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Mem. Inst. Oswaldo Cruz ; 112(9): 626-631, Sept. 2017. tab
Article in English | LILACS | ID: biblio-894874


BACKGROUND In Brazil, few studies have investigated the prevalence of infection with the precore (PC) and basal core promoter (BCP) mutants of the hepatitis B virus (HBV). OBJECTIVES This study aimed to analyse the frequency of PC and BCP mutations among patients infected with HBV and to evaluate the association between the variants and advanced hepatic disease. METHODS A total of 161 patients infected with HBV were studied. To identify PC and BCP mutations, a 501-bp fragment of HBV DNA was amplified and sequenced. FINDINGS PC and BCP regions from HBV strains were successfully amplified and sequenced in 129 and 118 cases, respectively. PC and BCP mutations were detected in 61.0% and 80.6% of the cases, respectively. The A1762T/G1764A variant was identified in 36.7% of the patients with grade 1 and 2 liver fibrosis (29/79) and in 81.8% of the patients with grade 3 and 4 liver fibrosis (9/11) (p < 0.01); in 76.9% of the patients with cirrhosis (10/13) and in 38.1% of the patients without cirrhosis (40/105) (p = 0.01); and in 77.8% of the patients with hepatocellular carcinoma (HCC) (7/9) and in 39.4% of the patients without HCC (43/109) (p = 0.03). MAIN CONCLUSIONS A high prevalence of HBV PC and BCP mutants was found. The A1762T/G1764A variant was independently associated with advanced forms of liver fibrosis, hepatic cirrhosis, and HCC.

Humans , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Viral Core Proteins/genetics , Hepatitis B virus/genetics , Hepatitis B, Chronic/virology , Liver Cirrhosis/virology , Genotype , Mutation
Braz. j. infect. dis ; 19(4): 390-398, July-Aug. 2015. tab, ilus
Article in English | LILACS | ID: lil-759273


Response-guided therapy is of limited use in developing countries because hepatitis C virus RNA detection by sensitive molecular methods is time- and labor-consuming and expen- sive. We evaluated early predictive efficacy of serum hepatitis C virus core antigen kinetics on sustained virologic response in patients with genotype 1 hepatitis C virus during pegylated interferon plus ribavirin treatment. For 478 patients recruited, hepatitis C virus RNAs were detected at baseline, and at weeks 4, 12, 24, 48, and 72 using Cobas TaqMan. Architect hepatitis C virus core antigen was performed at baseline, and weeks 4 and 12. Predictive values of hepatitis C virus core antigen on sustained virologic response were compared to hepatitis C virus RNA. In the first 12 weeks after treatment initiation the dynamic patterns of serum hepatitis C virus core antigen and hepatitis C virus RNA levels were similar in sustained virologic response, relapse, and null response patients groups. Although areas under the receiver operating characteristics curves of hepatitis C virus core antigen were lower than those of hepatitis C virus RNA at the same time points, modeling analysis showed that undetectable hepatitis C virus core antigen (rapid virological response based on hepatitis C virus core antigen) had similar positive predictive value on sustained virologic response to hepatitis C virus RNA at week 4 (90.4% vs 93.3%), and hepatitis C virus core antigen decrease greater than 1 log10 IU/mL (early virological response based on hepatitis C virus core antigen) had similar negative predictive value to hepatitis C virus RNA at week 12 (94.1% vs 95.Z%). Analysis on the validation group demonstrated a positive predictivevalue of 97.5% in rapid virological response based on hepatitis C virus core antigen and a negative predictive value of 100% in early virological response based on hepatitis C virus core antigen. In conclusion, hepatitis C virus core antigen is comparable to hepatitis C virus RNA in predicting sustained virologic response of chronic genotype 1 hepatitis C virus infected patients, and can be used to guide anti-hepatitis C virus treatment, especially in resource-limited areas.

Adult , Female , Humans , Male , Middle Aged , Antiviral Agents/therapeutic use , Hepacivirus/immunology , Hepatitis C Antigens/immunology , Hepatitis C, Chronic/drug therapy , Interferon-alpha/therapeutic use , Polyethylene Glycols/therapeutic use , Ribavirin/therapeutic use , Genotype , Hepatitis C, Chronic/immunology , Hepatitis C, Chronic/virology , Predictive Value of Tests , ROC Curve , Recombinant Proteins/therapeutic use , Time Factors , Viral Core Proteins/immunology
EJMM-Egyptian Journal of Medical Microbiology [The]. 2015; 24 (4): 113-118
in English | IMEMR | ID: emr-175730


Background: Hepatitis B virus [HBV] and hepatitis C virus [HCV] Infections are important and common causes of liver disease in end-stage renal failure [ESRF] in patients on haemodialysis [HD]. HBV is less endemic than HCV in Egypt [ranges from 2%-7%]. Although, the prevalence of HBV in haemodialysis patients has decreased significantly due to HBV vaccine and screening of blood donors, the immunosuppressive nature of renal disease often leads to chronicity of the HBV infection and an opportunity for nosocomial spread of the infection among dialysis patients. Haemodialysis patients are more risky to develop occult hepatitis B infection [OBI] due to an increased number of blood transfusions, frequent invasive procedures, difficulty in diagnosis of occult hepatitis B infection [OBI] and immunosuppression. Occult hepatitis B infection [OBI] is defined by the presence of HBV DNA in serum or liver tissue in the absence of HBsAg

Objective: to study the prevalence of occult HBV infection in HCV-positive and HCV negative patients on regular hemodialysis from Upper Egypt

Methodology: One Hundred hemodialysis patients with negative HBsAg were included in the study. These patients were divided into two groups: HCV positive and HCV negative, based on the results of anti-HCV by ELISA and HCV-RNA by PCR. HBV-DNA was studied using the real-time PCR method in both groups

Results: HBV DNA was detected in 7 of the 100 patients [7%] and HBcAb was detected in 22 patients [22%]. There were no statistically significant differences in the age, sex, duration of hemodialysis, biochemical parameters, HBcAb, or HBV DNA between patients with and without HCV infection

Conclusion: The prevalence of occult HBV infection [OBI] among Egyptian hemodialysis patients is 7% with no significant difference in the prevalence of OBI between hemodialysis patients with or without HCV infection and we suggest screening of all HD patients for OBI by testing anti-HBc and HBV DNA

Adult , Female , Humans , Male , Middle Aged , Liver Diseases/virology , Hepatitis B virus , Renal Dialysis , Hepacivirus , Hepatitis C Antibodies , Polymerase Chain Reaction , Viral Core Proteins , Hepatitis B/transmission
Article in Chinese | WPRIM | ID: wpr-280303


To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine.

Adenoviridae , Genetics , Metabolism , Animals , CD4-Positive T-Lymphocytes , Allergy and Immunology , Cross Protection , Female , Genetic Vectors , Genetics , Hepacivirus , Genetics , Allergy and Immunology , Hepatitis C , Allergy and Immunology , Virology , Humans , Interferon-gamma , Allergy and Immunology , Mice , Mice, Inbred BALB C , Recombinant Proteins , Genetics , Allergy and Immunology , Viral Core Proteins , Genetics , Allergy and Immunology , Viral Hepatitis Vaccines , Genetics , Allergy and Immunology , Viral Nonstructural Proteins , Genetics , Allergy and Immunology
Chinese Journal of Virology ; (6): 579-584, 2015.
Article in Chinese | WPRIM | ID: wpr-296244


The core protein (CP) of the classical swine fever virus (CSFV) is one of its structural proteins. Apart from forming the nucleocapsid to protect internal viral genomic RNA, this protein is involved in transcriptional regulation. Also, during viral infection, the CP is involved in interactions with many host proteins. In this review, we combine study of this protein with its disorders, structural/functional characteristics, as well as its interactions with the non-structural proteins NS3, NS5B and host proteins such as SUMO-1, UBC9, OS9 and IQGAP1. We also summarize the important part played by the CP in CSFV pathogenicity, virulence and replication of genomic RNA. We also provide guidelines for further studies in the CP of the CSFV.

Animals , Classical Swine Fever , Virology , Classical Swine Fever Virus , Genetics , Metabolism , Virulence , Genome, Viral , Swine , Viral Core Proteins , Chemistry , Genetics , Metabolism , Virulence
J. bras. nefrol ; 36(4): 496-501, Oct-Dec/2014. tab, graf
Article in English | LILACS | ID: lil-731149


Introduction: Polyphenols contained in natural sources such as grapes, have been considered pharmacological agents to combat oxidative stress and inflammation, common features in Chronic Kidney Disease patients. Objective: To evaluate the effects of grape powder supplementation on inflammatory and antioxidant biomarkers in hemodialysis (HD) patients. Methods: The double-blind placebo-controlled randomized clinical trial evaluated non-diabetic HD patients that received grape powder (500 mg of polyphenols/day) (n = 16, 9 men, 53.0 ± 9.8 years of age, 111.6 ± 58.2 HD months) or placebo (n = 16, 9 men, 52.7 ± 13.7 years of age, 110.4 ± 93.1 HD months) for five weeks. The glutathione peroxidase (GSH-Px) activity and C-reactive protein (CRP) levels were evaluated by ELISA method. Results: After the intervention period, the patients receiving grape powder showed an increase in the GSH-Px activity (16.5 (41.0) to 42.0 (43.3) nmol/min/ml) (p < 0.05) and they did not have the CRP levels increased as seen in placebo group (2.6 (0.28) to 2.8 (0.23 mg/L) (p < 0.05). Conclusion: The use of grape powder as phenolic source could play an important role as an antioxidant and anti-inflammatory agent in non-diabetic HD patients. .

Introdução: Polifenóis contidos em fontes naturais, como as uvas, têm sido considerados agentes farmacológicos no combate ao estresse oxidativo e inflamação, condições comuns na Doença Renal Crônica. Objetivo: Avaliar os efeitos da suplementação de farinha de uva sobre marcadores inflamatórios e antioxidantes em pacientes submetidos à hemodiálise (HD). Métodos: Estudo randomizado, duplo-cego, placebocontrolado, no qual foram avaliados pacientes não diabéticos em HD que receberam farinha de uva (500 mg de polifenóis/dia) (n = 16, 9 homens, 53,0 ± 9,8 anos, 111,6 ± 58,2 meses em HD) ou placebo (n = 16, 9 homens, 52,7 ± 13,7 anos, 110,4 ± 93,1 meses em HD) por cinco semanas. A atividade da glutationa peroxidase (GSH-Px) e os níveis plasmáticos de proteína C-reativa (PCR) foram mensurados por meio do método ELISA. Resultados: Após o período de intervenção, os pacientes que receberam farinha de uva apresentaram elevação na atividade da GSH-Px (16,5 (41,0) para 42,0 (43,3) nmol/min/ml) (p < 0,05) e não foi observada elevação nos níveis de PCR, como visto no grupo placebo (2,6 (0,28) para 2,8 (0,23) mg/L) (p < 0,05). Conclusão: O uso da farinha de uva como fonte de polifenóis pode desempenhar um importante papel anti-inflamatório e antioxidante em pacientes não diabéticos submetidos à HD. .

Humans , DNA-Binding Proteins , Gene Expression Regulation , Mutation , Nuclear Proteins , Trans-Activators/metabolism , Transcription Factors/metabolism , Binding Sites , Carcinoma, Hepatocellular , DNA, Viral/metabolism , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , Precipitin Tests , Plasmids/genetics , Protein Precursors/genetics , Protein Precursors/metabolism , Transfection , Tumor Cells, Cultured , Trans-Activators/genetics , Transcription Factors/genetics , Viral Core Proteins/genetics , Viral Core Proteins/metabolism
Chinese Journal of Hepatology ; (12): 590-593, 2014.
Article in Chinese | WPRIM | ID: wpr-313997


<p><b>OBJECTIVE</b>To explore the effects of hepatitis C virus (HCV) core protein on the activity of double-stranded RNA-dependent protein kinase (PKR).</p><p><b>METHODS</b>The human hepatoma cell line BEL-7402 was transfected with the HCV core gene-containing eukaryotic expression vector pCMH6K-Core (at various concentrations), or empty vector, or no vector; a group of cells was co-transfected with the luciferase reporter plasmid pGL3-promoter. The cells were treated with interferon (IFN) a-2b to induce the expression and activation of endogenous PKR, or left untreated to serve as controls. The effect of core protein on PKR phosphorylation was detected by western blotting. Luciferase activity was detected to reflect effects of the core protein on the synthesis of cellular proteins. The t-test and F test were used for statistical analyses.</p><p><b>RESULTS</b>In the case of IFNa stimulation, PKR phosphorylation levels were significantly lower in the HCV core protein expressing cells than in the cells transfected with empty plasmid or with no vector, but the total PKR expression level was not significantly different among these three groups of cells. Cells co-transfected with luciferase plasmid and the core protein expressing vector showed significantly higher levels of luciferase expression than the cells co-transfected with the empty vector. Moreover, the luciferase activity and core protein expression levels increased in a dose-dependent manner, with the luciferase activity of the cells treated with 0.5 mug, 1.0 mug and 1.5 mug pCMH6K-Core being 1.941 ± 0.199 times, 2.868 ± 0.275 times and 3.839 ± 0.338 times higher than that of the empty vector group (all P < 0.05).</p><p><b>CONCLUSION</b>In the human hepatoma cell line BEL-7402, the HCV core protein can inhibit the activity of endogenous PKR, thereby promoting cell protein synthesis.</p>

Cell Line, Tumor , Genes, Reporter , Genetic Vectors , Humans , Phosphorylation , Protein Biosynthesis , RNA-Binding Proteins , Metabolism , Transfection , Viral Core Proteins , Genetics , Metabolism
Chinese Journal of Virology ; (6): 206-210, 2013.
Article in Chinese | WPRIM | ID: wpr-339951


Avian influenza virus subtype H9N2 has been circulating in multiple terrestrial birds and repeatedly infecting mammals, including swines and humans to pose a significant threat to public health. The cross-species infection of human, replication activity and tissue tropism of avian influenza virus H9N2 was evaluated in this study. The results showed that surgically removed human lung tissue samples were infected ex vivo by avian influenza virus subtype H9N2 (Ck/GX/1875/04, Ck/GX/187/05) and seasonal human influenza virus H3N2 (A/ST/602/05). Examination of nucleoprotein expression replication in the infected human lung tissue samples showed that the replication of avian influenza virus H9N2 and seasonal human influenza virus H3N2 were mainly prevalent in alveolar epithelial cells, respiratory bronchiole epithelial cells and bronchial epithelial cells. Double-immunostaining for viral antigens and cellular markers indicated that avian influenza virus subtype H9N2 replicated in type 2 alveolar epithelial cells. These findings suggest that the H9N2 virus may be better adapted to the human host and replicates efficiently in human lung epithelial cells. Moreover, H9N2 avian influenza virus repeatedly infecting human, may favor gene evolution and the potential emergence of pandemic influenza virus.

Animals , Epithelial Cells , Virology , Humans , Influenza A Virus, H3N2 Subtype , Genetics , Physiology , Influenza A Virus, H9N2 Subtype , Genetics , Physiology , Influenza, Human , Virology , Lung , Cell Biology , Virology , RNA-Binding Proteins , Genetics , Metabolism , Viral Core Proteins , Genetics , Metabolism , Virus Replication
Chinese Journal of Virology ; (6): 386-391, 2013.
Article in Chinese | WPRIM | ID: wpr-339940


In order to study the proliferation inhibition effect of H5N1 subtype avian influenza virus (AIV) with small interfere RNA (siRNA), a total of 4 siRNAs were designed in accordance with the NP and PA genes of H5N1 subtype AIV, the siRNAs were then transfected to chicken embryo fibroblast(CEF), CEF was infected with H5N1 subtype AIV after 6 hrs. Virus titer of cell supernatant was tested at 16-56hrs post infection, and pathological changes of the cells was observed; mRNA levels of NP, PA, HA and p13-actin gene were tested at 36hrs post infection. The results showed that these 4 siRNAs could inhibit the prolif-eration of H5N1 subtype AIV in CEF in varying degrees, and one siRNA targeting PA was best per-formed. The experimental results also showed that the inhibition effect was decreased with the time prolonged. This research provides a basis for further studying RNAi on AIV prevention and control.

Actins , Genetics , Animals , Chick Embryo , DNA Primers , Genetics , Fibroblasts , Virology , Hemagglutination , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Hemagglutinins , Genetics , Humans , Influenza A Virus, H5N1 Subtype , Genetics , Physiology , RNA Interference , RNA-Dependent RNA Polymerase , Genetics , RNA, Small Interfering , Genetics , RNA-Binding Proteins , Genetics , Real-Time Polymerase Chain Reaction , Specific Pathogen-Free Organisms , Transfection , Viral Core Proteins , Genetics , Viral Proteins , Genetics , Virus Replication
Article in Chinese | WPRIM | ID: wpr-318101


<p><b>OBJECTIVE</b>To establish a hepatitis C virus (HCV) diagnostic assay using a protein array based on the quantum dots (QDs) encoded microbeads.</p><p><b>METHOD</b>Using QDs encoded microbeads array and immunofluorescence techniques, the highly purified HCV NS3, NS4, NS5 and Core protein were respectively immobilized on the surface of encoded beads, which were used for the detection of anti-HCV antibody in serum. To evaluate the microbeads protein array, 120 HCV positive and 50 HCV negative samples were tested, and compared with recombinant immunoblot assay(RIBA) results as golden standard. The sensitivity, specificity and accuracy value were calculated.</p><p><b>RESULTS</b>Compared 120 positive samples detected with RIBA, the sensitivity of microbeads array is 97.50% (117/120), the specificity is 96.0% (48/50), and accuracy is 97. 06% [(117 + 48)/(120 + 50)], The sensitivity of microbeads protein array is similar with RIBA methods. In the 120 positive samples tested with protein array, the positive rate of anti-HCV Core is 92. 50% (111/120) , the positive rate of anti-HCV NS3 is 89. 17% (107/120), the positive rate of anti-HCV NS4 is 70. 83% (85/120), the positive rate of anti-HCV NS5 is 52.50% (63/120).</p>

Adolescent , Adult , Aged , Child , Clinical Laboratory Techniques , Methods , Female , Fluorescent Antibody Technique , Methods , Hepacivirus , Chemistry , Hepatitis C , Blood , Diagnosis , Virology , Hepatitis C Antibodies , Blood , Chemistry , Humans , Male , Microspheres , Middle Aged , Protein Array Analysis , Methods , Quantum Dots , Viral Core Proteins , Chemistry , Young Adult
Article in Chinese | WPRIM | ID: wpr-274705


<p><b>OBJECTIVE</b>To explore the characteristics of the genetic variation of hemagglutinin( HA) and three internal genes coding for the nucleoprotein ( NP) , matrix protein ( M) and nonstructural protein ( NS) of influenza B virus.</p><p><b>METHODS</b>A total of 31 strains of influenza B virus were isolated in Zhejiang province from 1999 to 2012, and then were amplified and sequenced the genes of HAl , NP, M and NS. The phylogenetic tree was constructed, the nucleotide substitution rate of the above individual gene was estimated and the variation sites of amino acids were analyzed.</p><p><b>RESULTS</b>The 31 isolated strains of influenza B virus were divided into two distinct lineages Victoria and Yamagata in the phylogenetic tree of HAl gene,represented by B/Victoria/2/87 and B/Yamagata/16/88. Phylogenetic analysis of the NP gene showed that the NP gene of Victoria-like influenza B strains which were isolated after 2010 was highly homologous with Yamagata-like isolates, and thereby they were found to be on the same branch of the phylogenetic tree of the NP gene. Nucleotide substitution rates of HAl , NP, M and NS genes were estimated to be 2. 29 x 10 -3 ,1. 39 X 10-3 ,1. 78 X 10-3 ,1. 30 X 10-3 /site per year, respectively. Variations of amino acid of HAl domain of Victoria-like isolates mainly included K48E ,L58P ,N75K,K80R,K129N/S,N165K,S172P ,Sl97N/D and A202V; while those in Yamagata-like isolates were R48K, S1501, N166Y, N203S, G230D and D233N. Determined amino acid sequences of NP of Victoria-like influenza B isolates were similar to Yamagata-like isolates after 2010 and variations happened on four characteristic amino acid sites, naming A60D, I233V, N513S and V5341, compared with previous Victoria-like influenza B isolates.</p><p><b>CONCLUSION</b>Significant variation was found among influenza B strains isolated in Zhejiang province from 1999 to 2012. The surface HAl gene evolved more rapidly than internal genes. Gene reassortment and gene mutation were the main evolutionary mechanism of influenza B virus.</p>

China , Epidemiology , Evolution, Molecular , Genes, Viral , Genetic Variation , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Humans , Influenza B virus , Genetics , Influenza, Human , Epidemiology , Virology , Phylogeny , Reassortant Viruses , Genetics , Viral Core Proteins , Genetics , Viral Matrix Proteins , Genetics , Viral Nonstructural Proteins , Genetics
Article in Chinese | WPRIM | ID: wpr-274703


<p><b>OBJECTIVE</b>To explore the characteristics of the whole genome of the influenza H1N1 virus of the mild and severe cases in Beijing.</p><p><b>METHODS</b>A total of 21 samples of throat swabs were collected from surveillance-designated hospitals between June and December in 2009, including 10 severe cases (4 death cases) and 11 mild cases. RNA of the virus were extracted,and the amplified primers of the whole genome were designed.Reverse transcription and PCR were performed to the RNA and then the PCR product was sequenced by software to analyze the evolution of the viral genes and the variation of the amino acids.</p><p><b>RESULTS</b>Compared with the reference vaccine strain A/California/07/2009 (H1N1), the genetic nucleotide homology in the eight segments of the pandemic H1N1 virus in Beijing in 2009 was higher than 99%, without significant variation. Among them,the genetic distance of hemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) was comparatively far, separately 0.0050, 0.0040 and 0.0040.The gene of HA, P83S, the gene of NA, N248D, the gene of polymerase (PA), P224S and the gene of NP, V100I and L122Q were found to mutate in all the samples. Genes of HA, NA, NP, PA, PB 2 and nonstructural protein (NS1) in severe cases showed obviously clustered evolution. The mutation of gene S128P and S203T of HA, gene R269R and D547E of PA, gene T588I of PB 2 and gene I123V of NS mainly happened in severe cases, separately counting 6, 9, 6, 7, 9 and 6 cases. The relevance between the mutation happened in S203T of HA, R269K and D547E of PA and the severeness of the cases showed statistical significance (P < 0.05). The mutations of HA gene were mainly on the Ca and Cb antigene domains. No drug resistant mutation was found on NA gene but happened on matrix protein 2 (M2 gene). None of the mutations were found on the virulence related genes.</p><p><b>CONCLUSION</b>A high homology was found between the pandemic H1N1 virus in Beijing in 2009 and the reference vaccine strain A/California/07/2009(H1N1). Mutational sites related with the severe and fatal cases were found, but not the virulence related mutation.</p>

Base Sequence , China , Epidemiology , Genes, Viral , Genetic Variation , Genome, Viral , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Humans , Influenza A Virus, H1N1 Subtype , Genetics , Influenza, Human , Epidemiology , Virology , Neuraminidase , Genetics , RNA-Binding Proteins , Genetics , Viral Core Proteins , Genetics
Chinese Journal of Hepatology ; (12): 565-569, 2013.
Article in Chinese | WPRIM | ID: wpr-278039


<p><b>OBJECTIVE</b>To investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system.</p><p><b>METHODS</b>The PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein.</p><p><b>RESULTS</b>The transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein.</p><p><b>CONCLUSION</b>Soluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.</p>

Escherichia coli , Metabolism , Genetic Vectors , Hepacivirus , Recombinant Proteins , Genetics , Metabolism , Viral Core Proteins , Genetics , Metabolism , Viral Nonstructural Proteins , Metabolism
Chinese Journal of Virology ; (6): 265-272, 2013.
Article in Chinese | WPRIM | ID: wpr-356693


Nucleoprotein (NP) of influenza virus is highly conserved and type-specific. NP can trigger strong cell-mediated immune responses in host and is involved in the protection against the challenges with different subtype influenza viruses. Here, NP of an avian H5N1 (A/Hubei/1/2010, HB) was expressed by baculovirus surface-display technology and its immunogenicity as well as protective mechanism was investigated in mice infection model. Western blot and immunolabeled electron microscopy assay showed NP was displayed on baculovirus surface. ELISA results showed NP could induce high level of anti-NP IgG in the sera from NP-Bac-inoculated mice. Two cellular immune peptides (NP57-74 IQNSITIERMVLSAFDER and NP441-458 RTEIIKMMESARPEDLSF) were identified by IFN-gamma ELISPOT assay. NP57-66 and NP441-450 and NP protein could be able to trigger the activation of CD4+ and CD8+ T cells, and the response of CD8+ T was more predominant. The challenge study of mice-adapted virus A/PR/8/34 (H1N1) showed that NP-Bac could reduce viral load and attenuate the damage to lung tissue. 50% protection ratio against the virus could be detected.

Animals , Antibodies, Viral , Allergy and Immunology , Baculoviridae , Genetics , Metabolism , Cross Protection , Enzyme-Linked Immunospot Assay , Female , Humans , Immunity, Cellular , Influenza A Virus, H1N1 Subtype , Genetics , Allergy and Immunology , Influenza A Virus, H5N1 Subtype , Genetics , Allergy and Immunology , Influenza, Human , Allergy and Immunology , Virology , Mice , Mice, Inbred BALB C , RNA-Binding Proteins , Genetics , Allergy and Immunology , T-Lymphocytes , Allergy and Immunology , Viral Core Proteins , Genetics , Allergy and Immunology
Chinese Journal of Biotechnology ; (12): 1786-1795, 2013.
Article in Chinese | WPRIM | ID: wpr-242453


Hepatitis C virus (HCV), one of the major pathogens of viral hepatitis, causes significant hazards in humans. Interferon treatment in combination with ribavirin is used as the first line clinical treatment for HCV infection. However, good response to this treatment has only been observed in few patients and repeated recurrence has also been reported frequently. Therefore, new antiviral agents and therapies are in urgent demand. Here, we report a newly constructed Escherichia coli RNase P based M1GS ribozyme that can specifically and efficiently target the core gene of HCV. The guide sequence (GS) of this M1IGS was designed according to the sequence of the core coding region of HCV genome. The GS was then covalently linked to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The specification of this sequence-specific ribozyme, M1GS, was then examined using an in vitro cleavage assay. The cytotoxicity and its activity in inhibition of HCV gene expression and viral proliferation were further studied in vivo. Our results show that the reconstructed M1GS ribozyme displayed obvious catalytic activity in cleaving target mRNAs fragment in vitro. Notable reduction in the expression of HCV core protein and a 1 000-fold reduction in viral growth were also observed in cultured HCV infected Huh7.5.1 cells expressing the functional M1GS ribozyme. This study demonstrated a direct evidence for the antiviral activity of the customized M1GS-HCV/C141 ribozyme, and thus provided a promising new strategy for clinical treatment of HCV infection.

Antiviral Agents , Pharmacology , Escherichia coli , Genetics , Genetic Engineering , Hepacivirus , Genetics , Physiology , RNA, Catalytic , Genetics , Pharmacology , RNA, Guide , Genetics , Ribonuclease P , Genetics , Viral Core Proteins , Genetics
Rev. argent. microbiol ; 44(1): 0-0, mar. 2012. tab
Article in English | LILACS | ID: lil-639714


At the time of influenza A (H1N1) emergency, the WHO responded with remarkable speed by releasing guidelines and a protocol for a real-time RT-PCR assay (rRT-PCR). The aim of the present study was to evalúate the performance of the "Real Time Ready Influenza A/H1N1 Detection Set" (June 2009)-Roche kit in comparison to the CDC reference rRT-PCR protocol. The overall sensitivity of the Roche assay for detection of the Inf A gene in the presence or absence of the H1 gene was 74.5 %. The sensitivity for detecting samples that were only positive for the Inf A gene (absence of the H1 gene) was 53.3 % whereas the sensitivity for H1N1-positive samples (presence of the Inf A gene and any other swine gene) was 76.4 %. The specificity of the assay was 97.1 %. A new version of the kit (November 2009) is now available, and a recent evaluation of its performance showed good sensitivity to detect pandemic H1N1 compared to other molecular assays.

Durante la pandemia de influenza A (H1N1), la OMS recomendó algoritmos y protocolos de detección del virus mediante RT-PCR en tiempo real. El objetivo del presente estudio fue evaluar el desempeño del equipo que comercializa la empresa Roche, Real Time Ready Influenza A/H1N1 Detection Set (junio de 2009), en comparación con el protocolo de RT-PCR en tiempo real de los CDC. La sensibilidad global del ensayo de Roche para la detección del gen Inf A en presencia o ausencia del gen H1 fue 74,5 %. La sensibilidad para la detección de muestras positivas solo para el gen Inf A (ausencia del gen H1) fue 53,3 % y la sensibilidad para la detección de muestras positivas para H1N1 (presencia del gen Inf A y cualquier otro gen porcino) fue 76,4 %. La especificidad fue 97,1 %. Existe una nueva versión del equipo (noviembre 2009) que, según se ha descrito, presenta buena sensibilidad en comparación con otros ensayos moleculares para detectar H1N1 pandémica.

Humans , Influenza A Virus, H1N1 Subtype , Influenza, Human/diagnosis , Reagent Kits, Diagnostic , Argentina/epidemiology , Centers for Disease Control and Prevention, U.S. , Disease Outbreaks , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/epidemiology , Influenza, Human/virology , Nasal Cavity/virology , Pharynx/virology , Reproducibility of Results , RNA, Viral/genetics , RNA-Binding Proteins/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , United States , Viral Core Proteins/genetics
Scientific and Research Journal of Army University of Medical Sciences-JAUMS. 2012; 10 (1): 1-9
in Persian | IMEMR | ID: emr-128939


Hepatitis C virus [HCV] genome contains a large open reading frame coding for a polyprotein that is cleaved into ten proteins. Recently, a new protein, named core+1, has been described to be expressed through a ribosomal frame shift within the capsid-encoding sequence. To address these possibilities, core+1 was produced in E.coli and the purified protein was evaluated for the immunological properties. The core+1 corresponding nucleotide sequence was created by PCR-based induction of a +1 frame shift mutation within the core gene template. The amplicon was cloned into the pET-24a vector and expressed in E.coli host. The expressed protein was purified under denaturing condition and after refolding steps was characterized by SDS-PAGE and Western Blotting. The immunization potential of core+1 with various adjuvant [Freunds [C/IFA], Montanide ISA 206 and IMS 1312, pluronic acid [F127] and imiquimod [IMIQ] was assessed in Balb/c mice. ELISA-based assays were used to analyze the humoral immune responses. The yield of E.coli-derived core+1 was 5 mg/ L of culture media and antigenicity of this protein was confirmed by western blotting. All the core+1/adjuvant formulations significantly developed the anti-core+1 IgG responses in the immunized mice but C/IFA and ISA206 elicited the highest antibody titers. ISA 206 and IMS 1312 formulation of core+1 induced strong Th1/Th2 responses. Our results indicated that core+1 formulation with various adjuvants may elicit the different immune response profiles [Th1/Th2]. Thus core+1 might be a potential component of future HCV vaccine too

Animals, Laboratory , Viral Core Proteins , Adjuvants, Immunologic , Blotting, Western , Polymerase Chain Reaction , Mice, Inbred BALB C
Article in Chinese | WPRIM | ID: wpr-352330


<p><b>OBJECTIVE</b>To investigate the genetic variation and typing of hepatitis B virus (HBV) in patients with chronic hepatitis B in relation to HBeAg status.</p><p><b>METHODS</b>Fluorescence quantitative polymerase chain reaction (PCR) was employed to detect serum HBV DNA in patients with chronic hepatitis B negative for HBeAg. Real-time fluorescent PCR and PCR-reverse dot blot hybridization were used to detect HBV genotypes and mutations, respectively.</p><p><b>RESULTS</b>Of the 389 patients, 214 (55.01%) were positive and 175 (44.99%) were negative for HBV DNA; 102 (26.22%) had a HBV DNA copy number of 1×10(3), and 41 (10.54%) had a copy number of 1×10(4) (Χ(2)=226.6729, P<0.001). Of the 21 patients with a HBV DNA load of 1×10(5), 15 (71.43%) were found to have precore mutations, and 11 (52.38%) had basic core promoter (BCP) mutations; a higher HBV-DNA load was associated with an increased incidence of HBV mutations. In the 214 patients positive for HBV DNA, HBV genotypes A, B, C, D and the mixed type were found in 6 (2.80%), 84 (39.25%), 106 (49.53%), and 7 (3.27%), and 11 (5.14%) patients, who showed precore mutation rates of 16.67% (1 case), 36.90% (31 cases), 44.34% (47 cases), 0, and 0, and BCP mutation rates of 0, 19.05% ( 16 cases), 26.42% (28 cases), 0, and 0, respectively, demonstrating significant differences in HBV mutations between the genotype groups (P<0.001).</p><p><b>CONCLUSION</b>HBeAg-negative and HBV DNA-positive patients with chronic hepatitis B have a relatively low HBV replication level, and HBV DNA load is associated with HBV mutations. The B and C genotypes are more likely to have HBV mutations in HBeAg-negative patients.</p>

DNA, Viral , Blood , Female , Genotype , Hepatitis B e Antigens , Blood , Hepatitis B virus , Classification , Genetics , Hepatitis B, Chronic , Blood , Virology , Humans , Male , Mutation , Promoter Regions, Genetic , Viral Core Proteins , Genetics , Viral Load
Chinese Journal of Hepatology ; (12): 368-371, 2012.
Article in Chinese | WPRIM | ID: wpr-262000


<p><b>OBJECTIVE</b>To investigate the biological function of the hepatitis C virus (HCV)-encoded F protein in hepatocytes.</p><p><b>METHODS</b>The full-length F gene was amplified by PCR from HCV genotype 1a and cloned into plasmid pSEB-3Flag by restriction enzyme digestion and ligation. Hepatoma cell lines, Huh7 and SMMC7721, were transfected with the resultant recombinant pSEB-3Flag-F or the original pSEB-3Flag (negative control) and screened with the selective antibiotic, blasticidin. Stable F gene and protein expression was verified by RT-PCR analysis. Analysis of cell growth and cell cycle was carried out by MTS assay, crystal violet staining and flow cytometry.</p><p><b>RESULTS</b>Huh7 and SMMC7721 cells transfected with pSEB-3Flag-F plasmid (Huh7-F and SMMC7721-F, respectively) uniquely expressed the F gene and protein. The Huh7-F and SMMC7721-F cells showed significantly decreased proliferation rates, compared to the respective control groups. A similar HCV F-mediated growth-inhibiting activity was observed by the cell viability assay. Furthermore, cell cycle analysis revealed that the S-phase distribution was much lower in Huh7-F (47.12%) and SMMC7721-F (30.75%) cells than in the respective controls (55.35% and 33.23%, respectively) (P less than 0.05).</p><p><b>CONCLUSION</b>Stable expression of the HCV F gene reduced the in vitro proliferation rate of hepatoma cell lines, indicating that the F protein may function as a growth inhibitor of infected cells.</p>

Carcinoma, Hepatocellular , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Hepacivirus , Genetics , Humans , Liver Neoplasms , Metabolism , Pathology , Reverse Transcriptase Polymerase Chain Reaction , Viral Core Proteins , Genetics , Metabolism
Article in Chinese | WPRIM | ID: wpr-246177


<p><b>OBJECTIVE</b>To develop a best method of constructing influenza NP fusion gene containing enhanced green fluorescent protein (EGFP).</p><p><b>METHODS</b>The full-length NP gene of influenza A was amplified by RT-PCR and was inserted into an eukaryotic expression vector pEGFP-N1 in order to construct a fusion gene of pEGFP-N1-NP using three different methods. Method one, NP gene containing restriction endonucleases and pEGFP-N1 were both digested using the same restrict enzymes and ligated, yielding the fusion gene of pEGFP-N1-NP. Method two, NP gene was cloned into pMD19-T Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP; Method three, NP gene containing restriction endonucleases was cloned into pMD19-T Simple Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP.</p><p><b>RESULTS</b>The fusion gene of recombinant eukaryotic expression vector pEGFP-N1-NP was successfully constructed by using method three.</p><p><b>CONCLUSIONS</b>The full-length NP gene is obtained and its fusion gene of recombinant eukaryotic expression plasmid is successfully constructed. This study provides foundation for further understanding the biological function of NP protein and the mechanism of diseases induced by influenza A virus.</p>

Artificial Gene Fusion , Cloning, Molecular , Genetic Vectors , Green Fluorescent Proteins , Genetics , Polymerase Chain Reaction , RNA-Binding Proteins , Genetics , Viral Core Proteins , Genetics