scholarly journals A Novel Structurally Stable Multiepitope Protein for Detection of HCV

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Alexsandro S. Galdino ◽  
José C. Santos ◽  
Marilen Q. Souza ◽  
Yanna K. M. Nóbrega ◽  
Mary-Ann E. Xavier ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Recombinant Protein ◽  
Structural Data ◽  
Cd Spectroscopy ◽  
Serum Samples ◽  
His Tag ◽  
Alkaline Conditions ◽  
Virus Polyprotein ◽  
Structurally Stable ◽  
Immunodominant Epitopes

Hepatitis C virus (HCV) has emerged as the major pathogen of liver diseases in recent years leading to worldwide blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Accurate diagnosis for differentiation of hepatitis C from other viruses is thus of pivotal importance for proper treatment. In this work we developed a recombinant multiepitope protein (rMEHCV) for hepatitis C diagnostic purposes based on conserved and immunodominant epitopes from core, NS3, NS4A, NS4B, and NS5 regions of the virus polyprotein of genotypes 1a, 1b, and 3a, the most prevalent genotypes in South America (especially in Brazil). A synthetic gene was designed to encode eight epitopes in tandem separated by a flexible linker and bearing a his-tag at the C-terminal end. The recombinant protein was produced in Escherichia coli and purified in a single affinity chromatographic step with >95% purity. Purified rMEHCV was used to perform an ELISA which showed that the recombinant protein was recognized by IgG and IgM from human serum samples. The structural data obtained by circular dichroism (CD) spectroscopy showed that rMEHCV is a highly thermal stable protein at neutral and alkaline conditions. Together, these results show that rMEHCV should be considered an alternative antigen for hepatitis C diagnosis.

A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis

2019 ◽  
Vol 13 (4) ◽  
pp. 316-328
Author(s):  
Patrícia A.F. Ribeiro ◽  
Marilen Q. Souza ◽  
Daniel S. Dias ◽  
Alice C. M. Álvares ◽  
Laís M. Nogueira ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Recombinant Protein ◽  
Human Cytomegalovirus ◽  
De Novo ◽  
Structural Data ◽  
Elisa Test ◽  
World Population ◽  
Serum Samples ◽  
Serological Tests ◽  
Circular Dichroïsm

Background: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. Objective: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. Methods: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). Results: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. Conclusion: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

NS3 is a serine protease required for processing of hepatitis C virus polyprotein.

1993 ◽  
Vol 67 (7) ◽  
pp. 4017-4026 ◽  
Author(s):  
L Tomei ◽  
C Failla ◽  
E Santolini ◽  
R De Francesco ◽  
N La Monica
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
Virus Polyprotein

scholarly journals Utility of the Cobas® Plasma Separation Card as a Sample Collection Device for Serological and Virological Diagnosis of Hepatitis C Virus Infection

Diagnostics ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 473
Author(s):  
Fernando Velásquez-Orozco ◽  
Ariadna Rando-Segura ◽  
Joan Martínez-Camprecios ◽  
Paula Salmeron ◽  
Adrián Najarro-Centeno ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Viral Load ◽  
Hepatitis C ◽  
Virus Infection ◽  
Hepatitis C Virus Infection ◽  
Sample Collection ◽  
Serum Samples ◽  
Plasma Separation ◽  
Virological Diagnosis ◽  
A Prospective Study

Diagnosis and clinical management of people infected with hepatitis C virus (HCV) relies on results from a combination of serological and virological tests. The aim of this study was to compare the performance of dried plasma spots (DPS), prepared using the cobas® Plasma Separation Card (PSC), to plasma and serum from venipuncture, for HCV diagnosis. We carried out a prospective study using DPS and paired plasma or serum samples. Serum and DPS samples were analyzed by immunoassay using Elecsys® Anti-HCV II (Roche). Plasma and DPS samples were analyzed using the cobas® HCV viral load and cobas® HCV genotyping tests (Roche). All DPS samples that had high anti-HCV antibody titers in serum were also antibody-positive, as were five of eight samples with moderate titers. Eight samples with low titers in serum were negative with DPS. Among 80 samples with plasma HCV viral loads between 61.5 and 2.2 × 108 IU/mL, 74 were RNA-positive in DPS. The mean viral load difference between plasma and DPS was 2.65 log10 IU/mL. The performance of DPS for detection of serological and virological markers of hepatitis C virus infection was comparable to that of the conventional specimen types. However, the limits of detection were higher for DPS.

Hepatitis C virus core antigen assay: an alternative method for hepatitis C diagnosis

2016 ◽  
Vol 54 (2) ◽  
pp. 279-285 ◽  
Author(s):  
Lijuan Wang ◽  
Hong Lv ◽  
Guojun Zhang
Keyword(s):  
Hepatitis C ◽  
Positive Predictive Value ◽  
Predictive Value ◽  
Characteristic Curve ◽  
High Specificity ◽  
Receiver Operator Characteristic Curve ◽  
Core Antigen ◽  
Hcv Rna ◽  
Serum Samples ◽  
Alternative Method

Background The study aimed to evaluate a fully automated chemiluminescent immunoassay and compared it with a quantitative RNA assay and anti-HCV assay to verify the utility of this automated Ag assay as an alternative method for hepatitis C diagnosis. Methods A total of 229 serum samples previously tested for anti-HCV concentrations by the Architect Anti-HCV assay, were selected for HCV RNA testing by real time RT-PCR kit (Shanghai ZJ Bio-Tec Co., Ltd) and 125 specimens were tested for HCV Ag by the Architect HCV core Antigen kit. Results The log10 HCVAg and HCV RNA concentrations were highly correlated [ r = 0.834); with HCV RNA as the comparator test, HCVAg had 100% specificity, 100% positive predictive value (PPV) and 94.8% sensitivity. We found 1 pg/mL of total HCV core Ag is equivalent to approximately 6607HCV RNA international units (IU)/mL. Receiver operator characteristic curve analysis showed that the area under the curve of HCV core Ag (0.989) was greater than HCV Ab (0.871). HCV Ag concentrations and RNA-to-Ag ratio of the groups for HCV RNA concentrations ≤105 and >105 IU/mL were both significantly different from each other ( P < 0.05). Conclusion The Architect HCV core Ag assay may be an alternative method for hepatitis C diagnosis, performed on the same analytical platform and sample as the anti-HCV assay, shortening the diagnostic window period, demonstrating good correlation with HCV RNA assay with high specificity and positive predictive value.

scholarly journals Ribavirin Quantification in Combination Treatment of Chronic Hepatitis C

2003 ◽  
Vol 47 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Sylvie Larrat ◽  
Françoise Stanke-Labesque ◽  
Agnès Plages ◽  
Jean-Pierre Zarski ◽  
Germain Bessard ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Combination Treatment ◽  
High Performance ◽  
Solid Phase ◽  
Ammonium Phosphate ◽  
Serum Samples ◽  
Coefficients Of Variation ◽  
Immunodeficiency Virus

ABSTRACT Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C18-bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra- and interassay accuracies, precision, and stability at 4°C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 ± 1.06 μg/ml (n = 24) at week 12 of treatment (W12) and 3.24 ± 1.35 μg/ml (n = 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n = 11) than in patients with treatment failure (n = 13), with significant intergroup differences at W12 (P = 0.030) and W24 (P = 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.

scholarly journals Branched‐chain amino acids reduce hepatic iron accumulation and oxidative stress in hepatitis C virus polyprotein‐expressing mice

2014 ◽  
Vol 35 (4) ◽  
pp. 1303-1314 ◽  
Author(s):  
Masaaki Korenaga ◽  
Sohji Nishina ◽  
Keiko Korenaga ◽  
Yasuyuki Tomiyama ◽  
Naoko Yoshioka ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Branched Chain Amino Acids ◽  
Iron Accumulation ◽  
Hepatic Iron ◽  
Branched Chain ◽  
Virus Polyprotein ◽  
And Oxidative Stress

Prospective study of hepatitis C virus infection in hemodialysis patients by monthly analysis of HCV RNA and antibodies

2003 ◽  
Vol 49 (8) ◽  
pp. 503-507 ◽  
Author(s):  
Regina Moreira ◽  
João Renato Rebello Pinho ◽  
Jorge Fares ◽  
Isabel Takano Oba ◽  
Maria Regina Cardoso ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hemodialysis Patients ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Hepatitis C Virus Infection ◽  
Serum Samples ◽  
Hcv Genotypes ◽  
High Prevalence ◽  
Time Required

The aims of this study were to (i) evaluate the prevalence and the incidence of hepatitis C virus (HCV) infection in hemodialysis patients in two different centers in São Paulo (Brazil), (ii) determine the time required to detect HCV infection among these patients by serology or PCR, (iii) establish the importance of alanine aminotransferase determination as a marker of HCV infection, and (iv) identify the HCV genotypes in this population. Serum samples were collected monthly for 1 year from 281 patients admitted to hospital for hemodialysis. Out of 281 patients, 41 patients (14.6%) were HCV positive; six patients seroconverted during this study (incidence = 3.1/1000 person-month). In 1.8% (5/281) of cases, RNA was detected before the appearance of antibodies (up to 5 months), and in 1.1% (3/281) of cases, RNA was the unique marker of HCV infection. The genotypes found were 1a, 1b, 3a, and 4a. The presence of genotype 4a is noteworthy, since it is a rare genotype in Brazil. These data pointed out the high prevalence and incidence of HCV infection at hemodialysis centers in Brazil and showed that routine PCR is fundamental for improving the detection of HCV carriers among patients undergoing hemodialysis.Key words: HCV genotypes, hemodialysis, hepatitis C, PCR, prevalence, incidence.

scholarly journals NS3 Variability in Hepatitis C Virus Genotype 1A Isolates from Liver Tissue and Serum Samples of Treatment-Naïve Patients with Chronic Hepatitis C

Intervirology ◽  
2018 ◽  
Vol 61 (1) ◽  
pp. 1-8
Author(s):  
Deborah D’Aliberti ◽  
Irene Cacciola ◽  
Cristina Musolino ◽  
Giuseppina Raffa ◽  
Roberto Filomia ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Liver Tissue ◽  
Serum Samples ◽  
Virus Genotype ◽  
Genotype 1A ◽  
Treatment Naïve

scholarly journals Transfusion-transmitted human T-cell lymphotropic virus type I infection in Taiwan: a true risk and occasional coinfection with hepatitis C virus shown in a prospective study

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 934-940
Author(s):  
JT Wang ◽  
MT Lin ◽  
PJ Chen ◽  
JC Sheu ◽  
JT Lin ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Blood Transfusion ◽  
Prospective Study ◽  
Family Members ◽  
Type I ◽  
Serum Samples ◽  
Human T Cell ◽  
A Prospective Study

To study the incidence of human T-cell lymphotropic virus (HTLV) after blood transfusion in Taiwan, serum samples from 699 patients in a prospective study were examined for seroreactivity of anti-HTLV. By an enzyme immunoassay, 9 of the 699 recipients were repeatedly positive. Serial serum samples of these 9 patients were then confirmed with a Western blot analysis and with a polymerase chain reaction (PCR) assay for HTLV-I genome. Four were already positive for anti-HTLV before transfusion, 1 carried antibodies to HTLV-I transiently after transfusion, and only 4 cases had de nova seroconversions. These patients and their family members were called back and tested for HTLV- I genome in the peripheral blood mononuclear cell (PBMC) and plasma. All the serologically positive patients, except the “transient one,” were positive for HTLV sequences in the PBMCs. Viral sequences could also be detected in several serum or plasma samples. In the family members, only the spouse of a pretransfusion-positive patient was infected. These results suggested that approximately 0.6% of the blood recipients were infected by HTLV-I through transfusion in Taiwan, and that the frequency of intrafamilial HTLV-I transmission is low. We also observed the unusual coinfection by both HTLV-I and hepatitis C virus in 2 patients, and superinfection of hepatitis C virus after blood transfusion in 1 HTLV-I carrier. Cases of coinfection suggest a prevalence of both viruses in blood donors and warrant further screening.

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