HCV infection in order to diagnose infection, guide treatment decisions and assess the virological response to antiviral therapy. Anti- HCV antibody testing and HCV RNA testing are used to diagnose acute and chronic hepatitis C. The HCV genotype should be systematically determined before treatment, as it determines the indication, the duration of treatment, the dose of ribavirin and the virological monitoring procedure. HCV RNA monitoring during therapy is used to tailor treatment duration in HCV genotype 1 infection, and molecular assays are used to assess the end-of-treatment and, most importantly the sustained virological response, i.e. the endpoint of therapy.

Detection of hepatitis C virus core antigen for early diagnosis of hepatitis C virus infection in plasma donor in China

AIM： To evaluate the efficacy of a new hepatitis C virus （HCV） core antigen assay developed in China. METHODS： After the determination of HCV infection, 49 serial samples were selected from II regular plasma donors in 5 different plasma stations. To compare the performance of HCV core antigen detection and HCV PCR, these samples were genotyped, and each specimen was analyzed by ELISA for the detection of HCV core antigen and by qualitative HCV PCR. RESULTS： Among all of the sequential samples, the original 23 specimens were HCV RNA-negative, and 36 samples were HCV RNA-positive. Twenty-seven samples （75%） were HCV core antigen-positive from these HCV RNA-positive specimens. Conversely, 27 samples （93.2%） were found HCV RNA-positive in HCV core antigen- positive samples. Intervals between HCV RNA and HCV core antigen-positive, as well as between HCV core antigen-positive and HCV antibody-positive were 36.0 and 32.8 d, respectively. CONCLUSION： This HCV core antigen assay, developed in China, is able to detect much of anti-HCV-negative, HCV RNA-positive preseroconversion window period （PWP） plasma donations.

Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C

AIM To explore the status of extrahepatichepatitis C virus(HCV)infection and replicationin hepatitis C patients,and its potentialimplication in HCV infection and pathogenicity.METHODS By reverse-transcriptase poly-merase chain reaction(RT-PCR),in situhybridization(ISH)and immunohistochemistry,HCV RNA,HCV replicative intermediate(minus-strand of HCV RNA),and HCV antigens weredetected in 38 autopsy extrahepatic tissuespecimens(including 9 kidneys,9 hearts,9pancreas,5 intestines,2 adrenal glands,2spleens,1 lymph node,and 1 gallbladder)from 9hepatitis C patients,respectively;and thestatus of HCV replication in extrahepatic tissueswas studied.RESULTS By RT-PCR,all 9 patients werepositive for HCV RNA in kidney,heart,pancreas,and intestine,but only 6(66.7%)patients were positive for HCV replicativeintermediate.HCV RNA and HCV antigens weredetected in kidney,heart,pancreas,intestine,adrenal gland,lymph node,and gallbladder in 5(55.6%)and 6(66.7%)patients by ISH andimmunohistochemistry,respectively.HCV RNA and HCV antigens were not detected in theseextrahepatic organs in 3(33.3%)patients,although their livers were positive for HCV.HCVreplicative intermediate detected by RT-PCR wasconsistent with HCV RNA and HCV antigensdetected by ISH and immunohistochemistry(Kappa=0.42-0.75).HCV RNA and HCVantigens were detected in myocardial cells,epithelial cells of intestinal gladular,interstitialcells of kidney,epithelial cells of tubules andglomerulus,pancreas acinar cells and epithelialcells of pancreatic duct,epithelial cells ofmucous membrane sinus of gallbladder,cortexand medulla cells in adrenal gland,andmononuclear cells in lymph node.HCV RNA wasalso detected in bile duct epithelial cells,sinusoidal cells,and mononuclear cells in livertissues by ISH.CONCLUSION HCV can infect extrahepatictissues,and many various tissue cells maysupport HCV replication;extrahepatic HCVinfection and replication may be of'concomitantstate'in most of patients with hepatitis C.Theinfected extrahepatic tissues might act as areservoir for HCV,and play a role in both HCVpersistence and reactivation of infection.HCVas an etiologic agent replicating and expressingviral proteins in extrahepatic tissues itselfcontributes to extrahepatic syndromeassociated.HCV infection in a few patients withchronic HCV infection.

Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: Binding and entry

AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （El）. We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS： Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for i h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS： After 1 h of incubation, antibodies against C1, C2, and El detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION： Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

Hepatitis C virus micro-elimination:Where do we stand?

Hepatitis C virus (HCV) elimination by 2030, using direct-acting antiviraltreatments, has been promoted by the World Health Organization. Thisachievement is not attainable, however, particularly after the 2020 pandemic ofthe coronavirus disease 2019. Consequently, the more realistic objective ofeliminating HCV from population segments for which targeted strategies ofprevention and treatment are easily attained has been promoted in Europe, as avalid alternative. The underlying idea is that micro-elimination will ultimatelylead to macro-elimination. The micro-elimination strategy may target differentspecific populations and at-risk groups. Different settings, including prisons andhospitals, have also been identified as micro-elimination scenarios. In addition,dedicated micro-elimination strategies have been designed that are tailored at thegeographical level according to HCV epidemiology and individual country’sincome. The main elements of a valid and successful micro-elimination project arereliable epidemiological data and active involvement of all the stakeholders.Community involvement represents another essential component for a successfulprogram.

Desperately seeking hepatitis C virus

Spanish investigators described recently the so-called occult hepatitis C virus (HCV) infection, emphasizing the detection of genomic and antigenomic HCV RNA strands in liver and peripheral blood mononuclear cells. Therefore, the persistence of viral replication in occult HCV infection should be considered as a putative source of infection among family members and patients undergoing invasive procedures, transfusion or transplantation. Additionally, the most worrisome finding is that an occult HCV infection may persist in patients with sustained virological response.

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

AIM： To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus （HCV） internal ribosome entry sequences （IRES） on the response of chronic HCV genotype 4a patients to interferon therapy.METHODS： HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain Ⅲ was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustaIW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.RESULTS： Analysis of RNA secondary structure revealed that insertions in domain Ⅲ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain - were statistically more prevalent in sustained viral response patients （SVR, n = 14） as compared to breakthrough （BT, n = 5） patients.CONCLUSION： The influence of mutations within domain Ⅲ on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain Ⅲ.

SEN virus does not affect treatment response in hepatitis C virus coinfected patients but SEN virus response depends on SEN virus DNA concentration

AIM: To clarify the effect of SEN virus (SENV) infection on a combination therapy including interferon alfa (IFN-α) or pegylated-IFN with ribavirin in patients with chronic hepatitis and the effect of a combination therapy on SENV.METHODS: SENV DNA was determined by polymerase chain reaction in serum samples from 95 patients with chronic hepatitis C. Quantitative analysis was done for SENV H DNA.RESULTS: Twenty-one (22%) of 95 patients were positive for SENV DNA. There was no difference in clinical and biochemical parameters between patients with HCV infection alone and coinfected patients. The sustained response rate for HCV clearance after combination therapy did not differ between patients with SENV (52%) and without SENV(50%, n.s.). SENV DNA was undetectable in 76% of the initially SENV positive patients at the end of follow-up. SENV H response to combination therapy was significantly correlated with SENV DNA level (P=-0.05).CONCLUSION: SENV infection had no influence on the HCV sustained response rate to the combination therapy.Response rate of SENV to the combination therapy depends on SENV DNA level.

Efficacy and Safety of Glecaprevir/Pibrentasvir in Combination Therapy in Chronic Hemodialysis Patients with Genotype 2 Hepatitis C Virus Infection

Background: Glecaprevir (nonstructural protein 3/4A protease inhibitor) and Pibrentasvir (nonstructural protein 5A inhibitor) (G/P), a coformulated once-daily, all oral, ribavirin (RBV)-free, direct-antiviral regimen, was evaluated for safety and efficacy in chronic hemodialysis patients with genotype 2 hepatitis C virus infection. Methods: In this prospective, observational, single-center study at Masuko Memorial Hospital, between November 2017 and December 2018, a total of 8 HD patients with an HCV infection genotype 2 received G/P combination therapy. Age was an average of 67.1 (61 - 75) years and there were four men and two women. It was FIB4 INDX an average of 2.67 (1.5 - 3.34) before the start of therapy. It was quantity of HCV RNA an average of 4.43 (2.1 - 6.5). HCV RNA levels were measured by real-time RCR-based method (COBAS AmpiPrep/COBAS TaqMan HCV Test. 4 cases 12 weeks were 2 cases eight weeks for dosing period. Patients were excluded if they had evidence of hepatocellular carcinoma. This study was approved by the ethics committee of our hospital, while we obtained written consent from the participants after providing a thorough explanation of the contents and methods of this study. Results: 6 patients were available for total dose internal use. As for the HCV RNA of the fourth week, (100%) HCV RNA became negative after administration start of therapy. Rapid virologic response (RVR) achieved all cases. 5 patients achieved 12-week sustained virologic response (SVR12) and were following up the 1 patient. The itching appeared in two cases (33%), but there was symptom improvement in nalfurafine hydrochloride use treatment, and treatment continuation was possible. Conclusion: It is thought that G/P can be given to the HD patients’ safety, but we will accumulate a case in future, and it is thought to be necessary to examine utility and safety.

Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release

AIM To explore hepatitis C virus(HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODS A series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5 A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and celllysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets(LDs) with NS5 A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5 A was analyzed further.RESULTS The plasmids named JFH1-m E2, JFH1-mp7, JFH1-m NS4 B, JFH1-m NS5 A, JFH1-m E2/NS5 A, JFH1-mp7/NS5 A, JFH1-m NS4 B/NS5 A, JFH1-m E2/p7/NS5 A, and m JFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units(FFUs)/m L. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5 A(C2274 R, I2340 T, and V2440 L) and p7(H781 Y) were critical adaptive mutations. The effect of NS5 A(C2274 R, I2340 T, and V2440 L), p7(H781 Y), and NS4 B(N1931 S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release. However, the six adaptive mutations were not required for the LD localization of NS5 A proteins or the phosphorylation of NS5 A.CONCLUSION In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/m L, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.