37 research outputs found
A novel indirect immunofluorescence test for the detection of IgG and IgA antibodies for diagnosis of Hepatitis E Virus infections
No full textHepatitis E Virus (HEV) causes epidemic infections in regions of poor hygiene in the developing world. Over the last years, however, increasing numbers of autochthonous infections in industrialized countries have been described, leading to new interest in this pathogen. Currently available serological test formats to detect IgG and IgM antibodies are mainly based on bacterially expressed ORF2 and ORF3 antigens and often give ambiguous results. The objective of this study was the development of a different assay format for HEV diagnosis—a HEV immunofluorescence test (HEV-IFT) based on mammalian cells transiently expressing recombinant HEV ORF2 protein with a simple production and staining protocol and the investigation of its performance and methodical feasibility under diagnostic laboratory conditions. 31 sera of patients at different phases of HEV infection and 40 control sera from a non-endemic region were analyzed for anti-HEV IgG, IgM, and IgA antibodies. The HEV-IFT detected successfully anti-HEV IgG and IgA, but not anti-HEV IgM antibodies. In the study group the HEV-IFT was able to confirm HEV infections and to support diagnosis when ambiguous results were obtained by commercial assays. Signal localization and staining patterns helped to gather additional information about reactive antibodies present in patient sera. In conclusion the developed IFT for the detection of anti-HEV IgG and IgA antibodies can be used for diagnosis and for the serological confirmation of HEV infections.Fil: Osterman, Andreas. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; AlemaniaFil: Vizoso Pinto, María Guadalupe. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; ArgentinaFil: Jung, Jette. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; AlemaniaFil: Jaeger, Gundula. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; AlemaniaFil: Eberle, Josef. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; AlemaniaFil: Nitschko, Hans. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; AlemaniaFil: Baiker, Armin. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania. Bavarian Health and Food Safety Authority; Alemani
β-Carboline derivatives as novel antivirals for herpes simplex virus
No full textSeveral commercial and novel synthetic β-carbolines (βCs) were evaluated for their antiviral activity against herpes simplex virus type 1 (HSV-1) using an adapted MTS assay. Of 21 drugs tested, although 11 exerted antiviral activity at non-cytotoxic concentrations, only 3 of them [9-methyl-norharmane (9-Me-nHo), 9-methyl-harmane (9-Me-Ho) and 6-methoxy-harmane (6-MeO-Ho)] completely avoided virus-driven cytopathic effects. Half-maximal effective concentrations (EC 50 values) (4.9 ± 0.4, 5.9 ± 0.8 and 19.5 ± 0.3 µM, respectively) and selectivity indexes (88.8, 40.2 and 7.0, respectively) of the latter three βCs against HSV-1 were determined by MTS, flow cytometry and plaque reduction assays. The mode of action of these drugs was also evaluated. According to time-of-addition assays, the selected compounds were not virucidal and did not interfere with attachment or penetration of HSV-1, but interfered with later events of virus infection. Western blot studies showed that early and late protein expression was significantly delayed or even suppressed. Herpes simplex virus type 2 (HSV-2) was also inhibited by the selected substances in a similar manner. Interestingly, 6-MeO-Ho, 9-Me-Ho and 9-Me-nHo restricted HSV-1 ICP0 localisation to the nucleus during later stages of infection, possibly affecting its functionality in the cytoplasm where ICP0 normally inhibits antiviral signalling and promotes viral replication. In silico prediction of ADME (Absorption, Distribution, Metabolism and Excretion) properties showed that all compounds fulfilled Lipinski's rule and their calculated absorptions were >95%.Fil: Gonzalez, Maria Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Ludwig Maximilians Universitat; AlemaniaFil: Cabrerizo, Franco Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Baiker, Armin. Bavarian Health and Food Safety Authority; AlemaniaFil: Erra Balsells, Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Osterman, Andreas. Ludwig Maximilians Universitat; Alemania. German Center for Infection Research; AlemaniaFil: Nitschko, Hans. Ludwig Maximilians Universitat; Alemania. German Center for Infection Research; AlemaniaFil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina. Departamento Biomédico; Argentina. Ludwig Maximilians Universitat; Alemani
Evidencia serológica de la circulación del virus de la hepatitis E y prevalencia de anticuerpos contra la hepatitis A en una población indígena en el norte argentino
No full textIn 2005 a universal vaccination program against hepatitis A was introduced in Argentina. Nevertheless, there are still some unvaccinated marginal population groups. There are no data about the seroprevalence of hepatitis E in the northern region of Argentina mainly because of lack of awareness of this emergent pathogen. We aimed to determine the seroprevalence of hepatitis A, and hepatitis E in an indigenous population in northern Argentina. One hundred and twenty six (126) donor serum samples collected near San Salvador de Jujuy were analyzed for anti-HAV IgG and HEV IgG and IgM, alkaline phosphatase and transaminase values. Volunteers were interviewed about their living conditions, animal farming, consumption of tap water or river water, and level of education. Seroprevalence of specific anti-HAV antibodies was high (80.2%, 95% confidence interval, 72.1–86.7%) in children under 5 years of age, indicating early infection in life. Seroprevalence of anti-HEV antibodies was 5.6% (95% CI: 2.3–11.2%), being slightly higher than the values found in healthy patients from other regions of the country. Although we could not characterize the genotype of the circulating HEV strain, the clear epidemiological difference between seroprevalence of HAV and HEV in a community with poor sanitary conditions suggest that the circulating HEV strains spread through a different transmission route than HAV. Furthermore a significant correlation between anti-HEV IgG and swine farming was found (p < 0.05), which supports a zoonotic transmission path. We reassessed the epidemiological pattern of HAV infection and reported evidence of HEV infection for the first-time in a community belonging to the Guarani ethnic group, highlighting the need to include hepatitis E testing in routine diagnostics in the region.En 2005 se inició un programa de vacunación universal contra la hepatitis A en Argentina, pero todavía existen algunas poblaciones marginales no vacunadas. Además, los datos sobre la circulación de hepatitis E en el noroeste argentino son escasos. El objetivo de este trabajo fue determinar la seroprevalencia de la hepatitis A y la hepatitis E en una población autóctona del norte de Argentina. Se colectaron y analizaron 126 muestras de suero en habitantes de las yungas jujeñas; se determinaron transaminasas, fosfatasa alcalina y anticuerpos contra los virus de hepatitis A (HAV) (IgG) y hepatitis E (HEV) (IgG e IgM). Se obtuvieron los consentimientos informados y los voluntarios fueron entrevistados para identificar posibles factores de riesgo, como las condiciones de vida y la cría de animales, entre otros. La seroprevalencia de anticuerpos específicos anti-HAV fue alta (80,2%; intervalo de confianza [IC] 95%: 72,1-86,7%) en niños menores de 5 años, lo que indica infección temprana. La seroprevalencia de anticuerpos anti-HEV fue del 5,6% (IC 95%: 2,3-11,2%), ligeramente más alta que en otras regiones del país en pacientes sanos. Aunque no se caracterizó el genotipo circulante del HEV, la clara diferencia epidemiológica entre la seroprevalencia de ambos virus en una comunidad con malas condiciones sanitarias sugiere que la cepa circulante de HEV se transmite por una vía diferente que la del HAV. Además, encontramos una significativa correlación entre la cría de cerdo y la presencia de anticuerpos IgG anti-HEV (p < 0,05), lo que sugiere una vía de transmisión zoonótica. Reevaluamos el patrón epidemiológico de infección por el HAV y aportamos por primera vez una evidencia de infección por el HEV en una comunidad que pertenece a la etnia guaraní, por lo que destacamos la necesidad de incluir la detección de hepatitis E en la región.Fil: Remondegui, Carlos. Hospital Zonal General Agudos San Roque; ArgentinaFil: Ceballos, Susana. Hospital Zonal General Agudos San Roque; ArgentinaFil: Arce, Lorena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: Pintado, Eduardo. Hospital Zonal General Agudos San Roque; ArgentinaFil: Vidaurre, Rene. Hospital Paterson de San Pedro de Jujuy; ArgentinaFil: Nitschko, Hans. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania. German Center for Infection Research; AlemaniaFil: Osterman, Andreas. German Center for Infection Research; Alemania. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; AlemaniaFil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentin
In VivoProcessing of Pr160gag-polfrom Human Immunodeficiency Virus Type 1 (HIV) in Acutely Infected, Cultured Human T-Lymphocytes
No full textAbstractThe processing of the HIV-1 Pr160gag-polprecursor polyprotein was analyzed in freshly HIV-1-infected MT-4 cultured cells. Single intermediates of the processing cascade were characterized by immunoblotting using distinct antisera. A potent inhibitor of the HIV protease (PR), Ro 31-8959, was employed to block cleavage by the mature PR, thus allowing insights into initial stages of thegag-pol(auto)-catalytical processing. While most knowngag-polcleavages were blocked in the presence of the inhibitor, the cleavage site between thegag-NC and thepol-p6* domains was still cleaved even in presence of high amounts (1 μM) of inhibitor, leading to the accumulation of a novel 114-kDa polyprotein comprising p6*-PR-RT-IN. In the absence of inhibitor no accumulation of p114 was observed. In inhibitor-treated, HIV-1-infected cells a p6*-PR intermediate was also detected, indicating subsequent cleavage of the PR/RT scissile bond. These results demonstrate initial cleavage(s) of thegag-polprecursor hydrolyzed by a proteolytic activity different from the mature PR and indicate that p114 (p6*-PR-RT-IN) and p6*-PR intermediates could play an essential role in the PR activation process
The mRNAs of Prekallikrein, Factors XI and XII, and Kininogen, Components of the Contact Phase Cascade Are Differentially Expressed in Multiple Non-hepatic Human Tissues
No full textSummaryRecently RT-PCR studies had demonstrated the expression of plasma prekallikrein (PPK) mRNA in extrahepatic tissues. The questions arose whether that is illegitimate or regular expression, and whether the mRNAs of blood coagulation factors XI and XII, and high molecular weight kininogen, components of the contact activation cascade of blood coagulation are also expressed in non-hepatic tissues. These questions were addressed in the present study by employing quantitative RT-PCR. The relative mRNA levels of the respective proteins determined in 16 human tissues indicate legitimate extrahepatic transcription of at least three of the genes. Transcription of all genes was highest in the liver, but only PPK mRNA was detected in all 16 tissues, especially high levels in pancreas, kidney, testis, spleen and prostate. We conclude from these results that PPK is synthesized in significant amounts in non-hepatic tissues and that this locally synthesized PPK may have special local functions.</jats:p
The polyomaviruses WUPyV and KIPyV: a retrospective quantitative analysis in patients undergoing hematopoietic stem cell transplantation
No full textAbstract Background The polyomaviruses WUPyV and KIPyV have been detected in various sample types including feces indicating pathogenicity in the gastrointestinal (GI) system. However, quantitative viral load data from other simultaneously collected sample types are missing. As a consequence, primary replication in the GI system cannot be differentiated from swallowed virus from the respiratory tract. Here we present a retrospective quantitative longitudinal analysis in simultaneously harvested specimens from different organ sites of patients undergoing hematopoietic stem cell transplantation (HSCT). This allows the definition of sample types where deoxyribonucleic acid (DNA) detection can be expected and, as a consequence, the identification of their primary replication site. Findings Viral DNA loads from 37 patients undergoing HSCT were quantified in respiratory tract secretions (RTS), stool and urine samples as well as in leukocytes (n = 449). Leukocyte-associated virus could not be found. WUPyV was found in feces, RTS and urine samples of an infant, while KIPyV was repeatedly detected in RTS and stool samples of 4 adult patients. RTS and stool samples were matched to determine the viral load difference showing a mean difference of 2.3 log copies/ml (p Conclusions The data collected in this study suggest that virus detection in the GI tract results from swallowed virus from the respiratory tract (RT). We conclude that shedding from the RT should be ruled out before viral DNA detection in the feces can be correlated to GI symptoms.</p
Tracking Virus-Specific CD4+ T Cells during and after Acute Hepatitis C Virus Infection
No full textBackground. CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. Methodology/Prencipal Findings. Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. Conclusions/Significance. During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1 + patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists
HCMV spread and cell tropism are determined by distinct virus populations.
No full textHuman cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells
Variability of the Hepatitis B Surface Protein in HBV-Infected Liver T ransplant Recipients
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