Basal core promoter and precore mutations among hepatitis B virus circulating in Brazil and its association with severe forms of hepatic diseases

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.

Early predictive efficacy of core antigen on antiviral outcomes in genotype 1 hepatitis C virus infected patients

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.

Effects of grape powder supplementation on inflammatory and antioxidant markers in hemodialysis patients: A randomized double-blind study / Efeitos da suplementação de farinha de uva sobre marcadores inflamatórios e antioxidantes em pacientes em hemodiálise: Estudo duplo-cego randomizado

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. .

Performance of a commercial assay for the diagnosis of influenza A (H1N1) infection in comparison to the Centers for Disease Control and Prevention protocol of real time RT-PCR / Evaluación del desempeño de un equipo comercial para el diagnóstico de influenza A (H1N1) en comparación con el protocolo de RT-PCR en tiempo real diseñado por los Centros de Control y Prevención de Enfermedades (CDC)

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.

Recombinant in vitro assembled hepatitis C virus core particles induce strong specific immunity enhanced by formulation with an oil-based adjuvant

In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-γ producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Bioinformatic prediction of polymerase elements in the rotavirus VP1 protein

Rotaviruses are the major cause of acute gastroenteritis in infants world-wide. The genome consists of eleven double stranded RNA segments. The major segment encodes the structural protein VP1, the viral RNA-dependent RNA polymerase (RdRp), which is a minor component of the viral inner core. This study is a detailed bioinformatic assessment of the VP1 sequence. Using various methods we have identified canonical motifs within the VP1 sequence which correspond to motifs previously identified within RdRps of other positive strand, double-strand RNA viruses. The study also predicts an overall structural conservation in the middle region that may correspond to the palm subdomain and part of the fingers and thumb subdomains, which comprise the polymerase core of the protein. Based on this analysis, we suggest that the rotavirus replicase has the minimal elements to function as an RNA-dependent RNA polymerase. VP1, besides having common RdRp features, also contains large unique regions that might be responsible for characteristic features observed in the Reoviridae family.

Evolution of hepatitis C virus-specific T cell responses and cytokine production in chronic hepatitis C patients treated with high doses of interferon-alpha

OBJECTIVE: To assess the evolution of in vitro T cell response to hepatitis C virus (HCV) Core, E1, E2 and NS3 antigens in 10 patients with chronic hepatitis C, before, during and after a high dose interferon alpha (IFN-alpha) therapy, and to evaluate the influence of IFN-alpha on the in vivo and in vitro production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). METHODS: T cell response to HCV antigens was evaluated by lymphoproliferation assays. In vivo and in vitro cytokine production was evaluated at weeks 0, 4, 8, and 12 of IFN-alpha therapy by enzyme immunoassays. RESULTS: In general, of all HCV antigens tested throughout the follow-up, those belonging to the Core region were the most immunostiumlatory. This observation was valid in IFN-alpha responders as well as IFN-alpha non-responders. The lymphoproliferative response to HCV antigens increased during IFN-alpha therapy. Serum levels of TNF-alpha were significantly increased in HCV patients, and six out of ten patients showed increased IFN-gamma serum levels. A significant decrease of IFN-gamma levels was observed during therapy and the same trend was seen for TNF-alpha. Mitogen-stimulated TNF-alpha and IFN-gamma production before therapy did not differ from that of normal controls, however, the cytokine production was reduced at week 4 of therapy, corresponding with a clinical improvement. A return to pretreatment values was observed after 8 weeks of therapy. CONCLUSIONS: a) Core antigens are the most immunostimulatory HCV antigens at the T cell level in chronic hepatitis C patients; b) High dose IFN-alpha therapy induces an increase in lymphoproliferative response to HCV antigens; c) Serum levels of TNF-alpha are increased in HCV patients; d) High dose IFN-alpha therapy induces a decrease in serum levels of IFN-gamma; e) High dose IFN-alpha therapy induces a transiently decreased mitogen-induced TNF-alpha and IFN-gamma production.

Additives and protein-DNA combinations modulate the humoral immune response elicited by a hepatitis C virus core-encoding plasmid in mice

Humoral and cellular immune responses are currently induced against hepatitis C virus (HCV) core following vaccination with core-encoding plasmids. However, the anti-core antibody response is frequently weak or transient. In this paper, we evaluated the effect of different additives and DNA-protein combinations on the anti-core antibody response. BALB/c mice were intramuscularly injected with an expression plasmid (pIDKCo), encoding a C-terminal truncated variant of the HCV core protein, alone or combined with CaCl2, PEG 6000, Freund's adjuvant, sonicated calf thymus DNA and a recombinant core protein (Co.120). Mixture of pIDKCo with PEG 6000 and Freund's adjuvant accelerated the development of the anti-core Ab response. Combination with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core antibody response elicited by Co.120 alone or combined with pIDKCo was different regarding that induced by the pIDKCo alone. Our data indicate that the antibody response induced following DNA immunization can be modified by formulation strategies

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott(r)), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95 percent) sera from patients in the study group and negative in 38 of the 39 (97 percent) sera from those in the control group, showing an accuracy of 96 percent. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients

Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6 and 41.3 were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1). The rate of anti-c100/3 in patients of Group 2 was 71.15 and reached 86.5 for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8 of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7 (Group 2) was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells

Los genes clonados de las proteínas de nucleocápside, N, de los arenavirus Junín y LCM (choriomeningitis linfocitaria) se insertaron en el vector de expresión pKG4 regulado por el promotor tardío del virus SV40. Cuando estas construcciones se utilizaron para transfectar las líneas celulares BHK-21 (fibroblastos de hamster lactante) y CV-1 (fibroblastos de riñón de mono verde africano) se observó la expresión transiente de un polipéptido de tamaño e inmunoreactividad indistinguible de la proteína N sintetizada durante una infección viral. El análisis por inmunofluorescencia reveló un patrón de distribución intracelular semejante al observado en células infectadas. Este patrón presentó variaciones desde una tinción citoplásmica difusa hasta gránulos citoplásmicos dispersos o concentrados en la zona perinuclear. La asociación de la proteína N con gránulos basófilos es semejante a la descripta en el efecto citópático causado por los arenavirus en las células infectadas, y podría relacionarse con las características fisicoquímicas de la proteina N, que contiene numerosas secuencias de aminoácidos básicos capaces de interactuar con ácidos ribonucleicos celulares