Molecular characterization of hepatitis A virus isolates from environmental and clinical samples in Greece

<p>Abstract</p> <p>Background</p> <p>Hepatitis A virus (HAV) strains detected in environmental and clinical samples were analysed to characterize the genotypes of HAV circulating in Greece. Fifty (50) sewage samples were collected from Patras (South-Western Greece) and Alexandroupolis (North-Eastern Greece) from 2007 until 2009, accordingly. The clinical samples derived from an HAV outbreak involved populations from three neighbouring prefectures of North-Eastern Greece (Xanthi, Rodopi, and Evros). HAV particles were detected by nested RT-PCR, using a previously validated set of primers to amplify a 290-bp fragment encompassing the 5'-NTR. Positive HAV samples were confirmed by sequencing of the PCR product. To determine the relatedness between the different isolated sequences, a phylogenetic tree was constructed.</p> <p>Results</p> <p>Results showed a 100% prevalence of genotype I, and particularly subgenotype IA. The analyzed HAV strains were closely related between them with the percentage of nucleotide identity ranging between 96% and 100%.</p> <p>Conclusions</p> <p>The study revealed the major prevalence of circulating strains of IA genotype in Greece and underlined the usefulness of molecular methods for the detection and typing of viruses in both environmental and clinical samples. The present study is, to our knowledge, the first in Greece to depict the simultaneous molecular characterization of HAV strains isolated from both clinical and environmental samples.</p

Development of an optimized method for the detection of airborne viruses with real-time PCR analysis

<p>Abstract</p> <p>Background</p> <p>Airborne viruses remain one of the major public health issues worldwide. Detection and quantification of airborne viruses is essential in order to provide information regarding public health risk assessment.</p> <p>Findings</p> <p>In this study, an optimized new, simple, low cost method for sampling of airborne viruses using Low Melting Agarose (LMA) plates and a conventional microbial air sampling device has been developed. The use of LMA plates permits the direct nucleic acids extraction of the captured viruses without the need of any preliminary elution step. Molecular detection and quantification of airborne viruses is performed using real-time quantitative (RT-)PCR (Q(RT-)PCR) technique. The method has been tested using Adenoviruses (AdVs) and Noroviruses (NoVs) GII, as representative DNA and RNA viruses, respectively. Moreover, the method has been tested successfully in outdoor experiments, by detecting and quantifying human adenoviruses (HAdVs) in the airborne environment of a wastewater treatment plant.</p> <p>Conclusions</p> <p>The great advantage of LMA is that nucleic acids extraction is performed directly on the LMA plates, while the eluted nucleic acids are totally free of inhibitory substances. Coupled with QPCR the whole procedure can be completed in less than three (3) hours.</p

Molecular detection of multiple viral targets in untreated urban sewage from Greece

<p>Abstract</p> <p>Background</p> <p>Urban sewage virological analysis may produce important information about the strains that cause clinical and subclinical infections in the population, thus supporting epidemiological studies.</p> <p>Methods</p> <p>In the present study, a twenty one-month survey (November 2007 to July 2009) was conducted in order to evaluate the presence of human adenoviruses (hAdV), hepatitis A viruses (HAV), hepatitis E viruses (HEV), Noroviruses (NoV), and human Polyomaviruses (hPyV) in untreated sewage samples collected from the inlet of Patras' municipal biological wastewater treatment plant, located in southwestern Greece. Nucleic acid amplification techniques were applied for viral nucleic acid detection. Positive samples were confirmed by sequencing and comparative phylogenetic analysis was performed on the isolated viral strains.</p> <p>Results</p> <p>In total, viruses were detected in 87.5% (42/48) of sewage samples. AdVs, PyVs, HAV, and NoVs were detected in 45.8% (22/48), 68.8% (33/48), 8.3% (4/48), and 6.3% (3/48) of the samples collected from the plant's inlet, while HEV was not detected at all. Adenovirus types 8 (Ad8), 40 (Ad40) and 41 (Ad41) were recognized, while JC and BK polyomaviruses were recorded. Noroviruses were identified as GII.4. HAV was typed as genotype IA.</p> <p>Conclusions</p> <p>Our study demonstrates the advantages of environmental surveillance as a tool to elucidate the molecular epidemiology of community circulating viruses. We underline the need of environmental surveillance programs in countries such as Greece with inadequate and problematic epidemiological surveillance system and no environmental surveillance system currently in action.</p

NFE2L2 (nuclear factor, erythroid 2-like 2)

Review on NFE2L2 (nuclear factor, erythroid 2-like 2

Epidemiological investigation of two parallel gastroenteritis outbreaks in school settings

BACKGROUND: Two parallel gastroenteritis outbreaks occurred in an elementary school and a neighboring kindergarten in Kilkis, Northern Greece in 2012. The aim of the study was the investigation of these two parallel outbreaks as well as their possible source. METHODS: Two retrospective cohort studies were performed to identify the mode and the vehicle of transmission as well as the possible connection between them. RESULTS: Elementary school and kindergarten populations of 79.9% (119/149) and 51.1% (23/45) respectively, participated in the study. Case definition was satisfied by 65 pupils from the elementary school and 14 from the kindergarten. For elementary school, 53 cases were considered primary cases of the outbreak and were included in the analysis. Based on the results of the multivariate analysis, consumption of tap water was the only statistically significant independent risk factor of gastroenteritis (RR = 2.34, 95% C.I.: 1.55-3.53).; a finding supported by the shape of the epidemic curve which referred to a common point source outbreak with secondary cases. For kindergarten, no statistically significant risk factor was identified, and the epidemic curve supported a person-to-person transmission according univariate analysis. Norovirus GI and GII and human Adenovirus were detected by Real Time PCR in stool samples from seven children of elementary school, but stool samples were not collected by children of the kindergarten. CONCLUSIONS: Even though the etiological agent of the outbreak was not verified, combined epidemiological and laboratory results were in favor of a waterborne viral gastroenteritis outbreak at the elementary school, followed by a person to person spread at the kindergarten

ASIC1a affects hypothalamic signaling and regulates the daily rhythm of body temperature in mice

The body temperature of mice is higher at night than during the day. We show here that global deletion of acid-sensing ion channel 1a (ASIC1a) results in lower body temperature during a part of the night. ASICs are pH sensors that modulate neuronal activity. The deletion of ASIC1a decreased the voluntary activity at night of mice that had access to a running wheel but did not affect their spontaneous activity. Daily rhythms of thyrotropin-releasing hormone mRNA in the hypothalamus and of thyroid-stimulating hormone β mRNA in the pituitary, and of prolactin mRNA in the hypothalamus and pituitary were suppressed in ASIC1a-/- mice. The serum thyroid hormone levels were however not significantly changed by ASIC1a deletion. Our findings indicate that ASIC1a regulates activity and signaling in the hypothalamus and pituitary. This likely leads to the observed changes in body temperature by affecting the metabolism or energy expenditure

NFE2-Related transcription factor 2 coordinates antioxidant defense with thyroglobulin production and iodination in the thyroid gland

Background: The thyroid gland has a special relationship with oxidative stress. While generation of oxidative substances is part of normal iodide metabolism during thyroid hormone synthesis, the gland must also defend itself against excessive oxidation in order to maintain normal function. Antioxidant and detoxification enzymes aid thyroid cells to maintain homeostasis by ameliorating oxidative insults, including during exposure to excess iodide, but the factors that coordinate their expression with the cellular redox status are not known. The antioxidant response system comprising the ubiquitously expressed NFE2-related transcription factor 2 (Nrf2) and its redox-sensitive cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) defends tissues against oxidative stress, thereby protecting against pathologies that relate to DNA, protein, and/or lipid oxidative damage. Thus, it was hypothesized that Nrf2 should also have important roles in maintaining thyroid homeostasis. Methods: Ubiquitous and thyroid-specific male C57BL6J Nrf2 knockout (Nrf2-KO) mice were studied. Plasma and thyroids were harvested for evaluation of thyroid function tests by radioimmunoassays and of gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Nrf2-KO and Keap1-KO clones of the PCCL3 rat thyroid follicular cell line were generated using CRISPR/Cas9 technology and were used for gene and protein expression studies. Software-predicted Nrf2 binding sites on the thyroglobulin enhancer were validated by site-directed in vitro mutagenesis and chromatin immunoprecipitation. Results: The study shows that Nrf2 mediates antioxidant transcriptional responses in thyroid cells and protects the thyroid from oxidation induced by iodide overload. Surprisingly, it was also found that Nrf2 has a dramatic impact on both the basal abundance and the thyrotropin-inducible intrathyroidal abundance of thyroglobulin (Tg), the precursor protein of thyroid hormones. This effect is mediated by cell-autonomous regulation of Tg gene expression by Nrf2 via its direct binding to two evolutionarily conserved antioxidant response elements in an upstream enhancer. Yet, despite upregulating Tg levels, Nrf2 limits Tg iodination both under basal conditions and in response to excess iodide. Conclusions: Nrf2 exerts pleiotropic roles in the thyroid gland to couple cell stress defense mechanisms to iodide metabolism and the thyroid hormone synthesis machinery, both under basal conditions and in response to excess iodide.Fil: Ziros, Panos G. Lausanne University; SuizaFil: Habeos, Ioannis. Patras University; GreciaFil: Chartoumpekis, Dionysios V. University of Pittsburgh; Estados UnidosFil: Ntalampyra, Eleni. Universite de Lausanne; SuizaFil: Somm, Emmanuel. Universite de Lausanne; SuizaFil: Renaud, Cédric O.. Universite de Lausanne; SuizaFil: Bongiovanni, Massimo. Institute Of Pathology Locarno; SuizaFil: Trougakos, Ioannis P. Universidad Nacional y Kapodistríaca de Atenas; GreciaFil: Yamamoto, Masayuki. University Of Tohoku; JapónFil: Kensler, Thomas W.. University of Pittsburgh at Johnstown; Estados UnidosFil: Santisteban, Pilar. Universidad Autónoma de Madrid; EspañaFil: Carrasco, Nancy. University of Yale. School of Medicine; Estados UnidosFil: Ris Stalpers, Carrie. Academic Medical Center; Países BajosFil: Amendola, Elena. Universidad de Nápoles; ItaliaFil: Liao, Xiao-Hui. University of Chicago; Estados UnidosFil: Rossich, Luciano Esteban. Comisión Nacional de Energía Atómica de Argentina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Thomasz, Lisa. Comisión Nacional de Energía Atómica de Argentina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Juvenal, Guillermo Juan. Comisión Nacional de Energía Atómica de Argentina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Refetoff, Samuel. University of Chicago; Estados UnidosFil: Sykiotis, Gerasimos P.. Universite de Lausanne; Suiz

Environmental Surveillance. An Additional/Alternative Approach for Virological Surveillance in Greece?

The detection of viruses in the sewage of an urban city by nucleic acid amplification techniques allows the identification of the viral strains that are circulating in the community. The aim of the study was the application of such detection which gives useful data on the distribution, spread, and frequency of these viruses, supporting epidemiological studies of the related viral infections. A two year (2007–2009) survey was conducted in order to evaluate the presence of human adenoviruses (hAdV), hepatitis A viruses (HAV), hepatitis E viruses (HEV), noroviruses (NoV), and human polyomaviruses (hPyV) in sewage samples collected from the inlet of a municipal biological wastewater treatment plant located in southwestern Greece. PCR methods were used for this survey. In total, viruses have been detected in 87.5% (42/48) of the analyzed sewage samples. Analytically, DNA viruses, hAdVs and hPyVs have been detected in 45.8% (22/48) and 68.8% (33/48) of the samples, respectively. As it concerns RNA viruses, HAV was detected in 8.3% (4/48), NoVs in 6.3% (3/48), while HEV has not been detected at all. After sequencing, AdVs were typed as Ad8, Ad40 and Ad41, while both JC and BK hPyVs have been recognized. All NoVs have been identified as GII4, while HAV was typed as genotype IA. Similar long-term studies could be undertaken in countries such as Greece in order to offer a valuable and complementary tool to current problematic epidemiological surveillance systems. This study demonstrates the advantages of environmental surveillance as a tool to determine the epidemiology of viruses circulating in a given community. To our knowledge this was the first of its kind study performed in Greece in order to establish this new way of surveillance

The Transcriptomic Response of the Murine Thyroid Gland to Iodide Overload and the Role of the Nrf2 Antioxidant System

Background: Thyroid follicular cells have physiologically high levels of reactive oxygen species because oxidation of iodide is essential for the iodination of thyroglobulin (Tg) during thyroid hormone synthesis. Thyroid follicles (the functional units of the thyroid) also utilize incompletely understood autoregulatory mechanisms to defend against exposure to excess iodide. To date, no transcriptomic studies have investigated these phenomena in vivo. Nuclear erythroid factor 2 like 2 (Nrf2 or Nfe2l2) is a transcription factor that regulates the expression of numerous antioxidant and other cytoprotective genes. We showed previously that the Nrf2 pathway regulates the antioxidant defense of follicular cells, as well as Tg transcription and Tg iodination. We, thus, hypothesized that Nrf2 might be involved in the transcriptional response to iodide overload. Methods: C57BL6/J wild-type (WT) or Nrf2 knockout (KO) male mice were administered regular water or water supplemented with 0.05% sodium iodide for seven days. RNA from their thyroids was prepared for next-generation RNA sequencing (RNA-Seq). Gene expression changes were assessed and pathway analyses were performed on the sets of differentially expressed genes. Results: Analysis of differentially expressed messenger RNAs (mRNAs) indicated that iodide overload upregulates inflammatory-, immune-, fibrosis- and oxidative stress-related pathways, including the Nrf2 pathway. Nrf2 KO mice showed a more pronounced inflammatory–autoimmune transcriptional response to iodide than WT mice. Compared to previously published datasets, the response patterns observed in WT mice had strong similarities with the patterns typical of Graves’ disease and papillary thyroid carcinoma (PTC). Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) also responded to iodide overload, with the latter targeting mRNAs that participate mainly in inflammation pathways. Conclusions: Iodide overload induces the Nrf2 cytoprotective response and upregulates inflammatory, immune, and fibrosis pathways similar to autoimmune hyperthyroidism (Graves’ disease) and PTC

Differential Expression of MicroRNAs in Adipose Tissue after Long-Term High-Fat Diet-Induced Obesity in Mice

Obesity is a major health concern worldwide which is associated with increased risk of chronic diseases such as metabolic syndrome, cardiovascular disease and cancer. The elucidation of the molecular mechanisms involved in adipogenesis and obesogenesis is of essential importance as it could lead to the identification of novel biomarkers and therapeutic targets for the development of anti-obesity drugs. MicroRNAs (miRNAs) have been shown to play regulatory roles in several biological processes. They have become a growing research field and consist of promising pharmaceutical targets in various fields such as cancer, metabolism, etc. The present study investigated the possible implication of miRNAs in adipose tissue during the development of obesity using as a model the C57BLJ6 mice fed a high-fat diet