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1.
Future Microbiol ; 14: 749-755, 2019 06.
Article in English | MEDLINE | ID: mdl-31271060

ABSTRACT

Aim: Antibiotic resistance in Mycobacterium abscessus renders treatment poorly effective. Despite erm(41)-gene-mediated macrolide resistance, treatment with azithromycin or clarithromycin is recommended. It is contested whether macrolides differ in erm(41) induction. We determine whether this is the case. Methods:M. abscessus CIP104536 was used. Minimum inhibitory concentrations of clarithromycin and azithromycin were determined. Time-kill kinetics of M. abscessus exposed to azithromycin or clarithromycin were performed and RNA was isolated at predetermined intervals for erm(41) quantification. Results: Minimum inhibitory concentrations increased >30-fold. Time-kill kinetics showed a temporary bacteriostatic effect, abrogated by induced resistance. Erm(41) expression was increased following exposure to either macrolide for 7 days. Conclusion: Both macrolides induce resistance similarly, and this should not be an argument in choosing either macrolide for therapy.


Subject(s)
Anti-Bacterial Agents/pharmacology , Azithromycin/pharmacology , Clarithromycin/pharmacology , Drug Resistance, Bacterial/drug effects , Mycobacterium abscessus/drug effects , Transcriptional Activation/drug effects , Gene Expression Profiling , Macrolides/pharmacology , Microbial Sensitivity Tests , RNA, Bacterial/analysis , RNA, Messenger/analysis
3.
J Clin Microbiol ; 54(7): 1915-1917, 2016 07.
Article in English | MEDLINE | ID: mdl-27147723

ABSTRACT

We have assessed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification (Bruker) of nontuberculous mycobacteria from newly positive liquid cultures of respiratory samples. Twelve (22%) of 54 isolates were identified directly from liquid medium. After subculture and with manual laser operation, this rose to 49/54 isolates (91%). MALDI-TOF MS is less promising than previously suggested.


Subject(s)
Bacteriological Techniques/methods , Mycobacterium Infections, Nontuberculous/diagnosis , Mycobacterium Infections, Nontuberculous/microbiology , Nontuberculous Mycobacteria/chemistry , Nontuberculous Mycobacteria/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Sputum/microbiology , Humans , Sensitivity and Specificity
4.
Antimicrob Agents Chemother ; 60(2): 1097-105, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26643335

ABSTRACT

Multidrug therapy is a standard practice when treating infections by nontuberculous mycobacteria (NTM), but few treatment options exist. We conducted this study to define the drug-drug interaction between clofazimine and both amikacin and clarithromycin and its contribution to NTM treatment. Mycobacterium abscessus and Mycobacterium avium type strains were used. Time-kill assays for clofazimine alone and combined with amikacin or clarithromycin were performed at concentrations of 0.25× to 2× MIC. Pharmacodynamic interactions were assessed by response surface model of Bliss independence (RSBI) and isobolographic analysis of Loewe additivity (ISLA), calculating the percentage of statistically significant Bliss interactions and interaction indices (I), respectively. Monte Carlo simulations with predicted human lung concentrations were used to calculate target attainment rates for combination and monotherapy regimens. Clofazimine alone was bacteriostatic for both NTM. Clofazimine-amikacin was synergistic against M. abscessus (I = 0.41; 95% confidence interval [CI], 0.29 to 0.55) and M. avium (I = 0.027; 95% CI, 0.007 to 0.048). Based on RSBI analysis, synergistic interactions of 28.4 to 29.0% and 23.2 to 56.7% were observed at 1× to 2× MIC and 0.25× to 2× MIC for M. abscessus and M. avium, respectively. Clofazimine-clarithromycin was also synergistic against M. abscessus (I = 0.53; 95% CI, 0.35 to 0.72) and M. avium (I = 0.16; 95% CI, 0.04 to 0.35), RSBI analysis showed 23.5% and 23.3 to 53.3% at 2× MIC and 0.25× to 0.5× MIC for M. abscessus and M. avium, respectively. Clofazimine prevented the regrowth observed with amikacin or clarithromycin alone. Target attainment rates of combination regimens were >60% higher than those of monotherapy regimens for M. abscessus and M. avium. The combination of clofazimine with amikacin or clarithromycin was synergistic in vitro. This suggests a potential role for clofazimine in treatment regimens that warrants further evaluation.


Subject(s)
Amikacin/pharmacology , Anti-Bacterial Agents/pharmacology , Clarithromycin/pharmacology , Clofazimine/pharmacology , Mycobacterium avium/drug effects , Nontuberculous Mycobacteria/drug effects , Drug Interactions , Drug Synergism , Drug Therapy, Combination , Microbial Sensitivity Tests , Monte Carlo Method , Mutation , Mycobacterium avium/genetics , Mycobacterium avium/growth & development , Nontuberculous Mycobacteria/genetics , Nontuberculous Mycobacteria/growth & development
5.
Ann Clin Transl Neurol ; 1(8): 618-21, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25356431

ABSTRACT

Saffold cardiovirus, a newly discovered human cardiovirus, has close similarity with Theiler's murine encephalomyelitis virus (TMEV) which can cause a chronic demyelinating encephalomyelitis in mice. In this study, we tested whether Saffold cardiovirus infection of the brain is associated with multiple sclerosis (MS). Autopsy white matter samples from 19 MS and 9 normal brain donors were tested by polymerase chain reaction. All were negative. Paired cerebrospinal fluid and serum samples from 24 MS patients and 27 controls were tested for Saffold cardiovirus-specific oligoclonal bands, two patients and two controls reacted positive. We conclude that an association between Saffold cardiovirus and MS is highly improbable.

7.
J Clin Virol ; 58(3): 559-63, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24113293

ABSTRACT

BACKGROUND: The role of viral infections in preterm prelabor rupture of the membranes (PPROM) is not established. Studies on the presence of viral genomes in the amniotic fluid (AF) collected in pregnancies complicated by PPROM show contradictory outcomes. OBJECTIVES: To investigate AF samples of PPROM pregnancies for the presence of viral genomes. STUDY DESIGN: AF samples from patients with PPROM were collected during a 4-year (2008-2012) observational study. 174 women were included with selection criteria of singleton pregnancy, PPROM, and maternal age of 18 years and above. PCR was used for detection of human cytomegalovirus (HCMV), herpes simplex virus (HSV), parvovirus B19, human adenoviruses (HAdV), enteroviruses (EV) and human parechovirus (HPeV). The selection of these viral targets was based on literature regarding screening of AF for presence of viral genomes. RESULTS: Only a single sample was positive out of the 174 tested AFs, HCMV DNA was detected. CONCLUSIONS: PPROM is not associated with active viral infections.


Subject(s)
Amniotic Fluid/virology , Fetal Membranes, Premature Rupture/etiology , Viruses/isolation & purification , Adult , Female , Genome, Viral , Humans , Polymerase Chain Reaction , Pregnancy , Viruses/genetics , Young Adult
8.
J Virol Methods ; 190(1-2): 53-62, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23458694

ABSTRACT

During September and October 2010, the Dutch Public Health Institute detected an enterovirus (EV) 68 (EV68) epidemic in the Netherlands through general practitioner-based surveillance of acute respiratory infections. EV68 shares phenotypic and genotypic properties with human rhinovirus (HRV). Despite increased EV and HRV detections, Dutch clinical laboratories did not identify EV68. To assess the capability of Dutch clinical laboratories to detect EV68, ten laboratories with more than eight detected EV and HRV cases in September and October 2010 provided information about their detection algorithms and testing results for a 2010 Dutch EV68 strain. For EV detection mostly stool specimens (median 49%), respiratory specimens (median 27%) and cerebrospinal fluid (median 22%) were used. For HRV detection only respiratory specimens were used. Except for the Seeplex® RV15ACE EV-specific assay, all EV and 73% of HRV assays, including those of the Public Health Institute, were able to detect EV68. Two-step EV RT-PCR protocols were the most sensitive. Thus, laboratories might have misidentified EV68 as HRV. In addition, EV68 cases might have also been missed because patients with respiratory diseases are usually not tested for EV infection. Therefore, clinical laboratories should include EV detection in the differential diagnosis of patients presenting with respiratory symptoms.


Subject(s)
Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Enterovirus Infections/diagnosis , Enterovirus/isolation & purification , Respiratory Tract Infections/diagnosis , Cerebrospinal Fluid/virology , Enterovirus Infections/virology , Feces/virology , Humans , Laboratory Proficiency Testing , Netherlands , Respiratory Tract Infections/virology , Sensitivity and Specificity , Sputum/virology
9.
Diagn Microbiol Infect Dis ; 75(4): 431-3, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23375086

ABSTRACT

Ninety-nine clinical isolates previously identified as Klebsiella oxytoca were evaluated using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight isolates were identified as Raoultella spp., being 5 Raoultella spp. and 3 K. oxytoca, by 16S rRNA sequencing. These isolates were correctly identified by applying the 10% differential rule for the MALDI-TOF MS score values. This approach might be useful to discriminate Raoultella species from K. oxytoca.


Subject(s)
Bacteriological Techniques/methods , Enterobacteriaceae/chemistry , Enterobacteriaceae/classification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Humans , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
10.
Diagn Microbiol Infect Dis ; 74(4): 404-5, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22999333

ABSTRACT

Here, we report the first isolation of Wautersiella falsenii from the urine of an infant with a complicated urinary tract infection. W. falsenii was correctly identified by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The identification was confirmed by 16S polymerase chain reaction. Susceptibility test results of this isolate are reported. Ciprofloxacin treatment resulted in clinical and microbiological improvement.


Subject(s)
Flavobacteriaceae Infections/diagnosis , Flavobacteriaceae Infections/microbiology , Flavobacteriaceae/isolation & purification , Pyelonephritis/diagnosis , Pyelonephritis/microbiology , Urine/microbiology , Anti-Bacterial Agents/pharmacology , Bacteriological Techniques/methods , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Female , Flavobacteriaceae/chemistry , Flavobacteriaceae/genetics , Humans , Infant , Microbial Sensitivity Tests , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
11.
Sensors (Basel) ; 12(12): 17358-71, 2012 Dec 13.
Article in English | MEDLINE | ID: mdl-23443402

ABSTRACT

In this paper, a laboratory goniometer system for performing multi-angular measurements under controlled illumination conditions is described. A commercially available robotic arm enables the acquisition of a large number of measurements over the full hemisphere within a short time span making it much faster than other goniometers. In addition, the presented set-up enables assessment of anisotropic reflectance and emittance behaviour of soils, leaves and small canopies. Mounting a spectrometer enables acquisition of either hemispherical measurements or measurements in the horizontal plane. Mounting a thermal camera allows directional observations of the thermal emittance. This paper also presents three showcases of these different measurement set-ups in order to illustrate its possibilities. Finally, suggestions for applying this instrument and for future research directions are given, including linking the measured reflectance anisotropy with physically-based anisotropy models on the one hand and combining them with field goniometry measurements for joint analysis with remote sensing data on the other hand. The speed and flexibility of the system offer a large added value to the existing pool of laboratory goniometers.


Subject(s)
Anisotropy , Earth, Planet , Robotics , Climatic Processes , Humans
12.
Antiviral Res ; 92(1): 81-9, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21767571

ABSTRACT

Enhanced surveillance of infections due to the pandemic A(H1N1) influenza virus, which included monitoring for antiviral resistance, was carried out in the Netherlands from late April 2009 through late May 2010. More than 1100 instances of infection with the pandemic A(H1N1) influenza virus from 2009 and 2010 [A(H1N1) 2009] distributed across this period were analyzed. Of these, 19 cases of oseltamivir-resistant virus harboring the H275Y mutation in the neuraminidase (NA) were detected. The mean 50% inhibitory concentration (IC50) levels for oseltamivir- and zanamivir-susceptible A(H1N1) 2009 viruses were 1.4-fold and 2-fold, respectively, lower than for the seasonal A(H1N1) influenza viruses from 2007/2008; for oseltamivir-resistant A(H1N1) 2009 virus the IC50 was 2.9-fold lower. Eighteen of the 19 patients with oseltamivir-resistant virus showed prolonged shedding of the virus and developed resistance while on oseltamivir therapy. Sixteen of these 18 patients had an immunodeficiency, of whom 11 had a hematologic disorder. The two other patients had another underlying disease. Six of the patients who had an underlying disease died; of these, five had received cytostatic or immunosuppressive therapy. No indications for onward transmission of resistant viruses were found. This study showed that the main association for the emergence of cases of oseltamivir-resistant A(H1N1) 2009 virus was receiving antiviral therapy and having drug-induced immunosuppression or an hematologic disorder. Except for a single case of a resistant virus not linked to oseltamivir therapy, the absence of detection of resistant variants in community specimens and in specimens from contacts of cases with resistant virus suggested that the spread of resistant A(H1N1) 2009 virus was limited. Containment may have been the cumulative result of impaired NA function, successful isolation of the patients, and prophylactic measures to limit exposure.


Subject(s)
Drug Resistance, Viral , Influenza A Virus, H1N1 Subtype/drug effects , Influenza, Human/drug therapy , Influenza, Human/epidemiology , Oseltamivir/therapeutic use , Pandemics , Adolescent , Adult , Aged , Animals , Cell Line , Child , Child, Preschool , Female , Humans , Infant , Influenza A Virus, H1N1 Subtype/classification , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Influenza, Human/virology , Male , Middle Aged , Molecular Sequence Data , Mutation , Netherlands/epidemiology , Neuraminidase/genetics , Neuraminidase/metabolism , Phylogeny , Sentinel Surveillance , Viral Proteins/genetics , Viral Proteins/metabolism , Young Adult
13.
Prostate ; 71(4): 415-20, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-20878949

ABSTRACT

BACKGROUND: The occurrence of the retrovirus xenotropic murine leukemia virus (MLV)-related virus (XMRV) has been reported in prostate tissue of patients with prostate cancer (PrCa). Considering the potential great medical and social relevance of this discovery, we investigated whether this finding could be confirmed in an independent group of Dutch sporadic PrCa cases. METHODS: We investigated the occurrence of XMRV in fresh-frozen PrCa specimens of 74 PrCa patients from The Netherlands. Total RNA and DNA were isolated, subjected to cDNA synthesis, and analyzed by real-time PCR targeting conserved XMRV sequences. RESULTS: Spiking experiments showed that we were able to detect at least 10 copies of XMRV sequences in 100,000 cells by real-time PCR, demonstrating high sensitivity of the assay. XMRV sequences were detected in 3 out of 74 (i.e., 4%) PrCa specimens. The number of XMRV containing cells was extremely low (1 in 600-7,000 cells). This was corroborated by the fact that XMRV could not be detected in consecutive tissue sections of the initial XMRV-positive cases. CONCLUSIONS: XMRV was rarely detected, and at extremely low levels, in sporadic PrCa samples from Dutch patients. When XMRV would play a causal role in prostate carcinogenesis, integration of the provirus could be expected to be present in, at least, a proportion of tumor cells. Therefore, our data do not support the claim that there is an association between XMRV infection and PrCa in Dutch PrCa patients.


Subject(s)
Prostatic Neoplasms/virology , Xenotropic murine leukemia virus-related virus/isolation & purification , Humans , Male , Netherlands , Polymerase Chain Reaction , RNA, Viral/analysis
14.
J Immunol Methods ; 365(1-2): 58-66, 2011 Feb 28.
Article in English | MEDLINE | ID: mdl-21182842

ABSTRACT

Mutations in UNC13D cause the severe immune disorder familial haemophagocytic lymphohistiocytosis type 3 (FHL3). The gene product munc13-4 is expressed in hematopoietic cells and is essential for degranulation. Little information is available on genotype-phenotype relationships of UNC13D mutations. Some mutants may have residual functionality which qualifies them as promising targets for attempts to enhance function pharmacologically. A problem for such analysis is the scarcity of patient material. We established assays in the RBL-2H3 cell line to assess functionality of lentivirally transduced munc13-4 mutants. The basic principle of which is to silence endogenous rat munc13-4 and replace it with siRNA resistant YFP-tagged human variants. Localization, degranulation, and membrane binding kinetics can now easily be analyzed quantitatively. Such a system might also be useful to screen small molecular weight compounds for their ability to rescue degranulation in cells with reduced functional munc13-4.


Subject(s)
Genetic Complementation Test/methods , Lymphohistiocytosis, Hemophagocytic/genetics , Membrane Proteins/genetics , Mutation , Animals , Base Sequence , Cell Degranulation/genetics , Cell Degranulation/physiology , Cell Line , DNA, Complementary/genetics , Fluorescence Recovery After Photobleaching , Humans , Lentivirus/genetics , Lymphohistiocytosis, Hemophagocytic/physiopathology , Membrane Proteins/physiology , RNA, Small Interfering/genetics , Rats , Transduction, Genetic
15.
BMJ ; 340: c1018, 2010 Feb 25.
Article in English | MEDLINE | ID: mdl-20185493

ABSTRACT

OBJECTIVE: The presence of the retrovirus xenotropic murine leukaemia virus-related virus (XMRV) has been reported in peripheral blood mononuclear cells of patients with chronic fatigue syndrome. Considering the potentially great medical and social relevance of such a discovery, we investigated whether this finding could be confirmed in an independent European cohort of patients with chronic fatigue syndrome. DESIGN: Analysis of a well defined cohort of patients and matched neighbourhood controls by polymerase chain reaction. SETTING: Certified (ISO 15189) laboratory of clinical virology in a university hospital in the Netherlands. Population Between December 1991 and April 1992, peripheral blood mononuclear cells were isolated from 76 patients and 69 matched neighbourhood controls. In this study we tested cells from 32 patients and 43 controls from whom original cryopreserved phials were still available. MAIN OUTCOME MEASURES: Detection of XMRV in peripheral blood mononuclear cells by real time polymerase chain reaction assay targeting the XMRV integrase gene and/or a nested polymerase chain reaction assay targeting the XMRV gag gene. RESULTS: We detected no XMRV sequences in any of the patients or controls in either of the assays, in which relevant positive and negative isolation controls and polymerase chain reaction controls were included. Spiking experiments showed that we were able to detect at least 10 copies of XMRV sequences per 10(5) peripheral blood mononuclear cells by real time as well as by nested polymerase chain reaction, demonstrating high sensitivity of both assays. CONCLUSIONS: This study failed to show the presence of XMRV in peripheral blood mononuclear cells of patients with chronic fatigue syndrome from a Dutch cohort. These data cast doubt on the claim that XMRV is associated with chronic fatigue syndrome in the majority of patients.


Subject(s)
Fatigue Syndrome, Chronic/virology , Leukemia Virus, Murine/isolation & purification , Retroviridae Infections/complications , Adult , Case-Control Studies , DNA, Viral , Female , Humans , Leukocytes, Mononuclear/virology , Male , Middle Aged , Polymerase Chain Reaction , Retrospective Studies
16.
J Clin Virol ; 45(3): 179-84, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19540155

ABSTRACT

BACKGROUND: Late April 2009, human infection with variant influenza virus A(H1N1)v emerged in the Northern Americas posing a threat that this virus may become the next pandemic influenza virus. OBJECTIVES: To prepare laboratories for surge capacity for molecular diagnosis of patients suspected for A(H1N1)v infection in the Netherlands. STUDY DESIGN: A panel of 10 blinded specimens containing seasonal A(H1N1) or A(H3N2), or A/Netherlands/602/2009(H1N1)v influenza virus, or negative control was distributed to the outbreak assistance laboratories (OAL) together with influenza virus A (M-gene), swine influenza virus A (NP-gene) and influenza virus A(H1N1)v (H1v-gene) specific primers and probes and protocol (CDC Atlanta, USA). Laboratories were asked to implement and test this protocol. RESULTS: All OAL were able to detect A(H1N1)v using the CDC M-gene reagents, the majority with similar sensitivity as the in-house M-gene based assays. RT-PCRs used in routine diagnostic setting in the OAL specifically designed to detect H1, H3, or NS1 from seasonal influenza A viruses, did not or at very low level cross-react with A(H1N1)v. The CDC swine NP-gene and H1v-gene RT-PCRs showed somewhat reduced sensitivity compared to the CDC and in-house M-gene RT-PCRs. In contrast, in-house developed A(H1N1)v specific H1v-gene and N1v-gene RT-PCRs showed equal sensitivity to CDC and in-house M-gene RT-PCRs. CONCLUSIONS: The Dutch OAL are prepared for detection and specific identification of A(H1N1)v, although some level of cross-reactivity was observed with seasonal influenza viruses. Additionally, M-gene based generic influenza A virus detection is recommended to be able to detect emerging influenza A viruses in routine settings.


Subject(s)
Clinical Laboratory Techniques/standards , Communicable Disease Control/methods , Disease Outbreaks/prevention & control , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/diagnosis , Influenza, Human/virology , Animals , Cross Reactions , Health Services Research , Humans , Influenza A Virus, H3N2 Subtype/isolation & purification , Influenza, Human/epidemiology , Influenza, Human/prevention & control , Laboratories , Netherlands , Sensitivity and Specificity , Swine
17.
Cytometry A ; 73(2): 129-38, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18163486

ABSTRACT

Mitochondrial membrane potential (Deltapsi) is key to mitochondrial function and cellular survival. Here, we aimed to develop an automated protocol allowing sensitive quantification of Deltapsi in living cells at the level of individual mitochondria. Human skin fibroblasts were stained with the fluorescent cation tetramethyl rhodamine methyl ester (TMRM), which is sequestered by mitochondria according to their Deltapsi. Cells were visualized by videomicroscopy and the acquired images were processed to generate a mitochondria-specific mask. The latter was superimposed on the original image to allow quantification of TMRM fluorescence. Following validation, our approach revealed that mitochondria with different Deltapsi coexisted within the same cell. Furthermore, our method allowed reproducible detection of small (<10%) reductions in TMRM intensity induced by the complex III inhibitor antimycin A. Mitochondrial uncoupling by p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) greatly reduced mitochondrial TMRM fluorescence. Under these conditions faithful mask calculation and TMRM intensity analysis were still possible using a mitochondria-targeted green fluorescence protein (mitoAcGFP1), expressed in the cells using baculoviral transfection.


Subject(s)
Membrane Potential, Mitochondrial/physiology , Mitochondria/physiology , Cells, Cultured , Fibroblasts/ultrastructure , Fluorescent Dyes/chemistry , Green Fluorescent Proteins/metabolism , Humans , Mitochondria/ultrastructure , Rhodamines/chemistry
18.
J Virol ; 82(7): 3782-90, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18216106

ABSTRACT

The family Picornaviridae consists of a large group of plus-strand RNA viruses that share a similar genome organization. The nomenclature of the picornavirus proteins is based on their position in the viral RNA genome but does not necessarily imply a conserved function of proteins of different genera. The enterovirus 2B protein is a small hydrophobic protein that, upon individual expression, is localized to the endoplasmic reticulum (ER) and the Golgi complex, reduces ER and Golgi complex Ca(2+) levels, most likely by forming transmembrane pores, and inhibits protein trafficking through the Golgi complex. At present, little is known about the function of the other picornavirus 2B proteins. Here we show that rhinovirus 2B, which is phylogenetically closely related to enterovirus 2B, shows a similar subcellular localization and function to those of enterovirus 2B. In contrast, 2B proteins of hepatitis A virus, foot-and-mouth disease virus, and encephalomyocarditis virus, all of which are more distantly related to enteroviruses, show a different localization and have little, if any, effects on Ca(2+) homeostasis and intracellular protein trafficking. Our data suggest that the 2B proteins of enterovirus and rhinovirus share the same function in virus replication, while the other picornavirus 2B proteins support the viral life cycle in a different manner. Moreover, we show that an enterovirus 2B protein that is retained in the ER is unable to modify Ca(2+) homeostasis and inhibit protein trafficking, demonstrating the importance of Golgi complex localization for its functioning.


Subject(s)
Calcium/metabolism , Picornaviridae/physiology , Viral Proteins/metabolism , Animals , Cell Line , Chlorocebus aethiops , Endoplasmic Reticulum/virology , Golgi Apparatus/virology , Phylogeny , Picornaviridae/genetics , Protein Transport , Sequence Homology, Amino Acid
19.
J Biol Chem ; 281(20): 14144-50, 2006 May 19.
Article in English | MEDLINE | ID: mdl-16540472

ABSTRACT

Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca2+ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca2+ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca2+ into the stores. Measurement of intra-organelle-free Ca2+ in permeabilized cells revealed that the ability of 2B to reduce the Ca2+ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cell-free system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca2+-ATPase that transports Ca2+ from the cytosol into the stores. To investigate whether 2B specifically affects Ca2+ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H+ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.


Subject(s)
Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Viral Proteins/physiology , Adenosine Triphosphate/chemistry , Animals , Biological Transport , Calcium/metabolism , Cell Line , Chlorocebus aethiops , Hydrogen-Ion Concentration , Ions , Mutation , Sarcoplasmic Reticulum/metabolism , Viral Proteins/metabolism
20.
Trends Microbiol ; 13(2): 41-4, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15680759

ABSTRACT

Enteroviruses modify several cellular functions to ensure efficient replication. However, some of these alterations can trigger a defensive apoptotic host-cell program. To prevent premature abortion of their productive cycle, enteroviruses have developed anti-apoptotic countermeasures. Here, we discuss recent evidence that the enterovirus 2B protein exerts an anti-apoptotic activity that is related to its ability to form pores in endoplasmic reticulum (ER) and Golgi membranes, thereby reducing their Ca(2+) content and perturbing ER-mitochondrial Ca(2+) signaling.


Subject(s)
Apoptosis/physiology , Calcium/metabolism , Enterovirus Infections/metabolism , Enterovirus/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Calcium Signaling , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Enterovirus/genetics , Enterovirus Infections/pathology , Enterovirus Infections/virology , Golgi Apparatus/metabolism , Golgi Apparatus/virology , Humans , Molecular Sequence Data
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