A preliminary study into performing routine tube output and automatic exposure control quality assurance using radiology information system data.

Data are currently being collected from hospital radiology information systems in the North West of the UK for the purposes of both clinical audit and patient dose audit. Could these data also be used to satisfy quality assurance (QA) requirements according to UK guidance? From 2008 to 2009, 731 653 records were submitted from 8 hospitals from the North West England. For automatic exposure control QA, the protocol from Institute of Physics and Engineering in Medicine (IPEM) report 91 recommends that milliamperes per second can be monitored for repeatability and reproducibility using a suitable phantom, at 70-81 kV. Abdomen AP and chest PA examinations were analysed to find the most common kilovoltage used with these records then used to plot average monthly milliamperes per second with time. IPEM report 91 also recommends that a range of commonly used clinical settings is used to check output reproducibility and repeatability. For each tube, the dose area product values were plotted over time for two most common exposure factor sets. Results show that it is possible to do performance checks of AEC systems; however more work is required to be able to monitor tube output performance. Procedurally, the management system requires work and the benefits to the workflow would need to be demonstrated.

RNA integrity in post mortem human variant Creutzfeldt-Jakob disease (vCJD) and control brain tissue.

AIMS: To determine premortem and post mortem factors affecting quality and yield of RNA isolated from the unique archived brain material in the UK National Creutzfeldt-Jakob Disease Surveillance Unit Brain and Tissue Bank and to compare this to control brain tissue with no neurological disease. METHODS: In parallel and in replicate, RNA was prepared from the frontal parasagittal or subfrontal cortex of samples dissected from half brains (frozen intact) or from brain samples snap frozen or placed in RNALater. A total of 350 RNA samples from 78 human autopsy cases, 21 variant Creutzfeldt-Jakob disease, 26 other neurological diseases and 31 non-neurological diseases were studied. RESULTS: There was no difference in the quality or yield of RNA isolated from variant Creutzfeldt-Jakob disease, other neurological disease and non-neurological disease brains. RNA preparations from archived frozen half brains or snap frozen autopsy samples were generally of poor quality (RNA integrity number<5). There was a highly significant negative correlation between the number of times frozen half brains had been sampled and the quality of RNA. Samples stored in RNALater provided higher-quality RNA (RNA integrity number>5). Age at death, gender, post mortem interval and freezer storage time had no effect on RNA quality. CONCLUSION: Reasonable-quality RNA can be isolated from samples dissected from archived frozen human half brains but repeated sampling results in RNA degradation. Better-quality RNA is obtained from samples placed in RNALater than from snap frozen samples. The quality and yield of RNA are not affected by age at death, gender, post mortem interval of >6 h or freezer storage time.

Application of a statistical software package for analysis of large patient dose data sets obtained from RIS.

For the purpose of patient dose audit, clinical audit and radiology workload analysis, data from Radiology Information Systems (RIS) at many hospitals are collected using a database and the analysis was automated using a statistical package and Visual Basic coding. The database is a Structured Query Language database, which can be queried using an off-the-shelf statistical package, Statistica. Macros were created to automatically format the data to a consistent format between different hospitals ready for analysis. These macros can also be used to automate further analysis such as detailing mean kV, mAs and entrance surface dose per room and per gender. Standard deviation and standard error of the mean are also generated. Graphs can also be generated to illustrate the trends in doses between different variables such as room and gender. Collectively, this information can be used to generate a report. A process that once could take up to 1 d to complete now takes around 1 h. A major benefit in providing the service to hospital trusts is that less resource is now required to report on RIS data, making the possibility of continuous dose audit more likely. Time that was spent on sorting through data can now be spent on improving the analysis to provide benefit to the customer. Using data sets from RIS is a good way to perform dose audits as the huge numbers of data available provide the bases for very accurate analysis. Using macros written in Statistica Visual Basic has helped sort and consistently analyse these data. Being able to analyse by exposure factors has provided a more detailed report to the customer.

Semliki Forest virus strongly reduces mosquito host defence signaling.

The Alphavirus genus within the Togaviridae family contains several important mosquito-borne arboviruses. Other than the antiviral activity of RNAi, relatively little is known about alphavirus interactions with insect cell defences. Here we show that Semliki Forest virus (SFV) infection of Aedes albopictus-derived U4.4 mosquito cells reduces cellular gene expression. Activation prior to SFV infection of pathways involving STAT/IMD, but not Toll signaling reduced subsequent virus gene expression and RNA levels. These pathways are therefore not only able to mediate protective responses against bacteria but also arboviruses. However, SFV infection of mosquito cells did not result in activation of any of these pathways and suppressed their subsequent activation by other stimuli.

Inflammatory responses in the nervous system of mice infected with a street isolate of rabies virus.

Rabies virus causes severe encephalitis that is invariably fatal for the victim. However, the contribution of the virus and the host to damage of the CNS is unclear. In order to investigate this we studied the neuropathology and CNS gene expression patterns in a murine model of rabies using a 'street' isolate RV61. This virus was derived from a human case of disease. In this model, infection of the CNS progresses rapidly following inoculation in the periphery, leading to extensive virus replication in the brain and the development of disease. However, previous studies have found little evidence of inflammation and lymphocyte infiltration in many regions of the CNS of infected mice. During the current study virus replication was detected in the dorsal root ganglia (DRG), spinal cord, brain and salivary gland at 11 days postinfection (dpi). Mononuclear cell infiltration was observed in the DRG and to a lesser extent, the spinal cord. Immunolabelling demonstrated that T-lymphocytes were the dominant population of infiltrating cells. Murine innate immune response gene transcripts were detected in the brain as early as 5 dpi. At 11 dpi, coincidentwith the onset of disease, elevated levels of mRNA transcripts were recorded for type-1 (alpha and beta) and type-2 interferon (gamma) and certain chemokines (CCL5 and CXCL10) with chemotactic properties for T-cells. We suggest that damage to the DRG and spinal cord could be due to a combination of both virus infection and the infiltration of T-cells.

Reduced in vitro adherence of Staphylococcus species to feline corneocytes compared to canine and human corneocytes.

It is apparent that in-contact humans and animals exchange commensal staphylococci. Previous in vitro studies, however, indicate that staphylococci preferentially adhere to corneocytes from host species. This study compared adherence of meticillin-sensitive and -resistant Staphylococcus aureus (MSSA/MRSA), S. intermedius, S. felis and S. hominis to feline, canine and human corneocytes acquired from 10 healthy subjects using adhesive tape discs. Adherent bacteria were counted using an image processing and analysis programme. Mean adherence of MSSA (P = 0.0009), MRSA (P = 0.0162) and S. intermedius (P = 0.0117), but not S. felis or S. hominis, to feline corneocytes was significantly lower than that to canine and human corneocytes. All the isolates had similar adherence to both human and canine corneocytes. S. felis was the most adherent species to feline corneocytes followed by S. intermedius, and then MSSA, MRSA and S. hominis. For dogs and humans, S. intermedius and S. felis were the most adherent, followed by MRSA and MSSA, and then S. hominis. These results do not reveal any preferential adherence of staphylococci to canine or human corneocytes. Poor adherence to feline corneocytes could suggest that cats are relatively resistant to pyoderma and cross-species transmission of staphylococci.

Differential expression of erythroid genes in prion disease.

We previously reported reduced expression of erythroid-associated factor (ERAF) within haematopoietic tissues of rodent scrapie models, suggesting an unrecognized role for the erythroid lineage in prion disease. In the present study, we compared the expression of a panel of erythroid genes within four murine scrapie models and five virus infection models with parallels to prion disease pathogenesis. We report that differential expression of erythroid genes is not limited to ERAF, and is a common feature of murine scrapie, dependent on host expression of cellular prion protein. In contrast, erythroid gene expression was not altered following virus infection. Whilst these results further implicate cells of the erythroid lineage in the peripheral pathogenesis of prion disease, analysis of blood from BSE-infected cattle and scrapie-infected sheep reveals that the extent of differential expression of erythroid genes within peripheral blood is not sufficient to provide a discriminatory diagnostic test.

Virus tropism, distribution, persistence and pathology in the corpus callosum of the Semliki Forest virus-infected mouse brain: a novel system to study virus-oligodendrocyte interactions.

The temporal course of cellular pathology in virus-infected oligodendrocytes in vivo is not well defined. Here we study these events in the mouse brain using a novel system in which large numbers of oligodendrocytes can be reproducibly infected. In the mouse, following extraneural inoculation, the A7(74) strain of the alphavirus Semliki Forest virus (SFV) is efficiently neuroinvasive and central nervous system (CNS) infection leads to predominantly perivascular lesions of immune-mediated demyelination. This study demonstrates that direct intracerebral inoculation with SFV A7(74) or the SFV1 vector results in dramatic, selective and widespread infection of the major white matter tract of the brain, the corpus callosum. Mature oligodendrocytes are the predominant cell type infected. Subsequent events are complex; early virus-induced necrotic death of infected cells is followed by apoptotic death of adjacent apparently uninfected cells. A strong inflammatory response and considerable myelin loss are evident from 10 days and virus-positive cells are not observed after this time. In contrast, in athymic nu/nu mice, in the absence of T-cell responses, no inflammatory infiltrates are observed and virus-infected cells persist for over 30 days with extensive vacuolation but less demyelination. The change from an early destructive to a potentially persistent infection of oligodendrocytes is likely to reflect activation of innate immune responses. Activation of peripheral innate defences by inoculation of poly I : C prior to CNS virus infection abrogates the widespread corpus callosum infection. This widespread infection of the corpus callosum provides a novel in vivo system in which to study virus-oligodendrocyte interactions.

Identification of up-regulated genes by array analysis in scrapie-infected mouse brains.

The major neuropathological features of the transmissible spongiform encephalopathies (TSEs) are well documented, however, the underlying molecular events are poorly defined. We have applied cDNA expression arrays and quantitative RT-PCR to the study of gene expression in the brain, and more specifically in the hippocampus, of the well-characterized ME7/CV mouse model of scrapie. The number of genes showing consistent, scrapie-associated changes in expression was limited, and was primarily restricted to glial-associated genes. Increased expression of genes encoding glial fibrillary acidic protein, vimentin, complement component 1q (alpha and beta polypeptides), cathepsin D, clusterin and cystatin C was evident in the hippocampus from 170 days after inoculation (dpi), with expression increasing thereafter to terminal disease (225-235 dpi). Elevation of gene expression preceded clinical disease by approximately 30 days, and coincided with a 20-day period in the ME7/CV model during which 50% of the CA1 hippocampal neurones are lost. Increased expression of cystatin C, an inhibitor of lysosomal cysteine proteases, is a novel finding in the context of TSE neuropathology and was confirmed by Western analysis and immunocytochemistry.

Semliki forest virus infection of laboratory mice: a model to study the pathogenesis of viral encephalitis.

Semliki Forest virus (SFV) infection of the laboratory mouse provides an experimental system to study the pathogenesis of viral encephalitis. Following extraneural inoculation the virus is efficiently neuroinvasive and crosses the blood-brain barrier to initiate perivascular foci of infection in neurons and oligodendrocytes. The outcome of infection ranges from clinically unapparent mild encephalitis to fatal panencephalitis. SFV infections of the developing nervous system are always highly destructive and are generally fatal. In contrast, SFV infections of the mature nervous system can result in persistent infection with no apparent cell loss. This dramatic difference is attributable to developmental changes in the interactions between virus and CNS cells. Antibody responses clear the systemic infection and control the CNS infection. CD8+ T-cells are required to generate the lesions of inflammatory demyelination which can be a feature of the neuropathology. This article reviews the pathogenesis of SFV encephalitis, describing the neuropathology and the mechanisms which underlie it and which may be fundamental to many viral encephalitides.

Investigation of frequency of active Borna disease virus infection in Scottish blood donors.

BACKGROUND AND OBJECTIVES: Borna disease virus (BDV) can infect a wide range of vertebrate species causing neurological disease. In order to ensure the safety of blood supplies, it is essential to monitor blood for emerging pathogens. MATERIALS AND METHODS: One-hundred individual white cell pellets and pools representing 25 000 plasma donations from human blood were screened for BDV by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: BDV RNA was not detected in any of the samples. CONCLUSIONS: The results indicate that BDV is not widely spread in the UK human population and does not represent a risk as a transfusion-transmitted agent.

Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis.

Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) contains a tripartite negative-sense RNA genome. The smallest RNA segment, S, encodes the nucleocapsid protein N and a nonstructural protein, NSs, in overlapping reading frames. We have generated a mutant virus lacking NSs, called BUNdelNSs, by reverse genetics. Compared with the wild-type (wt) virus, BUNdelNSs exhibited a smaller plaque size and generated titers of virus approximately 1 log lower. In mammalian cells, the mutant expressed greatly increased levels of N protein; significantly, the marked inhibition of host cell protein synthesis shown by wt virus was considerably impaired by BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed BALB/c mice with a slower time course than wt and exhibited a reduced cell-to-cell spread, and titers of virus in the brain were lower. In addition, the abrogation of NSs expression changed Bunyamwera virus from a noninducer to an inducer of an interferon-beta promoter. These results suggest that, although not essential for growth in tissue culture or in mice, the bunyavirus NSs protein has several functions in the virus life cycle and contributes to viral pathogenesis.

Phenotypic characterisation and infection of ovine microglial cells with Maedi-Visna virus.

Maedi-Visna Virus (MVV) infection of the central nervous system (CNS) results in pathological changes, the mechanisms of which are poorly understood. MVV preferentially infects cell of the monocyte/macrophage lineage in vivo. The neuroparenchymal microglial cells are the resident tissue macrophages in the CNS and therefore likely targets for MVV infection. However, no information is currently available on the susceptibility of these cells to MVV infection or their contribution to neuropathological changes as a result of MVV infection. Highly enriched primary ovine microglial cell cultures were set up from brain tissues of lambs. These cells were amoeboid or bipolar with spikes, a morphology consistent with microglial cells of other species, and stained positive for CD1, CD11a, CD11c, CD14, MHC-class I, MHC-class II, and beta-N-acetyl galactose, but not with markers of astrocytes or oligodendrocytes. These sheep microglial cells were permissive for MVV infection. Productive MVV infection resulted in selective transcriptional up-regulation of the pro-inflammatory cytokines TNFalpha and IL-6. In contrast, there was no change in levels of transcripts for TGFbeta1, IL-1beta, GM-CSF, IL-10, or IL-12. These data provide the first evidence that ovine microglial cells can support productive infection with MVV, and that this leads to a selective up-regulation of proinflammatory cytokines. These may contribute to visna neuropathology.

Altruistic cell suicide and the specialized case of the virus-infected nervous system.

Depending on their differentiation state, vertebrate neurones can commit suicide after neurotropic virus infection. Such suicide might be an evolved strategy in multicellular organisms for limiting virus expansion. Regulation of suicide in this context operates by a programme similar to that activated during embryogenesis or in response to nervous-system injury and disease. In contrast to immature neurones that can readily initiate apoptosis following infection, mature neurones are generally highly resistant and can survive for long periods if they remain functional. Mature, infected neurones might gain competence to die owing to the attuned activation of pathways that sensitize the cell to subsequent stress. The consequence of either perturbation of function as a result of viral persistence or a chronic but progressive loss of infected neurones might be a failure of key neural functions.

Aurothiolates enhance the replication of Semliki Forest virus in the CNS and the exocrine pancreas.

The A7(74) strain of Semliki Forest virus (SFV) is avirulent and the L10 strain virulent in adult mice. A7(74) infection of adult mouse brain gives rise to small discrete foci of infection which, in immunocompetent animals, are cleared within 10 days. In contrast L10 infection results in a widespread and fatal central nervous system infection. Aurothiolates are linear, 2-coordinate complexes in which two ligands are covalently bound on either side of a gold nucleus in a +1 oxidation state (gold (I)). Pretreatment of A7(74) infected mice with two distinct aurothiolates (sodium aurothiomalate and aurothioglucose) resulted in significantly increased brain virus titers, and large confluent areas of infection in the brain similar to the pattern of infection seen with the L10 strain. The gold (I) moiety of aurothiolates was demonstrated to be the active component, since thiomalic acid when administered alone had no potentiating effect on the infection. Although both aurothiolates allowed productive replication and spread of A7(74) within the nature mouse brain, enhanced neuronal destruction was not apparent. There were no significant changes in virus distribution in any other tissue except for the exocrine pancreas and the myocardium where widespread infection of the acinar cells and occasional infected myocytes were observed.

Transneuronal spread of Semliki Forest virus in the developing mouse olfactory system is determined by neuronal maturity.

Many neurotropic virus infections have been shown to be virulent in neonatal and suckling mice but avirulent in weaned mice. The neurotropic alphavirus Semliki Forest virus is a well-studied example of this and importantly the age-related change in neurovirulence of this virus has been shown to be independent of specific immune responses. During the first two postnatal weeks many major physiological changes including axonogenesis, synaptogenesis and myelination occur within the rodent CNS. To investigate whether these changes affect virus replication, spread and virulence we have studied the course of infection in the mouse olfactory system. The olfactory system is well-characterized with regard to its development and neuroanatomy and represents an important route of entry of many neurotropic viruses. Following Semliki Forest virus infection, mice younger than 14 days-of-age died from a fulminant panencephalitis, whilst those 15 days and older survived and cleared the infection. Microscopic examination of brains from mice inoculated intranasally either bilaterally or unilaterally and stained by in situ hybridization to detect viral RNA revealed spread of infection along neurites in a circuit-specific manner. Spread in the main olfactory bulb and to primary, secondary and tertiary olfactory connections was observed. In neonatal mice virus rapidly spread throughout the olfactory system and the temporal progress of the infection correlated with the known connectivity patterns of this system. Both anterograde and retrograde axonal spread were observed. During the first three postnatal weeks the rate and extent of virus spread decreased with increasing age. Spread of infection between specific structures was closely related to neuronal maturation. As olfactory system connections matured transmission of virus was curtailed. In mice inoculated at six weeks or six months-of-age infection was minimal in and rarely observed beyond the continually renewed olfactory nerve layer. The ability of this virus to replicate and, or spread in the CNS is clearly linked to neuronal maturation.

Virus infection induces neuronal apoptosis: A comparison with trophic factor withdrawal.

Multicellular organisms can employ a number of defences to combat viral replication, the most dramatic being implementation of a cell autonomous apoptotic process. The overall cost to the viability of an organism of losing infected cells by apoptosis may be small if the dying cells can be substituted. In contrast, suicide of irreplaceable cells such as highly specialised neurons may have a more dramatic, even fatal consequence. Previous in vitro approaches to understanding whether neurotropic viruses cause neurons to apoptose have utilised transformed cell lines. These are not in the appropriate state of differentiation to provide an accurate indication of events in vivo. We have chosen to characterise the ability of a model CNS disease-causing virus, Semliki Forest virus (SFV), to infect and trigger apoptosis in primary cultures of nerve growth factor (NGF)-dependent sensory neurons. These cells are known to die when deprived of NGF and constitute a useful indicator of apoptosis. We observe that infection causes cell death which bears the morphological hallmarks of apoptosis, this occurs even in the present of survival promoting NGF and is concomitant with new virus production. Using the TUNEL (transferase dUTP nick end labelling) technique we show that SFV-induced apoptosis involves DNA fragmentation and requires caspase (CED-3/ICE cysteine protease) activation, as does apoptosis induced by NGF-deprivation. Extensive areas of apoptosis, as defined using a combination of ultrastructural analysis and TUNEL occur in infected neonatal mouse brains. The novel evidence that infection of primary neurons with SFV induces apoptosis with activation of one or more caspases defines a system for the further anlaysis of apoptosis regulation in physiologically relevant neurons.