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Contains fulltext : 172329.pdf (Publisher’s version ) (Closed access) Derailment of immune responses can lead to autoimmune type 1 diabetes, and this can be accelerated or even induced by local stress caused by inflammation or infection. Dendritic cells (DCs) shape both innate and adaptive immune responses. Here, we report on the responses of naturally occurring human myeloid BDCA1(+) DCs towards differentially stressed pancreatic beta cells. Our data show that BDCA1(+) DCs in human pancreas-draining lymph node (pdLN) suspensions and blood-derived BDCA1(+) DCs both effectively engulf beta cells, thus mimicking physiological conditions. Upon uptake of enterovirus-infected, but not mock-infected cells, BDCA1(+) DCs induced interferon (IFN)-alpha/beta responses, co-stimulatory molecules and proinflammatory cytokines and chemokines. Notably, induction of stress in beta cells by ultraviolet irradiation, culture in serum-free medium or cytokine-induced stress did not provoke strong DC activation, despite efficient phagocytosis. DC activation correlated with the amount of virus used to infect beta cells and required RNA within virally infected cells. DCs encountering enterovirus-infected beta cells, but not those incubated with mock-infected or stressed beta cells, suppressed T helper type 2 (Th2) cytokines and variably induced IFN-gamma in allogeneic mixed lymphocyte reaction (MLR). Thus, stressed beta cells have little effect on human BDCA1(+) DC activation and function, while enterovirus-infected beta cells impact these cells significantly, which could help to explain their role in development of autoimmune diabetes in individuals at risk.

Aims/hypothesisVirally induced inflammatory responses, beta cell destruction and release of beta cell autoantigens may lead to autoimmune reactions culminating in type 1 diabetes. Therefore, viral capability to induce beta cell death and the nature of virus-induced immune responses are among key determinants of diabetogenic viruses. We hypothesised that enterovirus infection induces a specific gene expression pattern that results in islet destruction and that such a host response pattern is not shared among all enterovirus infections but varies between virus strains.MethodsThe changes in global gene expression and secreted cytokine profiles induced by lytic or benign enterovirus infections were studied in primary human pancreatic islet using DNA microarrays and viral strains either isolated at the clinical onset of type 1 diabetes or capable of causing a diabetes-like condition in mice.ResultsThe expression of pro-inflammatory cytokine genes (IL-1-α, IL-1-β and TNF-α) that also mediate cytokine-induced beta cell dysfunction correlated with the lytic potential of a virus. Temporally increasing gene expression levels of double-stranded RNA recognition receptors, antiviral molecules, cytokines and chemokines were detected for all studied virus strains. Lytic coxsackievirus B5 (CBV-5)-DS infection also downregulated genes involved in glycolysis and insulin secretion.Conclusions/interpretationThe results suggest a distinct, virus-strain-specific, gene expression pattern leading to pancreatic islet destruction and pro-inflammatory effects after enterovirus infection. However, neither viral replication nor cytotoxic cytokine production alone are sufficient to induce necrotic cell death. More likely the combined effect of these and possibly cellular energy depletion lie behind the enterovirus-induced necrosis of islets.

Item does not contain fulltext Enteroviral infections go usually unnoticed, even during pregnancy, yet some case histories and mouse experiments indicate that these viruses may be transmitted vertically. More frequently, however, transmission occurs by (fecal) contamination during and shortly after birth. The aim of this study was to investigate the effect of maternal infection in mice (1) on gravidity outcome and (2) on subsequent challenge of the offspring with the same virus. CD1 outbred female mice were infected by the oral route with coxsackievirus B4 strain E2 or mock-infected at days 4, 10, or 17 of gestation. Weight and signs of sickness were noted daily. Pups were infected at day 25 after birth (4 days postweaning). Organs (brain, pancreas, and heart) were analyzed for viral RNA and histopathology. We observed that maternal infection at day 4 or day 17 of gestation had little effect on pregnancy outcome, whereas infection at day 10 affected dams and/or offspring. Infection of pups resulted in severe inflammation of the pancreas, but only when dams were previously infected, especially at day 17. The blood glucose levels were elevated. Because no trace of infection was found at the time of challenge, a role for immunopathology is suggested. 01 maart 2012

Contains fulltext : 88377.pdf (Publisher’s version ) (Open Access) Previous studies have shown that enteroviral RNA can be detected in blood at the onset of type 1 diabetes (T1D). The infection may play a role in triggering T1D and genetic host factors may contribute to this process. We investigated (1) whether enterovirus is present at the onset of T1D in peripheral blood mononuclear cells (PBMC), plasma, throat, or stool, and (2) whether enteroviral presence is linked with HLA-DR type and/or polymorphisms in melanoma differentiation-associated gene 5 (MDA5) and 2'-5' oligoadenylate synthetase 1 (OAS1), factors of antiviral immunity. To this end, PBMC, plasma, throat, and stool samples from 10 T1D patients and 20 unrelated controls were tested for the presence of enteroviruses (RT-PCR), for HLA-DR type, and polymorphisms in MDA5 and OAS1. Enterovirus RNA was detected in PBMC of 4/10 T1D patients, but none of 20 controls. Plasma was positive in 2/10 T1D patients and none of 20 controls, suggesting that enteroviruses found at the onset of T1D are mainly present in PBMC. All throat samples from positive T1D patients were virus-negative and only 1 fecal sample was positive. The negative results for all throat and most stool samples argues against acute infection. Enterovirus presence was linked with HLA-DR4, but not with polymorphisms in MDA5 or OAS1. 01 februari 2010

OBJECTIVEType 1 diabetes is a chronic endocrine disorder in which enteroviruses, such as coxsackie B viruses and echoviruses, are possible environmental factors that can trigger or accelerate disease. The development or acceleration of type 1 diabetes depends on the balance between autoreactive effector T-cells and regulatory T-cells. This balance is particularly influenced by dendritic cells (DCs). The goal of this study was to investigate the interaction between enterovirus-infected human pancreatic islets and human DCs.RESEARCH DESIGN AND METHODSIn vitro phagocytosis of human or porcine primary islets or Min6 mouse insuloma cells by DCs was investigated by flow cytometry and confocal analysis. Subsequent innate DC responses were monitored by quantitative PCR and Western blotting of interferon-stimulated genes (ISGs).RESULTSIn this study, we show that both mock- and coxsackievirus B3 (CVB3)-infected human and porcine pancreatic islets were efficiently phagocytosed by human monocyte–derived DCs. Phagocytosis of CVB3-infected, but not mock-infected, human and porcine islets resulted in induction of ISGs in DCs, including the retinoic acid–inducible gene (RIG)-I–like helicases (RLHs), RIG-I, and melanoma differentiation–associated gene 5 (Mda5). Studies with murine Min6 insuloma cells, which were also efficiently phagocytosed, revealed that increased ISG expression in DCs upon encountering CVB-infected cells resulted in an antiviral state that protected DCs from subsequent enterovirus infection. The observed innate antiviral responses depended on RNA within the phagocytosed cells, required endosomal acidification, and were type I interferon dependent.CONCLUSIONSHuman DCs can phagocytose enterovirus-infected pancreatic cells and subsequently induce innate antiviral responses, such as induction of RLHs. These responses may have important consequences for immune homeostasis in vivo and may play a role in the etiology of type 1 diabetes.

Dendritic cells (DCs) play a central role in instructing antiviral immune responses. DCs, however, can become targeted by different viruses themselves. We recently demonstrated that human DCs can be productively infected with echoviruses (EVs), but not coxsackie B viruses (CVBs), both of which are RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. We now show that phagocytosis of CVB-infected, type I interferon-deficient cells induces an antiviral state in human DCs. Uptake of infected cells increased the expression of the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5 as well as other interferon-stimulated genes and protected DCs against subsequent infection with EV9. These effects depended on recognition of viral RNA and could be mimicked by exposure to the synthetic double-stranded RNA analogue poly(I:C) but not other Toll-like receptor (TLR) ligands. Blocking endosomal acidification abrogated protection, suggesting a role for TLRs in the acquisition of an antiviral state in DCs. In conclusion, recognition of viral RNA rapidly induces an antiviral state in human DCs. This might provide a mechanism by which DCs protect themselves against viruses when attracted to an environment with ongoing infection.

Contains fulltext : 51866.pdf (Publisher’s version ) (Closed access) Coxsackie B viruses (CVB) and Echoviruses (EV) form a single species; Human enterovirus B (HeV-B), within the genus Enterovirus. Although HeV-B infections are usually mild or asymptomatic, they can cause serious acute illnesses. In addition, HeV-B infections have been associated with chronic immune disorders, such as type 1 diabetes mellitus and chronic myocarditis/dilated cardiomyopathy. It has therefore been suggested that these viruses may trigger an autoimmune process. Here, we demonstrate that human dendritic cells (DCs), which play an essential role in orchestration of the immune response, are productively infected by EV, but not CVB strains, in vitro. Infection does not result in DC activation or the induction of antiviral immune responses. Instead, EV infection rapidly impedes Toll-like receptor-mediated production of cytokines and upregulation of maturation markers, and ultimately causes loss of DC viability. These results describe for the first time the effect of EV on the function and viability of human DCs and suggest that infection of DCs in vivo can impede regulation of immune responses.

Contains fulltext : 51814.pdf (Publisher’s version ) (Closed access)

A systemic review is presented of all studies that have evaluated the inhibitory quotient (IQ). The IQ is defined as the ratio between (trough) drug concentration and level of drug resistance of the HIV isolate. From the studies presented, it can be concluded that for protease inhibitors (PIs) and efavirenz, the phenotypic IQ is associated with virological response. The genotypic IQ (GIQ) for PIs was also demonstrated to be associated with virological response. An intrinsic limitation of the GIQ is that it is only applicable for PIs, of which resistance is based on the cumulative effect of mutations. As the IQ can be modified by adjustment of the drug dosage, it may be of clinical value. Its application in patient care should therefore be further investigated.

Contains fulltext : 57742.pdf (Publisher’s version ) (Closed access) OBJECTIVES: To investigate the association of viral infections and febrile seizures (FS). STUDY DESIGN: From April 1998 to April 2002, a prospective, population-based study was carried out among general practitioners to assess the incidence of FS in their practices. Data thus obtained were compared with the incidence of common viral infections recorded in a national registry. Poisson regression analysis was performed to investigate whether the season or the type of infection was associated with the variation observed in FS incidence. RESULTS: Throughout the 4-year period, 267 of 303 (88%) of general practitioners in the Dutch province of Friesland participated in the study. The estimated observation period was approximately 160,000 patient-years. We registered 654 cases of FS in 429 children. The estimated incidence of FS was 2.4 in 1000 patient-years. Poisson regression analysis revealed a positive correlation between recurrent FS and influenza A ( P = .01). CONCLUSIONS: Our study suggests a relation between recurrent FS and influenza A. Influenza vaccination should be considered in all children with a history of FS.

Cytomegalovirus (CMV) is one of the most common causes of congenital infections in developed countries with reported incidences varying between 0.15% and 2.0%. The effects of congenital CMV infection may vary from a congenital syndrome to an asymptomatic course. Infants that are asymptomatic at birth may still present handicaps at a later age. It is generally accepted that symptoms of congenitally infected children are more severe after primary infection than after recurrent infection. In this article, we present two case reports which demonstrate that the outcome of recurrent maternal CMV infection may be severe. In the first case, early pregnancy serology showed positive IgG and IgM, but negative IgA, whereas at the time of diagnosed fetal death, 5 weeks later, there was only positive IgG. The second case showed positive IgG and negative IgM and IgA both in early pregnancy and after delivery. Since in both cases CMV was isolated from several organs, these findings are compatible with recurrent rather than primary CMV infection. In the reported patients, fetal death and necrotising enterocolitis occurred after a congenital CMV infection, with mothers having pre-existing immunity to CMV. In conclusion, these case reports and review of the literature emphasise that the outcome of recurrent maternal CMV infection may be severe and that congenital CMV infection should be considered in cases of pregnancy loss and necrotising enterocolitis with recurrent maternal CMV infection.

Item does not contain fulltext Cytomegalovirus (CMV) is one of the most common viral causes of congenital infection. A future decision to lower its incidence by vaccination will depend on epidemiological conditions within a country and on the safety of the vaccine to be used, because a life vaccine may cause latency and subsequent reactivation that still may harm the fetus. The aim was to review the epidemiological studies published so far, with respect to factors that affect the incidence of congenital CMV infection, and factors that may influence its outcome, such as preexisting maternal immunity. The study included the data of 19 studies that were retrieved from a MEDLINE search during the period 1977 to 1997. The incidence of congenital CMV infection varied between 0.15% and 2.0% and seemed to correlate with the level of preexisting immunity in the population. Although preexisting maternal immunity was reported to strongly reduce transmission, the severity of congenital CMV infection (symptoms at birth and or sequelae later in life) was not significantly greater after virus transmission due to a primary infection of the mother as compared with recurrence or reinfection. The data indicate that preexisting immunity of the mother does not significantly mitigate the outcome of congenital infection. Moreover, life vaccines may bear a serious risk when transmittable to the fetus. TARGET AUDIENCE: Obstetricians and Gynecologists, Family Physicians. LEARNING OBJECTIVES: After completion of this article, the reader will be able to describe the natural course of a CMV infection, to list the potential sequelae of a congenital CMV infection, to outline potential strategies to prevent transmission of CMV, and to summarize the diagnostic work up of a patient with a potential CMV infection.

Type I diabetes mellitus results from the autoimmune destruction of insulin producing beta cells in the pancreas. Certain viral infections, especially those caused by coxsackie B viruses and related enteroviruses, have been associated with the development of type I diabetes. The sequence homology between the coxsackie B4 virus nonstructural protein 2C (CVB4 p2C) and the major diabetes autoantigen glutamic acid decarboxylase (GAD65) provides a basis for the hypothesis of molecular mimicry. In this study, we investigated the prevalence of antibodies directed against nonstructural enterovirus proteins. In additon a correlation of antibodies against CVB4 p2C and GAD65 was studied in diabetes patients and in healthy controls. Antibody reactivity against CVB proteins was detected by immunoprecipitation of [35S]-methionine-labelled viral proteins and GAD65 antibodies were measured in a quantitive radio-immunoassay. It was shown that antibodies raised against the nonstructural proteins of CVB4 are very common in the population and a high degree of heterotypic cross-reactivity exists between different enterovirus types. CVB4 p2C specific antibodies were not only detectable in GAD65 antibody-positive diabetes patients but also in GAD54 antibody-negative healthy blood donors. Furthermore, GAD65 antibodies could not be detected in p2C-positive subjects who had various enterovirus infections, indicating that an antibody response to CVB4 p2C does not necessarily induce a cross-reactive immune response against GAD65. A correlation was not found between antibodies against GAD65 and p2C.

Contains fulltext : 70481.pdf (Publisher’s version ) (Closed access) An immunocompromized, VZV-vaccinated child had a breakthrough infection with VZV, acquired at a day-care centre during a chickenpox outbreak. Interestingly, the infection manifested as herpes zoster of 1 dermatome. Typing showed wild-type virus, which suggests that exogenous reinfection with a new strain may present as herpes zoster.

Contains fulltext : 51011.pdf (Publisher’s version ) (Closed access) For 95 protease inhibitor-experienced HIV-1-infected patients, the genotypic inhibitory quotient (GIQ; trough level/number of mutations) was calculated for lopinavir. Three different sets of mutations showed equal predictive value. However, the use of cumulative numbers of mutations for calculation of the GIQ showed significantly better association with the virological response. Furthermore, the predictive value of the GIQ was no different from that of the number of mutations alone.

Contains fulltext : 48025.pdf (Publisher’s version ) (Closed access) During 2 consecutive influenza seasons we investigated the presence of influenza virus, human herpesvirus (HHV) type 6, and HHV-7 in cerebrospinal fluid samples from 9 white children suffering from influenza-associated encephalopathy. We conclude that it is unlikely that neuroinvasion by influenza virus or reactivation of either HHV-6 or HHV-7 is involved.

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.

Contains fulltext : 110765.pdf (Publisher’s version ) (Open Access) Enteroviruses of the human enterovirus B species (HEV-Bs) (e.g., coxsackie B viruses [CVBs] and echoviruses) have been implicated as environmental factors that trigger/accelerate type 1 diabetes, but the underlying mechanism remains elusive. The aim of this study was to gain insight into the cytokines and chemokines that are produced by human pancreatic islets upon infection with CVBs. To this end, we studied the response of human islets of Langerhans upon mock or CVB3 infection. Using quantitative PCR, we showed that upon CVB3 infection, transcription of interferon (IFN), IFN-stimulated genes, and inflammatory genes was induced. Analysis of secreted cytokines and chemokines by Luminex technology confirmed production and secretion of proinflammatory cytokines (e.g., interleukin [IL]-6 and tumor necrosis factor-alpha) as well as various chemotactic proteins, such as IFN-gamma-induced protein 10, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and IL-8. Infection with other HEV-Bs induced similar responses, yet their extent depended on replication efficiency. Ultra violet-inactivated CVB3 did not induce any response, suggesting that virus replication is a prerequisite for antiviral responses. Our data represent the first comprehensive overview of inflammatory mediators that are secreted by human islets of Langerhans upon CVB infection and may shed light on the role of enteroviruses in type 1 diabetes pathogenesis. 01 augustus 2012

The applicability of the polymerase chain reaction (PCR) for the diagnosis of enteroviral infections is evaluated in this study. A general primer-mediated enterovirus specific PCR was used for the detection of enteroviral RNA in cerebrospinal fluid (CSF) samples from six patients with meningitis, 56 biopsy specimens from 16 patients with congestive heart failure, and two patients with a systemic enteroviral infection. Samples from three patients with meningitis were found positive and in each of eight patients with heart disease, enteroviral RNA was detected in at least one biopsy specimen. From both patients with a systemic enteroviral infection several different organs and tissues were found positive. After reviewing the literature, we concluded that the PCR might become a useful tool for the diagnosis of persistent enteroviral infections and enteroviral-induced meningitis, but that the applicability of PCR for the routine diagnosis of acute enteroviral infections is questionable.

Lombardi et al . (Reports, 23 October 2009, p. 585) reported detection of the human gammaretrovirus XMRV in the blood cells of patients with chronic fatigue syndrome (CFS). However, the patient description provided was incomplete. The inclusion of patients from a “CFS outbreak” previously linked with a viral infection, without confirmation in sporadic CFS cases, casts doubt on the role of XMRV in the pathogenesis of CFS.

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 105 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.

Since 1976, when general immunization against measles was introduced in the Netherlands, all new cases of subacute sclerosing panencephalitis (SSPE) were registered and detailed data about immunization, epidemiology and disease progression were collected on them. Up to 1991, 99 new patients have been registered of which 81 were born in this country and 18 elsewhere. From 1981 onwards, the incidence of SSPE among those born in the Netherlands decreased gradually from 13 cases per year to one case per year. This decrease is attributed to the large scale of immunization against measles. Three SSPE patients had been immunized against measles, all of them without a history of clinical measles. Epidemiology and risk factors of SSPE did not differ from those reported in other countries. An exceptional cluster of four patients in one town, who had measles in the same year, is reported. Progression of SSPE appeared to be age related. A total of 28 patients was treated with Inosiplex; no significant effect on survival in stage 3 of the disease was found.

The family Picornaviridae contains well-known human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and parechovirus). In addition, this family contains a number of viruses that infect animals, including members of the genus Cardiovirus such as Encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelits virus (TMEV). The latter are important murine pathogens that cause myocarditis, type 1 diabetes and chronic inflammation in the brains, mimicking multiple sclerosis. Recently, a new picornavirus was isolated from humans, named Saffold virus (SAFV). The virus is genetically related to Theiler's virus and classified as a new species in the genus Cardiovirus, which until the discovery of SAFV did not contain human viruses. By analogy with the rodent cardioviruses, SAFV may be a relevant new human pathogen. Thus far, SAFVs have sporadically been detected by molecular techniques in respiratory and fecal specimens, but the epidemiology and clinical significance remained unclear. Here we describe the first cultivated SAFV type 3 (SAFV-3) isolate, its growth characteristics, full-length sequence, and epidemiology. Unlike the previously isolated SAFV-1 and -2 viruses, SAFV-3 showed efficient growth in several cell lines with a clear cytopathic effect. The latter allowed us to conduct a large-scale serological survey by a virus-neutralization assay. This survey showed that infection by SAFV-3 occurs early in life (>75% positive at 24 months) and that the seroprevalence reaches >90% in older children and adults. Neutralizing antibodies were found in serum samples collected in several countries in Europe, Africa, and Asia. In conclusion, this study describes the first cultivated SAFV-3 isolate, its full-length sequence, and epidemiology. SAFV-3 is a highly common and widespread human virus causing infection in early childhood. This finding has important implications for understanding the impact of these ubiquitous viruses and their possible role in acute and/or chronic disease., Author Summary Recently, a new picornavirus was isolated from humans, named Saffold virus (SAFV). Picornaviruses are small RNA viruses with poliovirus as prototype. Saffold virus is genetically related to Theiler's virus, a member of the Cardiovirus genus, which until this recent discovery did not contain human viruses. Theiler's virus is an important mouse pathogen that causes chronic inflammation in the brains, closely resembling multiple sclerosis in humans. By analogy, SAFV may be a relevant human pathogen. Thus far, SAFVs have been sporadically detected by molecular techniques in respiratory and fecal specimens, but the epidemiology and clinical significance have remained unclear. Here we describe the first SAFV type 3 (SAFV-3) isolate, its growth characteristics in cell lines, full-length RNA-sequence, and epidemiology. Unlike the previously isolated SAFV-1 and SAFV-2, SAFV-3 grows well in cell lines, resulting in cell damage. This feature enabled us to conduct a large-scale serological survey for virus-neutralizing antibodies. This survey showed that SAFV-3 infection occurs early in life and that >90% of children >2 years and adults had antibodies. Neutralizing antibodies were found in serum samples collected in several countries in three continents. Hence, we concluded that SAFV-3 is a genuine and widespread human virus causing infection early in life.

Contains fulltext : 80397.pdf (Publisher’s version ) (Closed access) Based on a comparison of the phylogeny of two distant regions, evidence has been found for recombination within parechoviruses. However, recombination breakpoints could not be detected in this way. We searched for potential recombination breakpoints in parechovirus by analysis of complete parechovirus sequences, including a newly isolated strain. Bootscan analysis demonstrated that parechoviruses are mosaic viruses build of regions related to corresponding genomic regions of other parechoviruses. With a genetic algorithm for recombination detection, sites for recombination were found. Analysis of partial sequences, as defined by recombination breakpoints, showed phylogenetic segregation between regions.

We describe a renal transplant patient with a primary Toxoplasma gondii infection presenting as pneumonitis, with subsequent chorioretinitis and encephalitis. The diagnostic challenges of T. gondii infection in immunocompromised patients are discussed.

Contains fulltext : 59039.pdf (Publisher’s version ) (Open Access) Recent studies suggest that inflammation plays a central role in the pathogenesis of atherosclerosis, and IFN-gamma is a prominent proinflammatory mediator in this context. However, it is unclear what stimuli are responsible for initial stimulation of IFN-gamma synthesis in the vessel wall. In the present study, we demonstrate that Chlamydia pneumoniae is an important stimulus for IFN-gamma synthesis, and this production depends on release of endogenous IL-18, IL-12, and IL-1, but not of TNF. The production of the proinflammatory cytokines TNF and IL-1beta from PBMC by sonicated C. pneumoniae was mediated through TLR2-dependent pathways. In contrast, C. pneumoniae stimulated the production of IL-18 through MyD88-dependent, TLR2-, TLR4-, and CD14-independent pathways, mediated by posttranscriptional mechanisms not involving de novo protein synthesis. In conclusion, C. pneumoniae is a potent stimulus of IFN-gamma production, in addition to the proinflammatory cytokines TNF and IL-1beta, which may contribute to its proatherogenic effects. Most interestingly, C. pneumoniae is also a potent inducer of IL-18 production through pathways independent of TLR2 and TLR4.

Item does not contain fulltext Recent studies suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, and that cytokines play an important role in the initiation and progression of Chlamydia-induced inflammation. When freshly isolated peripheral blood mononuclear cells (PBMC) were stimulated for 24 h with sonicated C. pneumoniae, significant amounts of the pro-inflammatory cytokines TNF-alpha and IL-1beta and of the anti-inflammatory cytokine IL-10 were released into the supernatant. The addition of serum increased cytokine release induced by C. pneumonia two- to fivefold (p < 0.01). This effect was not due to complement, mannose-binding lectin (MBL) or lipopolysaccharide-binding protein (LBP). Incubation of PBMC with either anti-Toll-like receptor 4 (TLR4) or anti-CD14 blocking antibodies did not influence the production of cytokines induced by Chlamydia. The induction of cytokines by C. pneumoniae in macrophages from C3H / HeJ mice, known to have a defective TLR4, was identical to that measured in control macrophages from C3H / HeN mice. In contrast, incubation of PBMC with an anti-TLR2 blocking antibody significantly inhibited the production of TNF by 67 % and of IL-1beta by 72 %. In conclusion, C. pneumoniae stimulates cytokine production in a serum-dependent manner, but independently of complement, MBL and LBP. C. pneumoniae induces the pro-inflammatory cytokines TNF and IL-1beta through TLR2, but not TLR4 and CD14.

The secondary structures predicted for the enteroviral 3′ nontranslated region (3′NTR) all seem to indicate a conformation consisting of two (X and Y) hairpin structures. The higher-order RNA structure of the 3′NTR appears to exist as an intramolecular kissing interaction between the loops of these two hairpin structures. The enterovirus B-like subgroup possesses an additional stem-loop structure, domain Z, which is not present in the poliovirus-like enteroviruses. It has been suggested that the Z domain originated from a burst of short sequence repetitions (E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V. I. Agol, Nucleic Acids Res. 20:1739-1745, 1992). However, no functional features have yet been ascribed to this enterovirus B-like-specific RNA element in the 3′NTR. In this study, we tested the functional characteristics and biological significance of domain Z. A mutant of the cardiovirulent coxsackievirus group B3 strain Nancy which completely lacked the Z domain and which therefore acquired enterovirus C-like secondary structures exhibited a wild-type growth phenotype, as determined by single-cycle growth analysis with BGM cells. This result proves that the Z domain is virtually dispensable for viral growth in tissue cultures. Partial distortion of the Z domain structure resulted in a disabled virus with reduced growth kinetics, probably due to alternative conformations of the overall structure of the domain. Infection of mice showed that the recombinant coxsackievirus group B3 mutant which completely lacked the Z domain was less virulent. Pancreatic tissues from mice infected with wild-type virus and recombinant virus were equally affected. However, the heart tissue from mice infected with the recombinant virus showed only slight signs of myocarditis. These results suggest that the enterovirus B-like-specific Z domain plays a role in coxsackievirus-induced pathogenesis.

Enterovirus infections have been implicated in the development of type I diabetes mellitus. They may cause beta cell destruction either by cytolytic infection in the pancreas or indirectly by contributing to autoimmune reactivity. We sought evidence for these 2 mechanisms in a case of acute-onset diabetes mellitus that occurred during severe echovirus 9 infection. The virus was isolated and administered to cultured human beta cells. No viral proliferation was observed, and no beta cell death was induced, while parallel exposure to Coxsackie B virus serotype 3 resulted in viral proliferation and massive beta cell death. Although the viral protein 2C exhibited a sequence similar to that of the beta cell autoantigen glutamic acid decarboxylase (GAD(65)), no cross-reactive T cell responses were detected. The patient did not develop antibodies to GAD(65) either. Absence of evidence for direct cytolytic action or an indirect effect through molecular mimicry with GAD(65) in the present case raises the possibility of another indirect pathway through which enteroviruses can cause diabetes mellitus.

Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro-inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF-alpha, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF-binding protein and blockade of IL-1 receptors by IL-1 receptor antagonist revealed that TNF, IL-1 and IL-6 production was independent from each other, whereas IL-8 synthesis was strongly dependent on endogenous TNF and IL-1. In contrast, synthesis of MCP-1 and MIP-1alpha was dependent on endogenous TNF, but not IL-1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.

Enteroviruses haven been associated with autoimmune type 1 diabetes development, however the possible underlying mechanism remains unclear. Dendritic cells (DCs) are the major antigen-presenting cells (APCs) and initiate immune responses against pathogens, but also prevent (auto-)immune responses harmful to the host. We hypothesize that enterovirus- infected β-cells that are phagocytosed by DCs can unleash the immune system and eventuate in β-cell-autoimmunity in genetically susceptible individuals. Therefore we investigate the interaction between primary BDCA1+ DCs and enterovirus- infected pancreatic β-cells.

Summary A case of agammaglobulinaemia with hyper IgM (the ‘hyper-IgM syndrome') is described, in which a serological diagnosis of cytomegalovirus infection was made during repeated episodes of hepatitis. Three unrelated serological tests agreed with each other, but eventually it appeared that the reactions were non-specific. This shows the limitation of confirmation by tests which are used routinely.