Molecular evolution of GII-4 Norovirus strains.

BACKGROUND: Human Noroviruses (NoV) are the major cause of acute nonbacterial gastroenteritis and the leading cause of outbreaks of gastroenteritis worldwide. Genotype II-4 (GII-4) NoV has been shown to spread rapidly and is the most commonly detected strain worldwide, particularly in association with outbreaks. Previously, we have shown that circulating GII-4 NoV strains exist as populations of selectively neutral variants, and that the emergence of epidemic GII-4 NoV strains correlated with mutations in at least two key sites (Sites A and B) within the P2 domain of the surface exposed major capsid protein (VP1). METHODOLOGY: We developed a rapid pyrosequencing method for screening of the two Sites A and B and a homology based modelling system was used to predict the effects of amino acid substitutions at these sites on the antigenic properties of the virus (defined as surface motif types). PRINCIPLE FINDING/CONCLUSION: Here, we describe the characterisation of amino acid diversity at Sites A and B for 1062 GII-4 NoV strains from clinical specimen associated with outbreak of gastroenteritis (2000-2011) and 250 GII-4 NoV sequences from Genbank. Our data identified a high diversity of different Site A and B site combinations at amino acid level and amino acid diversity was higher at Site B than Site A. Site A motifs could be grouped into 3 clusters based on similar surface motif types. We predict that Site A is a major epitope on the virus surface, responsible for defining the antigenic profile, and a more subtle role for Site B, maintaining minor antigenic variation within the virus population

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >= 2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.Peer reviewe

Recommendations for enterovirus diagnostics and characterisation within and beyond Europe.

Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5'noncoding regions (5'NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5'NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden

Identification and characterization of genes associated with oral Candida albicans infections

Titel und Inhaltsverzeichnis 1 EINLEITUNG 2 MATERIAL UND METHODEN 3 ERGEBNISSE 4 DISKUSSION 5 ZUSAMMENFASSUNG 6 SUMMARY 7 LITERATURVERZEICHNISDer fakultativ humanpathogene Pilz C. albicans besiedelt als harmloser Kommensale Haut und Schleimhäute des Menschen. Als Pathogen spielt C. albicans als Erreger von oralen Candidosen vor allem bei immunsupprimierten Patienten eine wichtige Rolle. Kommt es zu Veränderungen der lokalen Mikroflora oder zu Abschwächungen des Immunsystems, wie es z.B. bei HIV+ Patienten der Fall ist, kann es zu oralen Infektionen durch C. albicans kommen. Diese manifestieren sich durch eine massenhafte Vermehrung der Pilzzellen sowie der Invasion in oberflächennahe Bereiche des Epithelgewebes. Die zellulären und molekularen Mechanismen der Interaktionen von C. albicans während der Kolonisierung oder der Infektion des oralen Epithelgewebes sind dabei noch weitgehend unklar. Um die komplexen Vorgänge der Wirt-Pathogen Interaktionen während der Infektion zu untersuchen, wurde eine experimentelle Infektion durchgeführt und analysiert. Dazu wurde ein in vitro generiertes orales Epithelgewebe (RHE) mit C. albicans infiziert und zu verschiedenen Zeitpunkten mikroskopisch analysiert. Basierend auf der mikroskopischen Analyse wurde die experimentelle Infektion in drei Phasen unterteilt: eine frühe Adhäsionsphase (1 h), eine mittlere, invasive Phase (3-6 h) und eine späte Phase (12-24 h), welche durch eine starke Schädigung des epithelialen Gewebes gekennzeichnet war. Die frühe Phase der Interaktion war charakterisiert durch eine unmittelbare Induktion der Hyphenbildung sowie einer starken Adhäsion der Pilzzellen an das epitheliale Gewebe. Analysen der elektronenmikroskopischen Aufnahmen (REM, TEM) zeigten weiterhin, dass die Invasion der C. albicans Hyphenzellen in das Gewebe dabei sowohl über passive Mechanismen (induzierte Endozytose) als auch über aktive Mechanismen (aktive Penetration) stattfand. Die späte Phase der experimentellen Infektion war charakterisiert durch ein dichtes Myzelgeflecht, welches sich invasiv im Gewebe ausbreitete und zu einer starken Schädigung des Gewebes führte. Basierend auf den mikroskopischen Untersuchungen erfolgte weiterführend die molekulare Analyse der Interaktionen. Dazu wurden das orale Epithelgewebe über verschiedene Zeitpunkte (1, 3, 6, 12 und 24 h) mit C. albicans infiziert und die Proben mit C. albicans spezifischen Mikroarrays hybridisiert. Um die Relevanz der Daten aus den experimentellen Infektionen zu überprüfen, wurden Mikroarray-Experimente mit in vivo isolierten Patientenproben durchgeführt und die erstellten Transkriptionsprofile mit den Transkriptionsprofilen der in vitro Infektionen verglichen. Trotzdem die Transkriptionsprofile der elf Patientenproben eine unerwartet starke Heterogenität aufwiesen, konnte eine Reihe von Genen identifiziert werden, welche sowohl in den in vitro als auch den in vivo Proben ähnlich reguliert waren. In dieser Gruppe waren Gene, welche mit dem Hefe/Myzel Übergang, einer adaptiven Anpassung an ein neutrales pH-Milieu, mit der Verwertung verschiedener Kohlenstoff-Quellen sowie der Bekämpfung von nitrosativem Stress assoziiert waren. Ein weiteres Ziel der Analyse der in vivo und in vitro erstellten Transkriptionsprofile war es, Gene zu identifizieren, welche möglicherweise mit der Virulenz von C. albicans assoziiert sind. Dazu wurden acht Gene ausgewählt, welche stark und spezifisch während der frühen oder der späten Phase der experimentellen Infektion sowie in den Patientenproben exprimierte waren. Diese wurden in C. albicans ausgeschaltet und die erstellten Mutantenstämme weiterführend analysiert. Bei der Analyse der Interaktion der erstellten Mutanten mit dem RHE-Modell waren vier der Mutanten leicht bis stark attenuiert in den Fähigkeiten, Gewebeschädigungen zu induzieren. Eine Mutante, in welcher das Gen EED1 ausgeschaltet worden war, zeigte einen nahezu avirulenten Phänotyp. Das Gen EED1, welches sowohl in den Patientenproben als auch während der experimentellen Infektion eine starke Expression gezeigt hatte, stellte sich weiterhin als essentiell für die Hyphenbildung in C. albicans heraus. Delta-eed1 Mutanten waren gekennzeichnet durch eine lediglich transiente Hyphenbildung und einem daraus resultierenden, auf oberflächliche Gewebebereiche begrenzten, invasiven Wachstum. Die weiterführende Proliferation der Delta-eed1 Mutante fand in den Epithelzellen und -gewebe dabei nur noch in der Hefeform statt, so dass keine invasive Ausbreitung mehr im Gewebe stattfinden konnte. Die in dieser Arbeit gewonnen Ergebnisse zeigen, dass die Hyphenmorphologie sowie die adaptive Interaktion der C. albicans Zellen mit dem Epithelgewebe eine wichtige Rolle bei der Initiation und der Etablierung einer oralen Infektion spielen.Candida albicans is normally a harmless commensal fungus of mucosal surfaces in healthy individuals but can cause several types of infections in predisposed patients, ranging from superficial to life-threatening disease. As the most common oral fungal pathogen of humans, C. albicans frequently causes oral infections in human immunodeficiency virus (HIV)-infected patients. During oral infections, fungal cells invade the oral mucosa and persist within the epithelium causing superficial lesions. However, the mechanisms by which C. albicans invades and persists within mucosal epithelium are not clear. To understand oral pathogenesis, cellular and molecular mechanisms of epithelial- fungus interactions were characterized using a model based on reconstituted human oral epithelial tissue (RHE). Based on the microscopical analysis of histological sections the experimental infection process was dissected into three different phases: an early attachment phase (1 h), a mid-invasion phase (3-6 h) and a late destruction phase (12 24 h). The early attachment phase was characterized by immediate hyphal formation and a strong adhesion of the fungal cells to the epithelial tissue. Furthermore, electron microscopy (SEM, TEM) revealed that hyphal formation facilitates epithelial invasion via both active (physical penetration) and passive (induced endocytosis) processes. The late phase was characterized by a dense network of hyphal cells invading and disseminating within the tissue. Only the late phase was correlated with strong tissue damage, reflected by the release of an epithelial marker enzyme (LDH). Based on these observations genome-wide transcript profiling of C. albicans was performed at five time-points (1, 3, 6, 12 and 24 h) aiming to identify phase-specific genes. To show that the data obtained from the in vitro infection model reflect the in vivo situation, transcriptional profiling of C. albicans cells isolated from patients suffering from oral Candida infections was also analyzed. Although the transcriptional profiles of the 11 patient samples showed an unexpected high heterogeneity, a set of genes was identified which were similarly regulated under in vitro and in vivo conditions. The expression profiles reflected the morphological switch and an adaptive response to neutral pH, non-glucose carbon sources and nitrosative stress. To identify genes, potentially associated with fungal virulence, eight genes were targeted to create knock-out mutants. Thereby only genes whose expression was phase-specific and/or expressed in both patients and the RHE model were chosen. All eight mutants were analyzed in the RHE model and under a number of in vitro growth conditions. Four of the mutants had moderate or strongly reduced abilities to damage epithelial tissue. One gene, up-regulated in both RHE infection and patients, named EED1, was essential for maintenance of hyphal elongation. Mutants lacking EED1 showed transient cell elongation on epithelial tissue, which enabled only superficial invasion of epithelial cells. Once inside an epithelial cell, delta-eed1 cells could proliferate as yeasts or pseudohyphae but remained trapped intracellular. These results suggest that the adaptive response and morphology of C. albicans play specific roles for host-fungal interactions during mucosal infections

Bistable Expression of CsgD in Biofilm Development of Salmonella enterica Serovar Typhimurium▿ †

Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms

Increased Expression of Toll-Like Receptors 4, 5, and 9 in Small Bowel Mucosa from Patients with Irritable Bowel Syndrome

The aim of our study was to compare patients with irritable bowel syndrome (IBS) and healthy controls regarding the expression of toll-like receptors 2, 4, 5, and 9 (TLR2, TLR4, TLR5, and TLR9), the primary mucosal receptors of bacterial components, in small and large bowel mucosa. Methods. We analysed biopsies from jejunum and sigmoid colon of 22 patients (17 females) with IBS aged 18–66 (median: 39) years and 14 healthy volunteers (12 females) aged 22–61 (median: 42) years. Eight patients had constipation-predominant IBS (C-IBS), 7 had diarrhoea-predominant IBS (D-IBS), and 7 had IBS without predominance of constipation or diarrhoea. We analysed mRNA levels for TLRs using quantitative PCR and distribution of TLRs in mucosa using immunohistochemistry. Results. We found increased mRNA expression of TLR4 (mean fold change 1.85±0.31 versus 1.0±0.20; p<0.05), TLR5 (1.96±0.36 versus 1.0±0.20; p<0.05) and TLR9 (2.00±0.24 versus 1.0±0.25; p<0.01) but not of TLR2 in the small bowel mucosa from patients with IBS compared to the controls. There was no significant difference in mRNA levels for TLRs in colon mucosa between patients and controls. Conclusion. Upregulation of TLR4, TLR5, and TLR9 suggests the involvement of bacteria or dysregulation of the immune response to commensal flora in small bowel mucosa in IBS patients

A Role for the EAL-Like Protein STM1344 in Regulation of CsgD Expression and Motility in Salmonella enterica Serovar Typhimurium▿

The bacterial second messenger cyclic di-GMP (c-di-GMP) regulates the transition between sessility and motility. In Salmonella enterica serovar Typhimurium, the expression of CsgD, the regulator of multicellular rdar morphotype behavior, is a major target of c-di-GMP signaling. CsgD expression is positively regulated by at least two diguanylate cyclases, GGDEF domain proteins, and negatively regulated by at least four phosphodiesterases, EAL domain proteins. Here, we show that in contrast to EAL domain proteins acting as phosphodiesterases, the EAL-like protein STM1344 regulated CsgD expression positively and motility negatively. STM1344, however, did not have a role in c-di-GMP turnover and also did not bind the nucleotide. STM1344 acted upstream of the phosphodiesterases STM1703 and STM3611, previously identified to participate in CsgD downregulation, where it repressed their expression. Consequently, although STM1344 has not retained a direct role in c-di-GMP metabolism, it still participates in the regulation of c-di-GMP turnover and has a role in the transition between sessility and motility

Frequencies of detection of Site B motifs 2000–2011.

<p>A single motif [STT] (green) was the most frequently detected throughout the analysis period. Between 2008 and 2009 the amount of diversity at Site B increased, with 15 and 13 different motifs detected, respectively. Amino acid motifs designated by standard IUPAC single-letter amino acid code.</p

Higher-than-average NoV activity correlates with shift in detected Site A surface motif type and increased amino acid diversity at Site B.

<p>(A) Frequency of detected surface motif types (SMT) of Site A, indicated in red (SMT1), green (SMT2) and blue (SMT3) (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041625#pone-0041625-g003" target="_blank">Figure 3</a>) changes over the time period analysed. (B) Frequency of SMT1 NoV strains with Site B motifs N-N (pink), NGT (purple), STT (red), and all other Site B amino acid motifs detected (dashed black). (C) Frequency of SMT2 NoV strains with Site B motif STT (green), and all other Site B amino acid motifs detected (dashed black). (D) Frequency of SMT3 NoV strains with Site B motif STT (blue), and all other Site B amino acid motifs detected (dashed black). Shaded columns highlight periods of high NoV activity in 2001/2002 (red), 2005/2006 (green) and 2009/2010 (blue). (E) Periods of high NoV are defined by the epidemiological trends observed over time. The data shown here presents epidemiological data (number of NoV-positive laboratory reports) for the period from 2007 to 2011 for England & Wales. Prior to 2007, the data is not comparable due to differences in reporting rates, and so is not shown. A clear increase in NoV-positive laboratory reports is seen in 2009/2010, indicating an epidemiologically-defined period of high NoV activity. Amino acid motifs designated by standard IUPAC single-letter amino acid code.</p