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1.  Human Parechovirus Infection, Denmark 
Emerging Infectious Diseases  2014;20(1):83-87.
HPeV should be tested for in all young children suspected to have HPeV or enterovirus infection.
Human parechoviruses (HPeVs) often cause severe illness among young children. National surveillance with routine testing of all cerebrospinal fluid, fecal, and tissue samples was conducted during January 2009–December 2012 in all counties in Denmark (6,817 samples from 4,804 children were screened for HPeV). We detected HPeV RNA in 202 (3.0%) specimens from 149 persons. Young infants were at highest risk for HPeV, and 9 (6%) of the HPeV-infected children died, probably of their HPeV illness. HPeV3 was the most common genotype identified, and 5 closely related clades of HPeV3 circulated in Denmark throughout the study period. Our study adds perspective on the prevalence and clinical and molecular virologic characteristics of HPeV infection.
doi:10.3201/eid2001.130569
PMCID: PMC3884717  PMID: 24377661
HPeV; parechovirus; meningitis; phylogenetic; Denmark; viruses; children; infants
2.  Tick-borne Encephalitis Virus, Zealand, Denmark, 2011 
Emerging Infectious Diseases  2013;19(7):1171-1173.
doi:10.3201/eid1907.130092
PMCID: PMC3903456  PMID: 23764123
Tick-borne; encephalitis; Denmark; flavivirus; Ixodes ricinus; emerging disease; vector-borne infections; ticks; ticks; viruses
3.  Co-Circulation and Persistence of Genetically Distinct Saffold Viruses, Denmark 
Emerging Infectious Diseases  2012;18(10):1694-1696.
doi:10.3201/eid1810.120793
PMCID: PMC3471643  PMID: 23017727
Safford virus; cardiovirus; evolution; phylogeny; co-circulation; persistence; Denmark; viruses; Suggested citation for this article: Christian A; Nielsen Y; Gyhrs ML; Holmes EC; Cui J. Co-circulation and persistence of genetically distinct Saffold viruses; Denmark. Emerg Infect Dis [Internet]. 2012 Oct [date cited]. http://dx.doi.org/10.3201/eid1810.120793
4.  Characterization of a Feedback-Resistant Mevalonate Kinase from the Archaeon Methanosarcina mazei▿ 
Applied and Environmental Microbiology  2011;77(21):7772-7778.
The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.
doi:10.1128/AEM.05761-11
PMCID: PMC3209144  PMID: 21908638
5.  Serious Invasive Saffold Virus Infections in Children, 2009 
Emerging Infectious Diseases  2012;18(1):7-12.
This virus might have caused previously unexplained cerebral infections and deaths in children.
The first human virus in the genus Cardiovirus was described in 2007 and named Saffold virus (SAFV). Cardioviruses can cause severe infections of the myocardium and central nervous system in animals, but SAFV has not yet been convincingly associated with disease in humans. To study a possible association between SAFV and infections in the human central nervous system, we designed a real-time PCR for SAFV and tested cerebrospinal fluid (CSF) samples from children <4 years of age. SAFV was detected in 2 children: in the CSF and a fecal sample from 1 child with monosymptomatic ataxia caused by cerebellitis; and in the CSF, blood, and myocardium of another child who died suddenly with no history of illness. Virus from each child was sequenced and shown to be SAFV type 2. These findings demonstrate that SAFV can cause serious invasive infection in children.
doi:10.3201/eid1801.110725
PMCID: PMC3310106  PMID: 22261113
Saffold virus; cardiovirus; viral encephalitis; encephalitis; cerebrospinal fluid; picornavirus infections; fatal outcome; viruses; Denmark; children
6.  A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine 
PLoS Pathogens  2010;6(9):e1001102.
A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.
Author Summary
Most pathogenic microorganisms infect in a stepwise manner: colonization of host surfaces is followed by invasion and injury of host tissues and, late in the infectious process, dissemination to other hosts occurs. During its residence in the host, the pathogen produces essential virulence determinants and often replicates rapidly, leading to a vast expansion of its biomass. Although this scenario is well established also for Vibrio cholerae, the cause of a potentially fatal diarrheal illness, it has not previously been possible to identify precisely when or where virulence determinants are produced in the intestine. We addressed this question by investigating the expression of virulence genes by individual V. cholerae during infection of the small intestine. Virulence genes were found to be powerfully expressed early in the infectious process by bacteria in close proximity to epithelial surfaces. Increased replication rates were also localized to epithelial surfaces. During later stages of the infection, the population of V. cholerae bifurcates into two fractions: one subpopulation continues to express virulence genes, whereas these genes are silenced in the other subpopulation. The genetic program controlling the continued production of virulence genes may mediate the persistence of a hyper-infectious subpopulation of bacteria in the stools of cholera patients.
doi:10.1371/journal.ppat.1001102
PMCID: PMC2940755  PMID: 20862321
7.  RpoS Controls the Vibrio cholerae Mucosal Escape Response 
PLoS Pathogens  2006;2(10):e109.
Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the “mucosal escape response,” this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.
Synopsis
Vibrio cholerae, a pathogenic microbe, causes a severe diarrhoeal disease mainly in Third World countries. Although the pathogenicity of this organism has been intensively studied for more than a century, most research has focused on the initial stages of the infection, especially colonization of the intestine and virulence gene expression. However, the last stages of the infectious process have received very little attention. In the present manuscript, the authors use the rabbit ileal loop model of cholera to show how this organism, late in the infection, detaches from the epithelial surface and migrates into the luminal fluid, a process the authors termed the “mucosal escape response.” This study identifies, for the first time, how the alternative starvation sigma factor RpoS regulates this process. Features of this genetic program include the dramatic induction of genes involved in motility and chemotaxis functions. This study furthermore identifies RpoS as an important regulator of virulence gene expression and shows that the mucosal escape response may coincide with diminished virulence gene expression. This work is essential for understanding a key and under-appreciated step in the life cycle of this important human pathogen: its exit from the intestine and how this serves to prepare it for transmission into environmental reservoirs or to new human hosts.
doi:10.1371/journal.ppat.0020109
PMCID: PMC1617127  PMID: 17054394
8.  Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms 
In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.
PMCID: PMC99748  PMID: 10473423
9.  Identification of a Novel Group of Bacteria in Sludge from a Deteriorated Biological Phosphorus Removal Reactor 
The microbial diversity of a deteriorated biological phosphorus removal reactor was investigated by methods not requiring direct cultivation. The reactor was fed with media containing acetate and high levels of phosphate (P/C weight ratio, 8:100) but failed to completely remove phosphate in the effluent and showed very limited biological phosphorus removal activity. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA was used to investigate the bacterial diversity. Up to 11 DGGE bands representing at least 11 different sequence types were observed; DNA from the 6 most dominant of these bands was further isolated and sequenced. Comparative phylogenetic analysis of the partial 16S rRNA sequences suggested that one sequence type was affiliated with the alpha subclass of the Proteobacteria, one was associated with the Legionella group of the gamma subclass of the Proteobacteria, and the remaining four formed a novel group of the gamma subclass of the Proteobacteria with no close relationship to any previously described species. The novel group represented approximately 75% of the PCR-amplified DNA, based on the DGGE band intensities. Two oligonucleotide rRNA probes for this novel group were designed and used in a whole-cell hybridization analysis to investigate the abundance of this novel group in situ. The bacteria were coccoid and 3 to 4 μm in diameter and represented approximately 35% of the total population, suggesting a relatively close agreement with the results obtained by the PCR-based DGGE method. Further, based on electron microscopy and standard staining microscopic analysis, this novel group was able to accumulate granule inclusions, possibly consisting of polyhydroxyalkanoate, inside the cells.
PMCID: PMC91172  PMID: 10049891
10.  Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool 
Biotechnology and Bioengineering  2014;111(8):1604-1616.
Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells. Biotechnol. Bioeng. 2014; 111: 1604–1616. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
doi:10.1002/bit.25233
PMCID: PMC4312910  PMID: 24827782
CRISPR Cas9; genome editing; CRISPy; Chinese hamster ovary cells; database

Results 1-10 (10)