Bluetongue virus assembly and morphogenesis.

Like other members of the Reoviridae, bluetongue virus faces the same constraints on structure and assembly that are imposed by a large dsRNA genome. However, since it is arthropod-transmitted, BTV must have assembly pathways that are sufficiently flexible to allow it to replicate in evolutionarily distant hosts. With this background, it is hardly surprising that BTV interacts with highly conserved cellular pathways during morphogenesis and trafficking. Indeed, recent studies have revealed striking parallels between the pathways involved in the entry and egress of nonenveloped BTV and those used by enveloped viruses. In addition, recent studies with the protein that is the major component of the BTV viroplasm have revealed how the assembly and, as importantly, the disassembly of this structure may be achieved. This is a first step towards resolving the interactions that occur in these virus 'assembly factories'. Overall, this review demonstrates that the integration of structural, biochemical and molecular data is necessary to fully understand the assembly and replication of this complex RNA virus.

Evaluation of the effect of radiotherapy for pituitary tumours on cognitive function and quality of life.

AIMS: Pituitary tumours are often treated with radiotherapy, which can cause cognitive impairment when given in high doses. It is assumed that current regimens do not cause damage, but this has not been established. The aim was to determine whether radiotherapy given to people with pituitary tumours was associated with cognitive impairment and reduced quality of life. MATERIAL AND METHODS: We retrospectively compared two outcome groups (patients with pituitary tumours who had undergone radiotherapy and surgery and patients with pituitary tumours who had surgery alone), and carried out standardised tests of cognitive function and quality of life. RESULTS: The data suggested that patients with pituitary tumours treated with surgery, with or without radiotherapy, had cognitive impairment compared with the normal population. Patients receiving radiotherapy performed significantly worse than those receiving only surgery on the Stroop test, a measure of executive function. They also scored significantly lower on the Physical Health composite of the SF36, although this difference was no longer significant when account was taken of baseline differences between the groups. There were no significant differences in other cognitive functions, mood, general well-being or the Mental Health Composite of the SF36. CONCLUSIONS: Patients treated for pituitary disease may have cognitive impairment. A decrease in cognitive function was found regardless of treatment type. The decrease seemed to be greater in the radiotherapy group and was mainly on executive function. This impairment of executive function could affect daily life. Further prospective studies are required to assess the effect of pituitary disease on cognitive function and the safety of radiotherapy.

Functional recovery of community stroke patients.

PURPOSE: The aims were to describe the functional abilities of and services provided to stroke patients not admitted to hospital a year after stroke and to investigate factors associated with receiving rehabilitation services. METHOD: A cohort of stroke patients who had not been admitted to hospital were notified by general practitioners. Patients were assessed at a month and a year after stroke on measures of impairment and disability. The rehabilitation received was recorded. There were 124 stroke patients notified by GP's who had not been admitted to hospital. Of these 70 were assessed at both one month and one year after stroke. RESULTS: Patients showed persistent impaired levels of disability one year after stroke, as measured by the Extended Activities of Daily Living (EADL) scale. There was no significant improvement in Barthel scores and only a slight improvement on the EADL from one month to one year after stroke. There were no patients with severe mobility or severe arm impairment. Most patients had some cognitive impairment but there was no significant change between a month and a year. The provision of rehabilitation services increased between a month and a year after stroke. Rehabilitation was provided to those patients with impaired motor function. CONCLUSIONS: Stroke patients not admitted to hospital have significant levels of disability which does not change substantially in the year after stroke. Rehabilitation was provided on the basis of physical function, rather than cognitive function.

A carotid artery aneurysm with a twist: case report and review.

BACKGROUND: Since their original description in 1687, aneurysms of the extracranial internal carotid artery (ICA) remain rare and account for less than 2% of all carotid operations (Zwolak et al. in: J Vasc Surg 1:415-422, 1984; El-Sabrout, Cooley in: J Vasc Surg 31:702-712, 2000). Cerebrovascular morbidity and mortality rates remain high without intervention (Zwolak et al. in: Vasc Surg 1:415-422, 1984). CASE REPORT: We report an unusual case of a saccular extracranial ICA aneurysm associated with kinking of the ICA and highlight the potential complications and risks associated with such an extreme anatomical deformation. CONCLUSION: Extracranial ICA aneurysm can be associated with unusual anatomical variations which can lead to unstable clinical symptomatology due to the variable presence of atherosclerotic material. Despite advances in diagnosis and treatment, surgical repair is not without risks and patients need to be informed of the potential complications.

The effect of modernising medical careers on foundation doctor career orientation in the Northern Ireland Foundation School.

OBJECTIVES: Modernising Medical Careers (MMC) emerged in response to acknowledged problems in training in the Senior House Officer grade. The objective of this study was to assess the effect of the Foundation Year 2 (F2) training programme on career orientation in the Northern Ireland Deanery. METHODS: A prospective survey-based study was conducted for all F2 doctors participating in the Northern Ireland Foundation Programme. Career orientation was investigated using the Specialty Choice Inventory 45 (SCI45) at the start (Q1) and end (Q2) of the F2 year. Specialty choice was collated after the outcome of specialty recruitment in 2008. RESULTS: There were 231 F2 doctors in programme during the first F2 year in 2006-2007. 147 (M=65, F=82) and 106 (M=55, F=51) completed questionnaires at Q1 and Q2. Male F2 doctors scored significantly higher in the action orientation (54.0 vs. 50.0, p<0.001) and need for assertiveness (53.0 vs. 48.0, p=0.005) subscales at both time points as well as Q1 detail is crucial (57.0 vs. 51.0, p=0.014) and Q2 independent specialty (53.0 vs. 46.0, p=0.016). Female F2 doctors scored significantly higher in the educating patients subscale at both time-points (44.0 vs. 46.0, p=0.009 and 46.0 vs. 47.0, p=0.03). Analysis of SCI45 subscale scores suggested that males tended to favour the surgical specialties while females favoured the care of the elderly and paediatric specialties. Overall only 29% of doctors were successfully appointed to a specialty in which they had expressed an interest at Q1 whilst 47.8% were selected to specialist training for their declared specialty interest at Q2. CONCLUSIONS: Despite introducing MMC with a coordinated UK wide specialty application process (MTAS), a detrimental effect on their career orientation was not evident. Pragmatic career choices based on lifestyle may be the reason why female doctors expressed a preference for care of the elderly and paediatrics while their male colleagues favoured acute, more surgically biased specialties.

Validation of a novel approach for the rapid production of immunogenic virus-like particles for bluetongue virus.

Bluetongue virus causes an emerging disease of ruminants, principally affecting sheep and cattle. Since 1998, there have been multiple separate outbreaks of bluetongue disease in Europe that have highlighted the need for a safe, efficacious, DIVA compliant vaccine. We report here a new baculovirus expression strategy which allowed pre-integration of the genes encoding the BTV inner capsid proteins at one baculovirus locus and those encoding the outer capsid proteins at a different locus. A modified baculovirus with two marker proteins to facilitate the phenotypic selection of recombinant viruses was developed. The utility of this approach is demonstrated by the production of BTV VLPs to a number of serotypes. For a proof of concept, VLPs of one serotype was then tested for protective immune response. VLPs were demonstrated to be safe, highly effective, immunogens in sheep, reducing post-challenge viraemia to levels below the threshold detection limit of quantitative RT-PCR when vaccinated animals were challenged with virulent virus.

Bluetongue vaccines.

Once thought largely restricted to India and Africa, the insect-borne livestock pathogen Bluetongue virus is now present on every continent with the exception of Antarctica. Outbreaks of the disease caused by the virus in Europe over the last decade, and the resulting impact on trade and agriculture, have focussed attention on the production of safe and effective vaccines. The determinants of protection for bluetongue are well defined but the variability of the virus, which exists as 24 immunologically distinct serotypes, means that even regions where large numbers of animals have been vaccinated remain at risk from new outbreaks of the virus.

Expression of functional elements inserted into the 35S promoter region of infectious cauliflower mosaic virus replicons.

We describe experiments directed towards development of cauliflower mosaic virus (CaMV) replicons for propagation of functional elements during infection of plants. Modifications and inserts were introduced into replaceable domains associated with the 35S promoter. The 35S enhancer (-208 to -56) was found to potentiate promoter activity when in reverse orientation sufficient to establish systemic infection. However, replacement of the 35S enhancer with that from the nos promoter caused loss of infectivity. A 31 bp oligonucleotide containing a polypurine tract specifying initiation of CaMV plus strand DNA synthesis was inserted into a 35S enhancer deletion mutant and propagated in plants. Analysis of progeny DNA showed the presence of an additional discontinuity at its new location in the 35S enhancer, indicating that the artificial primer had functioned correctly in an ectopic site. An intron and flanking sequences from the RNA leader of the Arabidopsis phytoene desaturase (pds) gene, when inserted into the 35S enhancer in forward orientation was very efficiently spliced during infection. The CaMV replicon carrying the pds gene fragment produced unusual infection characteristics, with plants showing early symptoms and then recovering. We conclude that infectious CaMV replicons can be used to carry a variety of elements that target both viral and host functions.

Genetic dissection of tomato bushy stunt virus p19-protein-mediated host-dependent symptom induction and systemic invasion.

The plus-sense single-stranded RNA of tomato bushy stunt virus (TBSV) encodes a 19-kDa protein, which is translated from a 3' proximal open reading frame (p19) that is entirely nested within the cell-to-cell movement gene (p22). Expression of the cytosolic p19-protein induces either a systemic lethal collapse in Nicotiana benthamiana and N. clevelandii, or necrotic local lesions on resistant N. tabacum. In spinach, the p19-protein is required at high abundance for efficient systemic invasion. This study aimed to determine whether these seemingly different host-dependent biological activities are governed by the same or separate regions on the 172 amino acid p19-protein. For this purpose, codons for charged amino acids predicted to be exposed on the surface of the polypeptide and presumably available for host-specific interactions, were targeted for mutagenesis. A total of 12 mutants were generated, which had no deficiencies in replication or cell-to-cell movement, and substitution of amino acids at the extreme N-terminal end or within the carboxyl 70 amino acids failed to cause a noticeable biological effect on plants. However, mutations dispersed between positions 43 and 85 on the N-terminal half prevented the onset of a systemic lethal necrosis on N. benthamiana and N. clevelandii. With one exception, the same mutants elicited mostly chlorotic, rather than necrotic, local lesions on N. tabacum. Mutations in the central region, which substituted Arg with Gly at positions 72 or 75-78, impaired the ability of TBSV to systemically invade spinach plants. However, substitution with Ala instead of Gly at position 72 had minimal effects on systemic spread in spinach, suggesting the possible influence of protein structure effects. The implications are that regions on the N-terminal portion of the p19-protein mediate interactions in a host-dependent manner and that a central region is required for all activities either by a direct effect of the amino acids or through maintenance of structural integrity.

Nitric oxide modulates the activity of tobacco aconitase.

Recent evidence suggests an important role for nitric oxide (NO) signaling in plant-pathogen interactions. Additional elucidation of the role of NO in plants will require identification of NO targets. Since aconitases are major NO targets in animals, we examined the effect of NO on tobacco (Nicotiana tabacum) aconitase. The tobacco aconitases, like their animal counterparts, were inhibited by NO donors. The cytosolic aconitase in animals, in addition to being a key redox and NO sensor, is converted by NO into an mRNA binding protein (IRP, or iron-regulatory protein) that regulates iron homeostasis. A tobacco cytosolic aconitase gene (NtACO1) whose deduced amino acid sequence shared 61% identity and 76% similarity with the human IRP-1 was cloned. Furthermore, residues involved in mRNA binding by IRP-1 were conserved in NtACO1. These results reveal additional similarities between the NO signaling mechanisms used by plants and animals.

Analysis of polypurine tract-associated DNA plus-strand priming in vivo utilizing a plant pararetroviral vector carrying redundant ectopic priming elements.

Initiation of DNA plus-strand synthesis in most reverse-transcribing elements requires primer generation by reverse transcriptase-associated RNase H at one or more template polypurine tracts (PPTs). We have exploited infectious clones of the plant pararetrovirus cauliflower mosaic virus carrying redundant ectopic plus-strand priming elements to study priming in vivo. Ectopic priming generated an additional discontinuity in progeny virion DNA during infection of plants. We found that altering the length of the 13-base pair PPT by +/-25% significantly reduced priming efficiency. A short pyrimidine tract 5' to the PPT, highly conserved among diverse reverse-transcribing elements, was shown to play an important role in PPT recognition in vivo. The predominant DNA plus-strand 5' end remained 3 nucleotides from the PPT 3' end in mutant primers that were longer or shorter than the wild-type primer. Use of an ectopic redundant primer to study replication-dependent priming was validated by demonstrating that it could rescue infectivity following destruction of the wild-type priming elements. We propose a model for plant pararetroviral plus-strand priming in which pyrimidines enhance PPT recognition during polymerase-dependent RNase H cleavages, and suggest that fidelity of primer maturation during polymerase-independent cleavages involves PPT length measurement and 3' end recognition by RNase H.

Nitric oxide and salicylic acid signaling in plant defense.

Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the H(2)O(2)-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to IkappaBalpha and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-1). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-1). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals.