Saffold cardiovirus and multiple sclerosis: no evidence for an association.

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.

Preterm prelabor rupture of membranes (PPROM) is not associated with presence of viral genomes in the amniotic fluid.

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.

September through October 2010 multi-centre study in the Netherlands examining laboratory ability to detect enterovirus 68, an emerging respiratory pathogen.

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.

Differentiation of Raoultella ornithinolytica/planticola and Klebsiella oxytoca clinical isolates by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

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.

First report of a Wautersiella falsenii isolated from the urine of an infant with pyelonephritis.

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.

Prevalence of human xenotropic murine leukemia virus-related gammaretrovirus (XMRV) in Dutch prostate cancer patients.

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.

Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort.

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.

Functional analysis of picornavirus 2B proteins: effects on calcium homeostasis and intracellular protein trafficking.

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.

Life cell quantification of mitochondrial membrane potential at the single organelle level.

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.

The coxsackievirus 2B protein increases efflux of ions from the endoplasmic reticulum and Golgi, thereby inhibiting protein trafficking through the Golgi.

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.

Enterovirus protein 2B po(u)res out the calcium: a viral strategy to survive?

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.

Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells.

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.

The coxsackievirus 2B protein suppresses apoptotic host cell responses by manipulating intracellular Ca2+ homeostasis.

Enteroviruses, small cytolytic RNA viruses, confer an antiapoptotic state to infected cells in order to suppress infection-limiting apoptotic host cell responses. This antiapoptotic state also lends protection against cell death induced by metabolic inhibitors like actinomycin D and cycloheximide. The identity of the viral antiapoptotic protein and the underlying mechanism are unknown. Here, we provide evidence that the coxsackievirus 2B protein modulates apoptosis by manipulating intracellular Ca(2+) homeostasis. Using fluorescent Ca(2+) indicators and organelle-targeted aequorins, we demonstrate that ectopic expression of 2B in HeLa cells decreases the Ca(2+) content of both the endoplasmic reticulum and the Golgi, resulting in down-regulation of Ca(2+) signaling between these stores and the mitochondria, and increases the influx of extracellular Ca(2+). In our studies of the physiological importance of the 2B-induced alterations in Ca(2+) signaling, we found that the expression of 2B suppressed caspase activation and apoptotic cell death induced by various stimuli, including actinomycin D and cycloheximide. Mutants of 2B that were defective in reducing the Ca(2+) content of the stores failed to suppress apoptosis. These data implicate a functional role of the perturbation of intracellular Ca(2+) compartmentalization in the enteroviral strategy to suppress intrinsic apoptotic host cell responses. The putative down-regulation of an endoplasmic reticulum-dependent apoptotic pathway is discussed.

Mutational analysis of different regions in the coxsackievirus 2B protein: requirements for homo-multimerization, membrane permeabilization, subcellular localization, and virus replication.

The coxsackievirus 2B protein is a small hydrophobic protein (99 amino acids) that increases host cell membrane permeability, possibly by forming homo-multimers that build membrane-integral pores. Previously, we defined the functional role of the two hydrophobic regions HR1 and HR2. Here, we investigated the importance of regions outside HR1 and HR2 for multimerization, increasing membrane permeability, subcellular localization, and virus replication through analysis of linker insertion and substitution mutants. From these studies, the following conclusions could be drawn. (i) The hydrophilic region ((58)RNHDD(62)) between HR1 and HR2 is critical for multimerization and increasing membrane permeability. Substitution analysis of Asn(61) and Asn(62) demonstrated the preference for short polar side chains (Asp, Asn), residues that are often present in turns, over long polar side chains (Glu, Gln). This finding supports the idea that the hydrophilic region is involved in pore formation by facilitating a turn between HR1 and HR2 to reverse chain direction. (ii) Studies undertaken to define the downstream boundary of HR2 demonstrated that the aromatic residues Trp(80) and Trp(82), but not the positively charged residues Arg(81), Lys(84), and Lys(86) are important for increasing membrane permeability. (iii) The N terminus is not required for multimerization but does contribute to the membrane-active character of 2B. (iv) The subcellular localization of 2B does not rely on regions outside HR1 and HR2 and does not require multimerization. (v) Virus replication requires both the membrane-active character and an additional function of 2B that is not connected to this activity.

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

The human La (SS-B) autoantigen interacts with DDX15/hPrp43, a putative DEAH-box RNA helicase.

The human La (SS-B) autoantigen is an abundantly expressed putative RNA chaperone, functioning in various intracellular processes involving RNA. To further explore the molecular mechanisms by which La functions in these processes, we performed large-scale immunoprecipitations of La from HeLa S100 extracts using the anti-La monoclonal antibody SW5. La-associated proteins were subsequently identified by sequence analysis. This approach allowed the identification of DDX15 as a protein interacting with La. DDX15, the human ortholog of yeast Prp43, is a member of the superfamily of DEAH-box RNA helicases that appeared to interact with La both in vivo and in vitro. The region needed for the interaction with La partly overlaps the DEAH-box domain of DDX15. Immunofluorescence data indicated that endogenous DDX15 accumulates in U snRNP containing nuclear speckles in HEp-2 cells. Surprisingly DDX15 also accumulates in the nucleoli of HEp-2 cells. Moreover, DDX15 and La seem to colocalize in the nucleoli. Regions of DDX15 involved in nuclear, nuclear speckle, and nucleolar localization are located within the N- and C-terminal regions flanking the DEAH-box. RNA coprecipitation experiments indicated that DDX15 is associated with spliceosomal U small nuclear RNAs in HeLa cell extracts. The possible functional implications of the interaction between La and DDX15 are discussed.

Multimerization reactions of coxsackievirus proteins 2B, 2C and 2BC: a mammalian two-hybrid analysis.

Recently, homomultimerization and heteromultimerization reactions of the poliovirus P2 region proteins were investigated using a yeast two-hybrid approach (Cuconati et al., Journal of Virology 72, 1297-1307, 1998). In this study, we investigated multimerization reactions of the 2B, 2C and 2BC proteins of the closely related coxsackie B3 virus (CBV3) using a mammalian two-hybrid system. This system allows the characterization of protein:protein interactions within a cellular environment that more closely mimics the native protein environment. Homomultimerization reactions were observed with the 2BC protein and, albeit weakly, with the 2B protein, but not with the 2C protein. To identify the determinants involved in the 2BC and 2B homomultimerization reactions, several mutants containing deletions or point mutations in the 2B region were tested. Disruption of the hydrophobic character of either the cationic amphipathic alpha-helix or the second hydrophobic domain of the 2B protein disturbed both the 2BC:2BC and the 2B:2B homomultimerization reactions. Disruption of either the cationic or the amphipathic character of the alpha-helix or deletion of the N-terminal 30 amino acids of the 2B protein, however, had no effect on the 2BC and 2B homomultimerization reactions. Heteromultimerization reactions were observed between proteins 2BC and 2B, and also between proteins 2BC and 2C, but not between the 2B and 2C proteins. The 2BC:2B and 2BC:2C heteromultimerization reactions were also mediated by hydrophobic determinants located in the amphipathic alpha-helix and the second hydrophobic domain. The nature of the interactions and their implications for the virus life-cycle are discussed.