Hepacivirus Research Articles

The new demands for HCV related clinical examinations for DAA treatment

Hepatitis C virus (HCV) is one of leading causes for chronic liver diseases, the treatment area has been changed rapidly since direct acting antiviral agents (DAAs) were approved to treat patients with chronic HCV infection. To cure hepatitis in 2030 is the goal set by World Health Organization. However, globally, especially in China, there are still many difficulties needing to be resolved on the cure road of chronic hepatitis C, such as large patient population, high percentage of untreated patients, nonstandard DAA treatment, low efficacy of special groups, and resistance-associated variants in baseline, etc. This comment will focus on the new demands for HCV related clinical examinations for DAA treatment, so as to improve the treatment effectiveness and to help achieve the goal of eliminating viral hepatitis. Key words: Hepacivirus; Hepatitis C; Antiviral agents; Clinical laboratory techniques

Analysis of the novel role of NS5A domain I in the assembly of infectious hepatitis C virus particles

Hepatitis C virus (HCV) is an enveloped virus with a positive-sense, single-stranded RNA of approximately 9.6 kb, a member of the genus Hepaci virus within the family Flaviviridae. The genome contains a single large open reading frame encoding a 3000 residue polyprotein. The non-structural 5A protein (NS5A) is a highly phosphorylated protein, whichis comprised of three domains (I, II and III). Previously, we demonstrated that two residues within NS5A domain I (V67 and P145) play critical roles in HCV assembly challenging the dogma that NS5A domain I exclusively participated in genome replication. In this study, we identified 8 surface exposed residues of domain Iwhich were located in close proximity to V67 and P145. The mutants were cloned into a JFH-1 derived subgenomic replicons (mSGR-luc-JFH1) to confirm whether they are required in genome replication. The results of luciferase assay suggested that I52A exhibited the same phenotype as V67A and P145A and is a further candidate for regulating assembly of HCV. In parallel we sought to investigate whether domain I was involved in the interaction of NS5A with cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase required for HCV replication. CypA can be inhibited by cyclosporin A (CsA), which also inhibits HCV genome replication. Surprisingly, all three mutant replicons (I52A, V67A and P145A) were more sensitive to CsA treatment than wildtype, suggesting that domain I does indeed interact with CypA. Ongoing studies will therefore investigate the roles of both domain I and CypA in genome replication and assembly of infectious HCV particles.

Diagnostic value of serum hyaluronic acid in hepatic fibrosis of chronic viral hepatitis

Objective To investigate the clinical value of serum hyaluronic acid(HA) in the diagnosis of hepatic fibrosis in chronic viral hepatitis. Methods From January 2015 to February 2016, 108 patients with chronic viral hepatitis diagnosed by liver biopsy in Yuncheng Central Hospitalwere selected, and 30 healthy subjects were selected as controls.Serum HA was measured and the sensitivity and specificity of serum HA in the diagnosis of liver fibrosis were analyzed.The relationship between serum HA and liver pathological grade and staging were explored. Results The levels of serum HA in acute jaundice hepatitis, chronic hepatitis, mild and chronic hepatitis, moderate and chronic hepatitis, severe hepatitis and liver cirrhosis were (138.76±14.92)ng/L, (90.21±6.73)ng/L, (106.29±12.35)ng/L, (365.48 ± 25.39)ng/L, (597.24± 48.11)ng/L, respectively, which were significantly higher than (64.26±8.24)ng/L in the control group, the difference was statistically significant(t=8.723, 6.275, 22.025, 65.798, 63.670, all P<0.05). With the increase of liver fibrosis, serum HA level was significantly increased.Serum HA and liver inflammation activity classification and liver fibrosis staging had certain correlation(r=0.48, 0.43, all P<0.05), and the specificity was high. Conclusion Serum HA has a good diagnostic value in the diagnosis of hepatic fibrosis in chronic viral hepatitis. Key words: Hepatitis; Hepacivirus; Liver cirrhosis; Hyaluronic acid; Diagnosis; Specificity

A new perspective of hepatitis markers opened up by exosomes

This review, based on the origin and the mechanism of release of exosomes, described the presence of HBV and HCV biomarkers in the exosomes and the mechanisms of secretion, and discussed the viral transmission, vaccination protection, and the influences on treatment and detection.Through basic research data, it preliminarily demonstrated the importance of nucleic acid detection being a gold standard, and pointed out that another new cause of the occult infection was existence of HBV and HCV in the exosomes.(Chin J Lab Med, 2018, 41: 496-498) Key words: Exosomes; Biomarkers; Hepatitis B virus; Hepacivirus

Analysis of genotype 1b hepatitis C virus NS5A resistance-associated variants and its related factors

Objective To analyze hepatitis C virus genotype (HCV GT)1b NS5A resistance-associated variants (RAV) and its related factors, and to provide references for direct-acting antivirals (DAA) agent selection and application. Methods From January 2017 to July 2017, 53 hepatitis C patients were selected from the Department of Infectious Diseases of Henan Province People's Hospital. The mutations of L31M and Y93H in NS5A RAV were analyzed in 43 HCV GT1b patients, and their correlations with hepatitis C virus, liver function, platelet and liver fibrosis diagnostic model [APRI, gamma-glutamyl transpeptidase to platelet ratio (GPR), FIb-4] were analyzed. The quantitative data were compared by two independent samples t test, and the qualitative data were compared by chi square test. Results Fifty-three subjects were enrolled, including 43 GT1b (9 males and 34 females) and 10 GT2a (2 males and 8 females). No other genotype was detected. The incidence of NS5A RAV in 43 HCV GT1b patients was 13.9% (6/43), of which L31M and Y93H were 1/43 (2.3%) and 5/43 (11.6%) with no significant difference (χ2=1.500, P=0.219). There were no significant differences in HCV RNA, ALT, AST, albumin, platelets and age between patients with or without mutation (all P>0.05). Conclusions The incidence of NS5A RAV in HCV GT1b patients is high, but not affected by virus, biochemical factors and liver fibrosis. The detection of NS5A RAV before HCV treatment is helpful for rational selection of DAA, which could reduce the drug resistance. Key words: Hepacivirus; Mutation; Resistance-associated variants

Effect of hepatitis C virus genotype on antiviral therapy in patients with human immunodeficiency virus/hepatitis C virus coinfection

Objective To investigate the effect of hepatitis C virus (HCV) genotype on antiviral therapy in patients with human immunodeficiency virus (HIV)/HCV coinfection in Henan province. Methods A total of 129 patients were coinfected with HIV and HCV, among whom, 70 were HCV 1b genotype and 57 HCV 2a genotype. And 131 patients were HIV single infection. Immunological failure rate, virological suppression, CD4+ T lymphocyte counts and liver and renal function after antiretroviral therapy (ART) were compared among the three groups. Flow cytometry was used to count CD4+ T lymphocytes and polymerase chain reaction amplification was used to detect HIV RNA. The liver and renal function were tested by automatic biochemical analysis. Statistical analysis was conducted by χ2 test, analysis of variance and LSD-t method. Results Immunological failure rate in HCV 1b group, HCV 2a group and HIV single infection group were 7.14% (5/70), 15.79% (9/57) and 9.92% (13/131), respectively. There was no significant statistical difference among the three groups (χ2=2.59, P>0.05). The CD4+ T lymphocyte counts in three groups were (614±258), (529±245), and (518±243) cells/μL, respectively. The difference was statistically significant (F=3.17, P 0.05). The levels of aspartate transaminase, alanine aminotransferase and total bilirubin in HCV 1b group and HCV 2a group were all significantly higher than those in HIV single infection group (F=27.38, 15.22 and 7.33, respectively, all P 0.05). Conclusions The main HCV genotypes in patients with HIV/HCV coinfection by blood transmission are HCV 1b and HCV 2a in Henan province. HIV/HCV coinfection does not affect the effect of ART, but could aggravate the liver damage in acquired immune deficiency syndrome patients. Key words: Human immunodeficiency virus; Hepacivirus; Genotype; Antiviral therapy; Effect analysis

Studies on interaction between host thioredoxin-interacting protein and hepatitis C virus infection

Objective To explore the relationship between host cell thioredoxin-interacting protein (TXNIP) and hepatitis C virus (HCV) replication, as well as the mechanism of how TXNIP promotes HCV replication. Methods The transcriptome shift of Huh7 cells after cell cultrued HCV (HCVcc) infection was analyzed by using Glue Grant Human Transcriptome Microarray. The differential expression of TXNIP was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput sequencing. The effect of TXNIP on HCV infection was tested by TXNIP overexpression and TXNIP knockdown. Results Transcriptome array demonstrated a 3.7 times fold upregulation of TXNIP mRNA in HCVcc infected Huh7 cell higher than those without HCVcc infection, which was further verified by high-throughput deep sequencing. The TXNIP mRNA level was significantly up-regulated at 36 h and 60 h post HCV infection (t=24.90 and 8.27, respectively, both P<0.01). In cell level analysis, knockdown of TXNIP by small interfering RNA (siRNA) could reduce HCV infection, while TXNIP overexpression could enhance HCV replication. Infection with HCVcc could increase the expression of TXNIP in Huh7 but not in Huh7.5.1 cells, and interferons α, β, and γ treatment could up-regulate TXNIP expressions in both cell lines. Conclusions HCV infection induces host cell TXNIP expression, and upregulated TXNIP enhances HCV replication. Host cell TXNIP plays an important role in HCV replication, infection and immune escape. Key words: Hepacivirus; Thioredoxin-interacting protein; Interferons

Analysis of genetic variations of hepatitis C virus 5'untranslated regions gene in human immunodeficiency virus-1/hepatitis C virus co-infected patients in Xinjiang

Objective To investigate the genetic dynamics of hepatitis C virus (HCV) 5'untranslated regions (UTR) gene in human immunodeficiency virus (HIV-1)/HCV co-infected patients in Xinjiang. Methods Anti-HCV was tested using enzyme linked immunosorbent assay in 212 HIV-1 infected patients in Xinjiang. RNA was extracted from HIV-1/HCV co-infected samples. Then HCV 5'UTR gene was amplified after reverse transcription using PrimeScript™ Ⅱ 1st Strand cDNA Synthesis kit. The sequences were then compared with HCV international standard strains to determine the HCV gene types. The gene distance of 4 sampling virus strains was calculated and the phylogenetic analysis was performed to establish the cladogram. Results One hundred and fifty-seven among the 212 HIV-infected patients were HCV antibody positive. Of them, 2 patients were infected through heterosexual transmission and the remaining 155 patients were injection drug users (IDU). HCV 5'UTR was amlipicated and geno-typed successfully in 131 samples. Five HCV genotypes were identified, including 3a (40/131, 30.53%), 3b (23/131, 17.56%), 1a (11/131, 8.40%), 1b (56/131, 42.75%), and 6a (1/131, 0.76%). The general homology was 87.5%-99.3% in 38 patients, and there were differences in homology in different subtypes. The homology of the 4 sampling sequences from the same sample was 99.3%-100.0%, with the variation of 0-0.7%. The intragroup gene deviation and deviation with the first sample tended to increase with time. The mean evolutionary rate of HCV 5'UTR in HIV-1 infected patients in Xinjiang was 1.62×10-3(95%CI: 1.52×10-3-5.38×10-3). The similarity and homology of several sequences obtained from the one participant were the highest. Conclusions The prevalence of HCV co-infection among HIV-1 infected patients in Xinjiang is high, especially among IDU. Subtype 1b of HCV is prevalent strain. Key words: Human immunodeficiency virus-1; Hepacivirus; 5'UTR; Genetic distance; Evolutionary rate

Correlation between HCV genotype and anti-HCV antibody level in hepatitis C patients

Objective To investigate the relationship between anti-HCV antibody level and hepatitis C virus genotype in the patients. Methods Total of 603 anti-HCV positive serum samples were collected during 2013 to 2014 by retrospective research method. HCV RNA were detected in anti-HCV positive samples by repeat test and the genotype were detected in HCV RNA positive samples.The distribution of anti-HCV level in different hepatitis C genotype patients was analyzed and the body's response to viral antibodies and viral genotype correlation with anti-HCV concentration interquartile range was explored. Rates among genotype groups were compared using chi-square test. Results Totally 412 of 603(68.33%) samples were anti-HCV positive by double reagent screening. 174(42.3%) samples were detected as HCV RNA positive. The distributions of different anti-HCV level in different genotype patients were 1a(n=8)1/8, 1/8, 4/8, 2/8; 1b(n=112)25.9%(29/112), 17.0%(19/112), 25.9%(29/112), 31.3%(35/112); 2a(n=14)3/14, 4/14, 5/14, 2/14; 3a(n=11)3/11, 6/11, 2/11, 0/11; 3b(n=16)4/16, 11/16, 1/16, 0/16; 6a(n=8)2/8, 2/8, 1/8, 3/8 with anti-HCV concentration interquartile range respectively. The anti-HCV concentration distribution was different in patients with different HCV genotypes. The anti-HCV concentration distribution in patients of 1b, 2a and 6a genotypes were evently, while anti-HCV level was relatively high in 1a (13.65) and relatively low in 3b (8.77). There were differences in different genotypes of antibody concentrations (χ2=35.2, P<0.05). Conclusions There was correlation between anti-HCV level and HCV genotype. Because there were fewer cases in some genotypes, it was necessary to investigate more samples to corfirm the above results.(Chin J Lab Med, 2016, 39: 95-98) Key words: Hepacivirus; Genotype; Hepatitis C Antibodies; RNA, Viral

Efficacy of peginterferon α-2a plus ribavirin for the treatment of patients with hepatitis B virus and hepatitis C virus coinfection: a paired study

Objective To analyze the virologic responses of peginterferon α-2a plus ribavirin (Peg IFNα-2a/RBV) for patients with hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfection by a paired study. Methods Sixteen patients with HBV/HCV coinfection and 32 patients with HCV monoinfection were allocated. The virological response and biochemical response were compared between the two groups after Peg IFNα-2a/RBV regimen treatment. Chi-square analysis or Fisher exact analysis was used to compare the responses. Logistic analysis was conducted to evaluate factors associated with sustained virological response (SVR). Results The median levels of HCV RNA were 6.54 lg IU/mL in 16 patients with HBV/HCV co-infection and 6.98 lg IU/mL in 32 patients of HCV monoinfection, which was statistically different (Z=4.124, P 0.05). The relapse rate in HBV/HCV-coinfected patients was significantly higher than that in HCV-monoinfected patients (35.7%[5/16] vs 8.7%[2/32]; χ2=4.142, P=0.042). In HBV/HCV coinfection group, 7 cases were HBV DNA detectable, 7 cases were hepatitis B e antigen (HBeAg) positive, 13 cases were abnormal alanine aminotransferase (ALT) levels and 12 cases were abnormal aspartate aminotransferase (AST) levels. After treatment, only 4 patients were HBV DNA detectable, 5 cases were HBeAg positive, 4 cases were abnormal ALT levels and 5 cases were abnormal AST levels (P=0.458, 0.716, 0.004 and 0.032, respectively). The median level of hepatitis B surface antigen (HBsAg) was 780 IU/mL and decreased to 250 IU/mL after treatment, while the difference did not reach statistical significance (Z=1.826, P=0.068). A total of 4 patients achieved complete virologic response for HBV, 3 achieved partial virologic response; 2 achieved HBeAg seroconversion. Logistic regression analysis showed that EVR was a positive factor for achieving SVR (OR=35.2, 95%CI=3.55-349.15, P=0.002). Conclusions HCV remains at low replication level in patients with HBV/HCV coinfection. Virological responses can be achieved by Peg IFNα-2a/RBV treatment. EVR might be a positive factor associated with SVR. Key words: Peginterferon α-2a; Ribavirin; Hepatitis B virus; Hepacivirus; Superinfection

Epidemiological characteristics and therapeutic strategies of hepatitis C virus genotype 6 in Guangxi area

Objective To investigate the epidemiological characteristics and therapeutic strategies of patients infected with hepatitis C virus (HCV) genotype 6 in Guangxi area. Methods Serum samples were collected from 150 patients with serologic HCV RNA positive in Guangxi, China. Reverse transcription nested polymerase chain reaction (PCR) was employed to amplify HCV NS5B fragments and the DNA products were sequenced. The sequences obtained were compared with the sequences deposited in GenBank to construct a phylogenetic tree. Among the patients who accomplished 48-week treatment of interferon plus ribavirin and 24-week follow-up after stopping medication, 10 cases were infected with genotype 6a and 28 cases with genotype 1 HCV. The virological responses were evaluated at week 4, week 12, week 24 of treatment and week 24 after the end of the treatment. Results Among all recruited 150 cases, 21 (14.0%) cases were HCV genotype 6 including two subtypes 6a (n=20) and 6d (n=1). Genotype 6 HCV mainly affected intravenous drug users, especially with age of ≤40 years old. Phylogenetic tree showed that there was very close evolutionary distance between HCV 6 strains of Guangxi and Hongkong, China strains (Y12083, DQ 480515) and Vietnam strain (EU246930). All of 10 HCV genotype 6a patients who completed 48 weeks of antiviral therapy achieved sustained virological response (SVR). The rate of SVR was higher than that of genotype 1 patients, but without statistically different significance (10/10 vs 75.0%, P>0.05). Conclusion HCV genotype 6 in Guangxi area mainly affects young intravenous drug users with age of ≤40 years old, which has high homology with Hongkong, China and Vietnam standard strains. Patients with HCV 6 genotype infection treated with interferon plus ribavirin for 48 weeks usually achieve favorable SVR. Key words: Guangxi region; Hepacivirus; HCV genotype 6; Phylogenetic tree; Characteristics; Therapy

Correlation Analysis Between Protein Phosphatase-1 Regulatory Inhibitor Subunit-11 Polymorphism and Hepatitis C Virus Spontaneous Clearance

Objective To explore the association of protein phosphatase-1 regulatory inhibitor subunit-11 (PPP1R11) polymorphism and hepatitis C virus (HCV) spontaneous clearance.Methods From January 2009 to May 2013,a total of 328 blood donors whose blood samples were anti-HCV positive with enzyme-linked immunosorbent assay (ELISA) were included into this study.According to follow-up results,they were divided into spontaneous clearance group whose HCV RNA were negative but anti-HCV recombinant immunoblot assay results were positive (n =134),and chronic infection group whose HCV RNA were positive (n=198).Polymerase chain reaction with sequence specific primer(PCR-SSP) was performed to determine the genotype of rs3807031 of PPP1R11.Statistical software SPSS was used to analyze the association between genotype of rs3807031 and HCV spontaneous clearance.Results The average age of donors in spontaneous clearance group was (28.8± 10.2) years old,which was significantly lower than that of chronic infection group whose average age was (31.0±9.0) years old (t=2.135,P =0.034).Higher percentage of female was observed in spontaneous clearance group compared to chronic infection group (35.8％ vs 21.6％ ; x2 =7.994,P=0.005).Importantly,rs3807031 A allele composed 30.6％ and 22.4％,while C allele composed 69.4％ and 77.6％ in spontaneous clearance and chronic infection group,respectively.The frequencies of rs3807031 alleles were statistically different between these two groups (x2 =5.538,P =0.023).Multivariate unconditional logistic regression analysis showed that gender (OR=1.957,95％CI:1.118-3.224,P=0.008) and genotype of rs3807031 (OR=0.650,95％CI:0.450-0.973,P =0.021) are independent factors associated with HCV spontaneous clearance.Conclusions PPP1R11 rs3807031 is associated with HCV spontaneous clearance,which has the potential to predict the spontaneous clearance of HCV infection.Our results provide information to the research on the mechanism and molecular marker of HCV spontaneous clearance. Key words: Hepatitis C, chronic; Hepacivirus; Genotype; Polymorphism, single nucleotide

Virus and malignant lymphoma

Virus infection is a major cause of lymphoma.Research shows that Epstein-Barr virus,human immunodeficiency virus,hepatitis C virus and hepatitis B virus involve in the occurrence and development of lymphoma through different ways and mechanisms.It is important significance that viral infection research elucidates the molecular mechanisms of lymphoma and explores therapy ways. Key words: Epstein-Barr virus infections ; HIV ; Hepacivirus; Hepatitis B virus; Lymphoma

Analysis of the results of external quality assessment for hepatitis C virus RNA tests

Objective To evaluate the performance of HCV RNA detection in the first EQA program in 2012 and analyze possible problems in clinical laboratories. Methods The panel consisting of 5 samples was distributed to 927 laboratories.Each panel contains one negative sample and 4 positive samples,which were virus-like particles calibrated by international standard.The percent agreements of all the laboratories for qualitative and quantitative results were calculated.Genomic means (GM) and standard deviations (s) of all laboratories and each reagent were calculated.The overall GM and the GM of each reagent were compared with expected results and correlation curves were calculated. Results The percent agreements of sample 1211, 1212, 1213, 1214 for qualitative results were 99.5% (403/405), 98.5% (400/406), 100.0% (405/405), 100.0% (406/406), respectively. The percent agreement of the negative sample was 99% (401/405). The percent agreements of sample 1211, 1212 and 1213 for quantitive results were similar,which were 93.8% (549/585),92.3% (541/586) and 94.5% (554/586). However,the agreement of sample 1214 was only 87.7% (514/586) and the agreement of sample 1214 for reagent A was 67.2% (92/137). The overall GM agreed with expected results, while GMs of reagent C, E and G deviated from expected results.GMs of sample 1211,1212,1213 and 1214 reported by labs using reagent C were 4.22, 3.56, 5.16 and 5.90, respectively.GMs of sample 1211,1212,1213 and 1214 reported by labs using reagent E were 4.52, 3.78, 5.55 and 6.29, respectively. GMs of sample 1211, 1212, 1213 and 1214 reported by labs using reagent G were 4.83, 4.36, 5.72 and 6.56, respectively. Conclusions The overall results of HCV RNA qualitative and quantitative detection are satisfactory.However,some problems still exist,such as deviation of GM of some reagents,the interassay variability,systematic deviation and accidental deviation,which show that the quality of reagents should be improved.(Chin J Lab Med,2013,36:271-276) Key words: Hepacivirus; RNA; viral; Qualitycontrol

5-Acetyl-2-arylbenzimidazoles as antiviral agents. Part 4

Within a project aimed at discovering new Flaviviridae inhibitors, new variously substituted 2-phenylbenzimidazoles were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against viruses representatives of the three genera of the Flaviviridae family, i.e.: Pestivirus (BVDV), Flavivirus (YFV) and Hepacivirus (HCV). Title compounds were also tested against RNA viruses representative of other single-stranded, positive-sense (ssRNA+) negative-sense (RNA−), or double-stranded (dsRNA) genomes, as well as against representatives of two DNA virus families.Nine compounds showed activity against BVDV (EC50 = 0.8–8.0 μM), compound 31 being the most potent (EC50 = 0.80 μM) and selective (SI = CC50/EC50 = >100). When tested in an HCV replicon assay, compound 31 resulted again the most potent, displaying an EC50 value of 1.11 μM and an SI of 100. Besides inhibiting BVDV, two compounds (35 and 38) showed a moderate activity also against YFV (EC50 = 13 μM). Interestingly, 35 was moderately active also against RSV (EC50 = 25 μM).

Quinoline tricyclic derivatives. Design, synthesis and evaluation of the antiviral activity of three new classes of RNA-dependent RNA polymerase inhibitors

In this study three new classes of linear N-tricyclic compounds, derived by condensation of the quinoline nucleus with 1,2,3-triazole, imidazole or pyrazine, were synthesized, obtaining triazolo[4,5-g]quinolines, imidazo[4,5-g]quinolines and pyrido[2,3-g]quinoxalines, respectively. Title compounds were tested in cell-based assays for cytotoxicity and antiviral activity against RNA viruses representative of the three genera of the Flaviviridae family, that is BVDV (Pestivirus), YFV (Flavivirus) and HCV (Hepacivirus). Quinoline derivatives were also tested against representatives of other RNA virus families containing single-stranded, either positive-sense (ssRNA+) or negative-sense (RNA−), and double-stranded genomes (dsRNA), as well as against representatives of two DNA virus families. Some quinolines showed moderate, although selective activity against CVB-5, Reo-1 and RSV. However, derivatives belonging to all classes showed activity against BVDV. Among the most potent were the bis-triazoloquinoline 1m, the imidazoquinolines 2e and 2h, and the pyridoquinoxalines 4h, 4j and 5n (EC50 range 1–5μM). When tested in a replicon assay, compound 2h was the sole derivative to also display anti-HCV activity (EC50=3.1μM). In enzyme assays, 1m, 2h, 5m and 5n proved to be potent inhibitors of the BVDV RNA-dependent RNA polymerase (RdRp), while only 2h also inhibited the recombinant HCV enzyme.

Development of a novel quantitative real-time assay using self-reporting duplex mutation primers for detection of HCV

Objective To establish a novel real-time PCR method to detect HCV RNA using Selfreporting duplex mutation primers.Methods The recombinant vector pMD18-T-HCV 5′-NCR was used as the calibrator.The Self-reporting duplex mutation primers were designed according to the gene sequence.And then the PCR reaction system was optimized and evaluated.The specificity,sensitivity and reproducibility of real-time PCR were estimated,The serum specimens from 90 cases(30 cases of HCV,30 cases of other viral hepatitis and 30 healthy volunteers) were tested with this real-time PCR; Results were compared with those obtained using a commercial TaqMan kit.Results The assay was established.It showed linearity over a wide range from 20 - 109 IU/ml.Intra-experimental coefficients of variation(CVs) were 1.37% -4.59%,and inter-experimental CVs were 1.58% -4.81%,respectively.There was no significant difference of HCV genome number tested by the two methods(R2 = 0.95) in 30 hepatitis C patients; HCV DNA was not detected in any serum samples of 30 healthy volunteers by the two methods.The specificity was 100%(60/60).All the samples in patients with clinically confirmed HCV infections showed HCV RNA positive.There wass good correlation between the quantitaive results and results obtained using the commercial TaqMan kit.Conclusions It is demonstrated that real-time PCR is a reliable,accurate and feasible assay for HCV.The establishment of this assay provided alternative technology for clinical diagnosis or therapeutic drug monitoring in the field of HCV infection and epidemiologic survey. Key words: Hepacivirus; Polymerase chain reaction; RNA,viral

External quality assessment for the quantitative detection of HCV RNA in China

Objective Anti-RNase virus-like particles containing HCV RNA 5'-UTR were used as positive samples in national external quality assessment ( EQA ) to evaluate the competency of clinical Laboratories for the quantification of HCV RNA and analyze the possible problems of domestic kits. Methods The quality control samples with target values in EQA panels were distributed nationally twice by National Center of Clinical Laboratory (NCCL) to participating laboratories for the quantification of HCV RNA in 2008 and 2009. Each panel consisted of 5 samples. All participants were required to carry out the detection and to return results in expected time. Positive samples were virus-like particles which had been calibrated against the WHO HCV International Standard (NIBSC96/798)and the results of positive samples from participants should be in the range of target value of logarithm ± 0. 5. The 2nd panel in 2008 contained the common HCV genotypes and the 2nd panel in 2009 contained serial diluted samples of genotype 1b. The results of positive samples detected with 3 different lots reagent (21001,21078 and 21097) from the 2nd EQA in 2009 were statistically analyzed using the analysis of variance, then Dunnett'S T3 and Tamhane'S T2 were used if heterogeneity of variance was found. Results There was 390 participating laboratories in 2008 and 428/426 in 2009. The percentages of laboratories within the range of target value of logarithm ± 0. 5 for varied genotypes were different. The percentages of laboratories for 1b were more than 91%, for 2a were 93.7% and 74. 2% ,for6 were 83.3% and 80. 3%. The CVfor the low-level sample was higher than that for the high-level sample in the same year. The numbers of laboratories reporting false-negative samples in 2008and the 2nd in 2009 were 5, 1 and 10 respectively. Statistical differences were found among the results of four quality control serum samples using 3 different reagents( F = 288.23, 324. 79, 291.98 and 261.16,P ＜0. 01 ). Conclusion The competency for detecting low concentration samples and samples with genotype 2a or 6 needs to be improved. Key words: Hepacivirus; RNA,viral; Vinon; Quality control

The tracability and uniform unit of hepatitis C virus RNA quantification by domestic made real-time fluorescence quantitative PCR method

Objective To determine the conversion equation for X(copies/ml, lg) quantity values of domestic HCV RNA quantitative fluorescence amplification assays approved by SFDA and Y( IU/ml, lg)reference values of standard substance. Methods The second generation WHO International Standard (NIBSC code:96/798) was mixed with human AB blood type serum to create 7 different dilutions which included 100 000, 50 000, 25 000, 10 000, 5 000, 2 500 and 1 000 IU/ml. Two different batches of each three domestic hepatitis C virus RNA real-time fluorescence quantitative PCR assays and 2 different batches of each assay were employed to detect the 7 different concentration samples with real-time PCR. Each test was performed 4 times repeatedly. Results The correlations between X( copies/ml,lg) values of domestic HCV RNA assays and Y(IU/ml,lg) reference values of standard substance were as follow,Assay A:Y =0. 902 0 X+0.284 9,R2 =0.953 3,P＜0. 01,n =56;Assay B: Y=0. 875 7 X +0.562 4,R2 =0.956 5,P＜0.01,n =56; Assay C: Y = 0. 843 8 X + 0. 560 5, R2 = 0. 945 8, P ＜ 0. 01, n = 56. Conclusions All the conversion equations are different among the quantity value of three assays and the reference values of standard substance, that suggests it is necessary to perform more stringent traceability analysis for the quantity values of 3 assays. Through standardizing the quantity values preliminarily, the conversion equation can enhance the comparability between the quantity values of different assays, and provide a standard of HCV RNA virus load detection for clinical diagnosis and treatment monitoring of HCV infection. Key words: Hepacivirus; RNA,viral; Reagent kits,diagnostic; Polymerase chain reaction

Comparative study of different screening markers for hemodialysis patients infected with hepatitis C virus

Objective To assess the application value of different detection markers in the screening of HCV infection in hemodialysis (HD) patients. Methods 230 chronic renal failure patients undergoing hemodialysis were enrolled in the study. HCV Ag/anti-HCV, anti-HCV, HCV Ag, and HCV RNA were detected in the 230 serum samples. HCV infection ratio assessed by these markers were compared. Results Thirty-seven case (16.1%) of 230 chronic renal failure patients undergoing haemodialysis were HCV Ag/anti-HCV positive, 28 case (12.2% ) were anti-HCV positive, 10 case (4.3%) patients were HCV Ag positive and 18 case (7.8%) were HCV RNA positive. The positive rate of HCV was higher with HCV Ag/anti-HCV assay than that with anti-HCV assay, but there was no significant difference between them(16.1% vs. 12.2% ,χ2= 1.451,P=0.228). The positive rate with HCV Ag/anti-HCV (16.1%) assay was higher than HCV Ag assay (4.3%) and HCV RNA assay (7.8%), the differences were statistically significant (χ2=17.276, P=0.000; χ2=7.455, P=0.006). All anti-HCV positive samples (S/CO: 10.16-167.41) were also positive by HCV Ag/anti-HCV assay, while one HCV Ag positive sample was not detectable by HCV Ag/anti-HCV assay. Conclusions Compared with anti-HCV, HCV Ag and HCV BNA, the HCV Ag/anti-HCV assay provides a higher detection rate for HCV infection in HD patients. Key words: Hepatitis C; Renal dialysis; Hepacivirus