The extracts and pure major constituents of Chios mastic gum (resin of Pistacia lentiscus var. chia) were tested for their activities against Helicobacter pylori. A total mastic extract without polymer (TMEWP) was prepared after removal of the contained insoluble polymer in order to ameliorate solubility and enhance in vivo activity. Administration of TMEWP to H. pylori SS1-infected mice over the period of 3 months with an average dose of 0.75 mg/day led to an approximately 30-fold reduction in the H. pylori colonization (1.5 log CFU/g of tissue). However, no attenuation in the H. pylori-associated chronic inflammatory infiltration and the activity of chronic gastritis was observed. To further characterize potential active mastic constituents, the TMEWP was separated into an acidic and a neutral fraction. Both were extensively characterized by nuclear magnetic resonance and mass spectroscopy to elucidate the structure of the components contained within each fraction. After chromatographic separation, the acid fraction gave the major triterpenic acids, while the neutral fraction gave several triterpenic alcohols and aldehydes. Mastic extracts and isolated pure triterpenic acids were tested for in vitro activity against a panel of 11 H. pylori clinical strains. The acid fraction was found to be the most active extract (minimum bactericidal concentration [MBC], 0.139 mg/ml), and the most active pure compound was isomasticadienolic acid (MBC, 0.202 mg/ml [0.443 mM]). Our results show that administration of TMEWP may be effective in reducing H. pylori colonization and that the major triterpenic acids in the acid extract may be responsible for such an activity.

Cytotoxin-associated gene A (CagA) diversity with regard to EPIYA-A, -B, -C, or -D phosphorylation motifs may play an important role in Helicobacter pylori pathogenesis, and therefore determination of these motifs in H. pylori clinical isolates can become a useful prognostic tool. We propose a strategy for the accurate determination of CagA EPIYA motifs in clinical strains, based upon one-step PCR amplification using primers that flank the EPIYA coding region. We thus analyzed 135 H. pylori isolates derived from 75 adults and 60 children Greek patients. A total of 34 cases were found to be EPIYA PCR negative and were consequently verified as cagA negative by cagA-specific PCR, empty-site cagA PCR, and Western blotting. Sequencing of the remaining 101 PCR-positive amplicons confirmed that an accurate prediction of the number of EPIYA motifs on the basis of size distribution of the PCR products was feasible in all cases. Furthermore, our assay could identify closely related H. pylori subclones within the same patient, harboring different numbers of EPIYA repeats. The prevalence of CagA proteins with three EPIYA motifs (ABC) or four EPIYA motifs (ABCC) was the same within the adult and children groups. However, CagA species with more than four EPIYA motifs were observed exclusively within adults (8.6%), suggesting that CagA-positive strains may acquire additional EPIYA-C motifs throughout adulthood. Our strategy requires no initial cagA screening of the clinical isolates and can accurately predict the number of EPIYA repeats in single or multiple closely related subclones bearing different numbers of EPIYA motifs in their CagA, which may coexist within the same patient.

; Applied Molecular Virology, Institut Pasteur Korea, Seongnam-si, South Korea g Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infectionbased assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-␣) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCVhost cell interaction. H epatitis C virus (HCV) infection causes a wide range of clinical manifestations, from a healthy carrier state to acute and chronic hepatitis that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Nearly 3% of the world's population is chronically infected with HCV (1, 2), and current therapeutic approaches are not broadly effective (3).HCV is a positive-strand RNA virus with a 9.6-kb genome that is flanked at both termini by conserved, nontranslated regions (NTRs), required for RNA translation and replication. The 5= NTR comprises an internal ribosome entry site (IRES) that directs the expression of a polyprotein precursor (4, 5). The polyprotein is cleaved into structural (core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins that, in association with cellular factors, form a membrane-associated replicase complex. This copies the viral positive-strand RNA into a negative-strand intermediate that serves as the template for the synthesis of progeny genomes. The alternative reading frame (ARFP) or coreϩ1 and minicore proteins, with as-yet-unknown functions, appear to be synthesized from the core region by alternative translation mechanisms (6, 7).Studies of the...

There was an increase in severe and fatal influenza cases in Greece during the 2011-2015 post-pandemic period. To investigate causality, we determined neuraminidase (NA) inhibitor susceptibility and resistance-conferring NA and hemagglutinin (HA) mutations in circulating influenza type A viruses during the pandemic (2009-2010) and post-pandemic periods in Greece. One hundred thirty-four influenza A(H1N1)pdm09 and 95 influenza A(H3N2) viruses submitted to the National Influenza Reference Laboratory of Southern Greece were tested for susceptibility to oseltamivir and zanamivir. Antiviral resistance was assessed by neuraminidase sequence analysis, as well as the fluorescence-based 50 % inhibitory concentration (IC) method. Five influenza A(H1N1)pdm09 viruses (2.2 %) showed significantly reduced inhibition by oseltamivir (average IC 300.60nM vs. 1.19nM) by Gaussian kernel density plot analysis. These viruses were isolated from immunocompromised patients and harbored the H275Y oseltamivir resistance-conferring NA substitution. All A(H1N1)pdm09 viruses were zanamivir-susceptible, and all A(H3N2) viruses were susceptible to both drugs. Oseltamivir-resistant viruses did not form a distinct cluster by phylogenetic analysis. Permissive mutations were detected in immunogenic and non immunogenic NA regions of both oseltamivir- resistant and susceptible viruses in the post-pandemic seasons. Several amino acid substitutions in the HA1 domain of the HA gene of post-pandemic viruses were identified. This study indicated low resistance to NAIs among tested influenza viruses. Antiviral resistance emerged only in immunocompromised patients under long-term oseltamivir treatment. Sequential sample testing in this vulnerable group of patients is recommended to characterise resistance or reinfection and viral evolution.

Viruses are the major cause of pediatric respiratory tract infection and yet many suspected cases of illness remain uncharacterized. This study aimed to determine the distribution of several respiratory viruses in children diagnosed as having influenza-like illness, over the winter period of 2005-2008. Molecular assays including conventional and real time PCR protocols, were employed to screen respiratory specimens, collected by clinicians of the Influenza sentinel system and of outpatient pediatric clinics, for identification of several respiratory viruses. Of 1,272 specimens tested, 814 (64%) were positive for at least one virus and included 387 influenza viruses, 160 rhinoviruses, 155 respiratory syncytial viruses, 95 adenoviruses, 81 bocaviruses, 47 parainfluenza viruses, 44 metapneumoviruses, and 30 coronaviruses. Simultaneous presence of two or three viruses was observed in 173 of the above positive cases, 21% of which included influenza virus and rhinovirus. The majority of positive cases occurred during January and February. Influenza virus predominated in children older than 1 year old, with type B being the dominant type for the first season and subtypes A/H3N2 and A/H1N1 the following two winter seasons, respectively. Respiratory syncytial virus prevailed in children younger than 2 years old, with subtypes A and B alternating from year to year. This is the most comprehensive study of the epidemiology of respiratory viruses in Greece, indicating influenza, rhinovirus and respiratory syncytial virus as major contributors to influenza-like illness in children.