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Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in‐house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV‐A71, echovirus 30, coxsackie A virus 21, and EV‐D68), HPeV3, and specificity controls. Reported results from 48 in‐house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In‐house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10−5). EV‐specific PCRs showed low cross‐reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in‐house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point‐of‐care tests. © 2019 The Authors. Journal of Medical Virology published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Summary Background Enteroviruses can cause severe infections, especially in young children. Non-polio enterovirus infections are not notifiable in most countries in the EU and European Economic Area (EEA) region, and surveillance varies substantially between countries. We collected and analysed available enterovirus data across EU and EEA countries to assess the current epidemiological situation and need for standardising surveillance. Methods Aggregated data on any enterovirus detected between Jan 1, 2015, and Dec 31, 2017, through national enterovirus reference laboratories were requested from representatives in all 31 EU and EEA countries. Information collected included enterovirus types detected by month, patient age group, symptom, and specimen type. We also collected sequence data on viral capsid sequences for the three most clinically relevant enterovirus types, as identified from the data. Findings Aggregated data were provided by representatives from 24 (77%) of 31 countries. 9914 (66%) of 14 999 enterovirus infections with information about age were in children younger than 5 years, and 3197 (45%) of 7139 individuals for whom symptoms were reported had neurological symptoms. Other symptoms were non-specific fever (in 1607 [23%] patients), respiratory symptoms (in 1197 [17%] patients), hand, foot, and mouth disease (in 528 [7% patients), and myocarditis (in 39 [1%] patients). 68 deaths were temporally associated with enterovirus infection. Typing for 11 559 (67%) of 17 136 specimens revealed 66 enterovirus types. Coxsackievirus A6 was the most frequently detected enterovirus type (in 1556 [13%] of 11 559 typed enteroviruses), and 292 (65%) of 448 patients with coxsackievirus A6 infection with available clinical data presented with hand, foot, and mouth disease. Echovirus 30 was the second most frequently detected enterovirus type, representing 1412 (12%) of 11 559 typed enteroviruses, and 384 (82%) of 467 individuals with echovirus 30 infection with available clinical data had neurological symptoms. Sequences available from 18 countries showed circulation of newly emerging strains of enterovirus A71 and enterovirus D68. Interpretation To our knowledge, this study is the largest investigation of enterovirus circulation in EU and EEA countries and confirms the availability of non-polio enterovirus data in the region. Our study highlights the wide circulation of non-polio enteroviruses in Europe, mostly affecting young children and leading to neurological symptoms. Collecting data on morbidity and mortality related to enterovirus infections, as well as harmonising case definition for surveillance, should be encouraged. Funding None.

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 change1.85±0.31versus1.0±0.20;p<0.05), TLR5 (1.96±0.36versus1.0±0.20;p<0.05) and TLR9 (2.00±0.24versus1.0±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.

Background Tajikistan had a major diphtheria outbreak (∼10,000 cases) in the 1990s, which was controlled after nationwide immunization campaigns with diphtheria-tetanus toxoid in 1995 and 1996. Since 2000, only 52 diphtheria cases have been reported. However, in coverage surveys conducted in 2000 and 2005, diphtheria-tetanus-pertussis vaccine coverage was lower than administratively reported estimates raising concerns about potential immunity gaps. To further assess population immunity to diphtheria in Tajikistan, diphtheria antibody testing was included in a large-scale nationwide serosurvey for vaccine-preventable diseases conducted in connection with a poliomyelitis outbreak in 2010. In addition, the serosurvey provided an opportunity to assess population immunity to tetanus. Methods Residents of all regions of Tajikistan aged 1–24 years were included in the serosurvey implemented during September–October 2010. Participants were selected through stratified cluster sampling. Specimens were tested for diphtheria antibodies using a Vero cell neutralization assay and for tetanus antibodies using an anti-tetanus IgG ELISA. Antibody concentrations ≥0.1 IU/mL were considered seropositive. Results Overall, 51.4% (95% CI, 47.1%–55.6%) of participants were seropositive for diphtheria and 78.9% (95% CI, 74.7%–82.5%) were seropositive for tetanus. The lowest percentages of seropositivity for both diseases were observed among persons aged 10–19 years: diphtheria seropositivity was 37.1% (95% CI, 31.0%–43.7%) among 10–14 year-olds, and 35.3% (95% CI, 29.9%–41.1%) among 15–19 year-olds; tetanus seropositivity in respective age groups was 65.3% (95% CI, 58.4%–71.6%) and 70.1% (95% CI, 64.5%–75.2%). Conclusions Population immunity for diphtheria in Tajikistan is low, particularly among 10–19 year-olds. Population immunity to tetanus is generally higher than for diphtheria, but is suboptimal among 10–19 year-olds. These findings highlight the need to improve routine immunization service delivery, and support a one-time supplementary immunization campaign with diphtheria-tetanus toxoid among birth cohorts aged 1–19 years in 2010 (3–21 years in 2012) to close immunity gaps and prevent diphtheria outbreaks.

Summary The transcriptional regulator CsgD of Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major regulator of biofilm formation required for the expression of csgBA, which encodes curli fimbriae, and adrA, coding for a diguanylate cyclase. CsgD is a response regulator with an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix–turn–helix DNA binding motif, but the mechanisms of target gene activation remained unclear. To study the DNA-binding properties of CsgD we used electrophoretic mobility shift assays and DNase I footprint analysis to show that unphosphorylated CsgD-His6 binds specifically to the csgBA and adrA promoter regions. In vitro transcription analysis revealed that CsgD-His6 is crucial for the expression of csgBA and adrA. CsgD-His6 is phosphorylated by acetyl phosphate in vitro, which decreases its DNA-binding properties. The functional impact of D59 in vivo was demonstrated as S. Typhimurium strains expressing modified CsgD protein (D59E and D59N) were dramatically reduced in biofilm formation due to decreased protein stability and DNA-binding properties in the case of D59E. In summary, our findings suggest that the response regulator CsgD functions in its unphosphorylated form under the conditions of biofilm formation investigated in this study.

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.

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.

Basic research on the biology and immunology of microbial infection requires appropriate model systems. Currently most such studies involve animal studies which are a focus of ethical controversy. Possible alternatives, especially for localized infections, are provided by models using in vitro reconstituted human epithelium or epidermis (RHE). In recent years, these model systems have been successfully established to evaluate the effectiveness of topical anti-infectives, to characterize the role of fungal virulence factors, and to study the immune responses during localized Candida albicans infections. Most recently, these models have been supplemented with immune cells such as lymphocytes, polymorphonuclear leukocytes, mast cells or dendritic cells, to study their role during the course of infection and to characterize the interaction between the skin barrier and accessory immune cells. Although the most experience is with Candida albicans RHE infections, such model systems can also be used to study infections with other fungi or bacteria.

Summary Candida albicans is the most common oral fungal pathogen of humans, but the mechanisms by which C. albicans invades and persists within mucosal epi- thelium are not clear. To understand oral pathogen- esis, we characterized the cellular and molecular mechanisms of epithelial-fungus interactions using reconstituted human oral epithelium (RHE). We observed that hyphal formation facilitates epithelial invasion via both active (physical penetration) and passive (induced endocytosis) processes. Genome wide transcript profiling of C. albicans experimental RHE infection was compared with that from 11 patient samples with pseudomembranous candidiasis to identify genes associated with disease development in vivo. Expression profiles reflected the morphologi- cal switch and an adaptive response to neutral pH, non-glucose carbon sources and nitrosative stress. We identified several novel infection-associated genes with unknown function. One gene, upregulated in both RHE infection and patients, named EED1, was essential for maintenance of hyphal elonga- tion. Mutants lacking EED1 showed transient cell elongation on epithelial tissue, which enabled only superficial invasion of epithelial cells. Once in- side an epithelial cell, Deed1 cells could proliferate as yeasts or pseudohyphae but remained trapped intracellularly. Our results suggest that the adaptive response and morphology of C. albicans play specific roles for host-fungal interactions during mucosal infections.

INTRODUCTION It is increasingly difficult to differentiate measles viruses (MeVs) relating to certain outbreaks on the basis of the nucleoprotein (N) gene sequence only, as the diversity of circulating MeV strains has decreased. We studied genomic regions that could provide better molecular discrimination between epidemiologically linked and unlinked MeV variants identified in Sweden during 2013-2014. METHODS The hemagglutinin (H) gene and hypervariable region between the fusion and matrix genes (MF-HVR) from 53 MeV-positive samples were amplified and sequenced. Data on phylogenetic clustering of MeVs on the basis of N, H, and MF-HVR sequences were compared to epidemiological data. RESULTS MeVs were genotyped: 27 were B3, and 26 were D8. One genotype B3 cluster based on the N gene sequence contained epidemiologically unrelated viruses from 4 outbreaks, whereas analysis of H and MF-HVR sequences separated them into phylogenetic clusters consistent with the epidemiological data. Similarly, the single cluster of viruses with a genotype D8 N gene could be divided into the 5 outbreak groups on the basis of the phylogeny of MF-HVR sequences. CONCLUSIONS A detailed picture of MeV circulation with more-defined links between outbreaks was obtained by sequencing the H gene and MF-HVR. Further identification and better genetic characterization of MeVs internationally is essential in identifying sources and routes of MeV spread within and beyond Europe in the elimination end game.

Several studies have indicated that colonic microbiota may exhibit important differences between patients with irritable bowel syndrome (IBS) and healthy controls. Less is known about the microbiota of the small bowel. We used massive parallel sequencing to explore the composition of small bowel mucosa-associated microbiota in patients with IBS and healthy controls. We analysed capsule biopsies from the jejunum of 35 patients (26 females) with IBS aged 18-(36)-57 years and 16 healthy volunteers (11 females) aged 20-(32)-48 years. Sequences were analysed based on taxonomic classification. The phyla with the highest total abundance across all samples were: Firmicutes (43%), Proteobacteria (23%), Bacteroidetes (15%), Actinobacteria (9.3%) and Fusobacteria (7.0%). The most abundant genera were: Streptococcus (19%), Veillonella (13%), Prevotella (12%), Rothia (6.4%), Haemophilus (5.7%), Actinobacillus (5.5%), Escherichia (4.6%) and Fusobacterium (4.3%). We found no difference among major phyla or genera between patients with IBS and controls. We identified a cluster of samples in the small bowel microbiota dominated by Prevotella, which may represent a common enterotype of the upper small intestine. The remaining samples formed a gradient, dominated by Streptococcus at one end and Escherichia at the other.

This protocol describes the setup, maintenance and characteristics of models of epithelial Candida infections based on well-established three-dimensional organotypic tissues of human oral and vaginal mucosa. Infection experiments are highly reproducible and can be used for the direct analysis of pathogen–epithelial cell interactions. This allows detailed investigations of Candida albicans wild type or mutant strain interaction with epithelial tissue or the evaluation of the host immune response using histological, biochemical and molecular methods. As such, the models can be utilized as a tool to investigate cellular interactions or protein and gene expression that are not complicated by non-epithelial factors. To study the impact of innate immunity or the antifungal activity of natural and non-natural compounds, the mucosal infection models can be supplemented with immune cells, antimicrobial agents or probiotic bacteria. The model requires at least 3 days to be established and can be maintained thereafter for 2–4 days.

Streptococcus agalactiae is a major cause of bacterial pneumonia, sepsis, and meningitis in human neonates. During the course of infection, S. agalactiae adheres to a variety of epithelial cells but the underlying mechanisms are only poorly understood. The present report demonstrates the importance of the fibrinogen receptor FbsA for the streptococcal adherence and invasion of epithelial cells. Deletion of the fbsA gene in various S. agalactiae strains substantially reduced their binding of soluble fibrinogen and their adherence to and invasion of epithelial cells, indicating a role of FbsA in these different processes. The adherence and invasiveness of an fbsA deletion mutant were partially restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade epithelial cells, suggesting that FbsA is a streptococcal adhesin. Flow cytometry experiments revealed a dose-dependent binding of FbsA to the surface of epithelial cells. Furthermore, tissue culture experiments exhibited an intimate contact of FbsA-coated latex beads with the surfaces of human epithelial cells. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to epithelial cells but that FbsA does not mediate the bacterial invasion into host cells. Our results also indicate that fibrinogen-binding epitopes within FbsA are involved in the adherence of S. agalactiae to epithelial cells.

Group B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat-encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA -proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen-binding motif with the consensus sequence G-N/S/T-V-L-A/E/M/Q-R-R-X-K/R/W-A/D/E/N/Q-A/F/I/L/V/Y-X-X-K/R-X-X.

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.

Diphtheria, caused by toxigenic strains of Corynebacterium diphtheriae, is an ancient disease with high incidence and mortality that has always been characterized by epidemic waves of occurrence. Whilst towards the beginning of the 1980s, many European countries were progressing towards the elimination of diphtheria, an epidemic re-emergence of diphtheria in the Russian Federation and the Newly Independent States of the former Soviet Union demonstrated a continuous threat of the disease into the 1990s. At present, the epidemic is under control and only sporadic cases are observed in Europe. However, the circulation of toxigenic strains is still observed in all parts of the world, posing a constant threat to the population with low levels of seroprotection. More recently, Corynebacterium ulcerans has been increasingly isolated as emerging zoonotic agent of diphtheria from companion animals such as cats or dogs, indicating the enduring threat of this thought-to-be controlled disease.

The transcriptional regulator CsgD of Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major regulator of biofilm formation required for the expression of csgBA, which encodes curli fimbriae, and adrA, coding for a diguanylate cyclase. CsgD is a response regulator with an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif, but the mechanisms of target gene activation remained unclear. To study the DNA-binding properties of CsgD we used electrophoretic mobility shift assays and DNase I footprint analysis to show that unphosphorylated CsgD-His(6) binds specifically to the csgBA and adrA promoter regions. In vitro transcription analysis revealed that CsgD-His(6) is crucial for the expression of csgBA and adrA. CsgD-His(6) is phosphorylated by acetyl phosphate in vitro, which decreases its DNA-binding properties. The functional impact of D59 in vivo was demonstrated as S. Typhimurium strains expressing modified CsgD protein (D59E and D59N) were dramatically reduced in biofilm formation due to decreased protein stability and DNA-binding properties in the case of D59E. In summary, our findings suggest that the response regulator CsgD functions in its unphosphorylated form under the conditions of biofilm formation investigated in this study.

The human pathogenic yeast Candida albicans can cause an unusually broad range of infections reflecting a remarkable potential to adapt to various microniches within the human host. The exceptional adaptability of C. albicans is mediated by rapid alterations in gene expression in response to various environmental stimuli and this transcriptional flexibility can be monitored with tools such as microarrays. Using such technology it is possible to (1) capture a genome-wide portrait of the transcriptome that mirrors the environmental conditions, (2) identify known genes, signalling pathways and transcription factors involved in pathogenesis, (3) identify new patterns of gene expression and (4) identify previously uncharacterized genes that may be associated with infection. In this review, we describe the molecular dissection of three distinct stages of infections, covering both superficial and invasive disease, using in vitro, ex vivo and in vivo infection models and microarrays.

Candida albicans is the most common oral fungal pathogen of humans, but the mechanisms by which C. albicans invades and persists within mucosal epithelium are not clear. To understand oral pathogenesis, we characterized the cellular and molecular mechanisms of epithelial-fungus interactions using reconstituted human oral epithelium (RHE). We observed that hyphal formation facilitates epithelial invasion via both active (physical penetration) and passive (induced endocytosis) processes. Genome wide transcript profiling of C. albicans experimental RHE infection was compared with that from 11 patient samples with pseudomembranous candidiasis to identify genes associated with disease development in vivo. Expression profiles reflected the morphological switch and an adaptive response to neutral pH, non-glucose carbon sources and nitrosative stress. We identified several novel infection-associated genes with unknown function. One gene, upregulated 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, Deltaeed1 cells could proliferate as yeasts or pseudohyphae but remained trapped intracellularly. Our results suggest that the adaptive response and morphology of C. albicans play specific roles for host-fungal interactions during mucosal infections.

Transglucosidases play a significant role in fungal cell wall biosynthesis. We identified three as yet undescribed genes encoding β-glucan transglucosidases, homologues of the pH-regulated PHR1 and PHR2, in the genome of the pathogenic yeast Candida albicans. Transcript levels of the gene PGA4 encoding a putative GPI-anchored protein were elevated in C. albicans wild-type cells during infection of reconstituted human epithelial and mouse liver tissue, and transiently increased after induction of hyphal formation with serum. The serum-specific increase in PGA4 transcript was found to be dependent on the transcription factors Ras1p, Cyr1p, and Tec1p. The remaining C. albicans Phr homologues, PHR3 and PGA5, showed low expression levels. Unlike PHR1 and PHR2, the expression of PHR3, PGA4, and PGA5 was not dependent on the pH of the growth medium. Neither PHR3 deletion nor PGA4 disruption resulted in a distinct growth or morphology phenotype. A PGA4 disruption strain was found to have wild-type capacity of infecting reconstituted oral epithelial tissue. Our data suggest that PGA4, and potentially PHR3 and PGA5, are expressed under distinct conditions, which differ from those of PHR1 and PHR2.

Group B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat-encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA -proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen-binding motif with the consensus sequence G-N/S/T-V-L-A/E/M/Q-R-R-X-K/R/W-A/D/E/N/Q-A/F/I/L/V/Y-X-X-K/R-X-X.

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. ispartof: Journal of Clinical Virology vol:101 pages:11-17 ispartof: location:Netherlands status: published

Background Enteroendocrine cells (EEC) are highly specialized cells producing signalling molecules vital to the normal functions of the gut. Recently, we showed altered protein distribution in Chlamydia infected EEC in vitro. The aim of this study was to perform a microarray analysis of the response pattern of EEC from both large and small bowel to infection in vitro, using Chlamydia trachomatis infection as a model. Methods Two human EEC lines: LCC-18, derived from a neuroendocrine colonic tumour, and CNDT-2, derived from a small intestinal carcinoid, were infected using cultured C. trachomatis serovar LGV II strain 434 (ATCC VR-902B). Penicillin G was used to induce persistent infection. We used microarray analysis (Affymetrix GeneChip®) for studying changes in gene expression at different stages of infection. Results Twenty-four hours after active and persistent infection, 66 and 411 genes in LCC-18 and 68 and 170 genes in CNDT-2 cells, respectively showed mean expression ratios >2-fold compared to non-infected cells. These genes encoded factors regulating apoptosis, cell differentiation, transcription regulation, cytokine activity, amine biosynthesis and vesicular transport. We found significant differences in gene transcription levels between persistently infected and non-infected cells in 10 genes coding for different solute carrier transporters (SLC) and in 5 genes related to endocrine function (GABARAPL1, GRIP1, DRD2, SYT5 and SYT7). Conclusions Infected EEC cells exhibit cell-type specific patterns related to vesicular transport, secretion and neurotransmitters. EEC play a pivotal role in regulation of gut motility and an impairment of enteroendocrine function can contribute to motility disorders.