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1.
Sci Rep ; 14(1): 5998, 2024 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472287

RESUMO

Clinical gait analysis is a crucial step for identifying foot disorders and planning surgery. Automating this process is essential for efficiently assessing the substantial amount of gait data. In this study, we explored the potential of state-of-the-art machine learning (ML) and explainable artificial intelligence (XAI) algorithms to automate all various steps involved in gait analysis for six specific foot conditions. To address the complexity of gait data, we manually created new features, followed by recursive feature elimination using Support Vector Machines (SVM) and Random Forests (RF) to eliminate low-variance features. SVM, RF, K-nearest Neighbor (KNN), and Logistic Regression (LREGR) were compared for classification, with a Majority Voting (MV) model combining trained models. KNN and MV achieved mean balanced accuracy, recall, precision, and F1 score of 0.87. All models were interpreted using Local Interpretable Model-agnostic Explanation (LIME) method and the five most relevant features were identified for each foot condition. High success scores indicate a strong relationship between selected features and foot conditions, potentially indicating clinical relevance. The proposed ML pipeline, adaptable for other foot conditions, showcases its potential in aiding experts in foot condition identification and planning surgeries.


Assuntos
Inteligência Artificial , Análise da Marcha , Algoritmos , , Aprendizado de Máquina
2.
Artigo em Inglês | MEDLINE | ID: mdl-36497613

RESUMO

Occupational exoskeletons are a promising solution to prevent work-related musculoskeletal disorders (WMSDs). However, there are no established systems that support heavy lifting to shoulder height. Thus, this work presents a model-based analysis of heavy lifting activities and subsequent exoskeleton concept optimization. Six motion sequences were captured in the laboratory for three subjects and analyzed in multibody simulations with respect to muscle activities (MAs) and joint forces (JFs). The most strenuous sequence was selected and utilized in further simulations of a human model connected to 32 exoskeleton concept variants. Six simulated concepts were compared concerning occurring JFs and MAs as well as interaction loads in the exoskeleton arm interfaces. Symmetric uplifting of a 21 kg box from hip to shoulder height was identified as the most strenuous motion sequence with highly loaded arms, shoulders, and back. Six concept variants reduced mean JFs (spine: >70%, glenohumeral joint: >69%) and MAs (back: >63%, shoulder: >59% in five concepts). Parasitic loads in the arm bracing varied strongly among variants. An exoskeleton design was identified that effectively supports heavy lifting, combining high musculoskeletal relief and low parasitic loads. The applied workflow can help developers in the optimization of exoskeletons.


Assuntos
Exoesqueleto Energizado , Articulação do Ombro , Humanos , Fenômenos Biomecânicos , Ombro/fisiologia , Articulação do Ombro/fisiologia , Extremidade Superior , Remoção , Eletromiografia
3.
Artigo em Inglês | MEDLINE | ID: mdl-35897411

RESUMO

Musculoskeletal disorders (MSDs) induced by industrial manual handling tasks are a major issue for workers and companies. As flexible ergonomic solutions, occupational exoskeletons can decrease critically high body stress in situations of awkward postures and motions. Biomechanical models with detailed anthropometrics and motions help us to acquire a comprehension of person- and application-specifics by considering the intended and unintended effects, which is crucial for effective implementation. In the present model-based analysis, a generic back-support exoskeleton model was introduced and applied to the motion data of one male subject performing symmetric and asymmetric dynamic manual handling tasks. Different support modes were implemented with this model, including support profiles typical of passive and active systems and an unconstrained optimal support mode used for reference to compare and quantify their biomechanical effects. The conducted simulations indicate that there is a high potential to decrease the peak compression forces in L4/L5 during the investigated heavy loaded tasks for all motion sequences and exoskeleton support modes (mean reduction of 16.0% without the optimal support mode). In particular, asymmetric motions (mean reduction of 11.9%) can be relieved more than symmetric ones (mean reduction of 8.9%) by the exoskeleton support modes without the optimal assistance. The analysis of metabolic energy consumption indicates a high dependency on lifting techniques for the effectiveness of the exoskeleton support. While the exoskeleton support substantially reduces the metabolic cost for the free-squat motions, a slightly higher energy consumption was found for the symmetric stoop motion technique with the active and optimal support mode.


Assuntos
Exoesqueleto Energizado , Fenômenos Biomecânicos , Eletromiografia/métodos , Humanos , Remoção , Masculino , Postura
4.
Prosthet Orthot Int ; 46(2): 202-205, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-34932513

RESUMO

BACKGROUND: Individuals with lower limb loss often wear a gel liner and enclosed socket for connecting to a terminal prosthetic device. Historically, a significant limitation to traditional liners and sockets is that they are thermal insulators, thereby trapping heat and moisture within, which can lead to numerous deleterious issues, including loss of suspension and residual limb skin problems, and, in turn, reductions in mobility, function, and overall quality of life. To mitigate these issues, new approaches are therefore needed to enhance the residual limb climate (e.g. breathability and air permeability), allowing the dispersal of heat and moisture from within the liner and socket. METHODS: In this study, a multidisciplinary team sought to establish the feasibility of an innovative prosthetic liner-socket system, designed to improve residual limb climate by capitalizing on passive (i.e. nonpowered) ventilation to reduce temperature/moisture and improve socket comfort for persons with transtibial amputations. Focus group meetings, along with an iterative design approach, were implemented to establish innovative design and development concepts that led to a passively ventilated liner-socket system. CONCLUSIONS: Ex vivo design has supported the feasibility of developing a passively ventilated liner-socket. To build on these successes, future development and human subjects testing are needed to finalize a commercially viable system. Implementing a passively ventilated liner-socket system that improves residual limb health and comfort, without compromising function or mobility of the user, into standard clinical care may encourage a more active lifestyle and enhance the quality of life for individuals after lower limb loss.


Assuntos
Membros Artificiais , Qualidade de Vida , Cotos de Amputação , Humanos , Extremidade Inferior , Desenho de Prótese
5.
Wearable Technol ; 3: e17, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-38486892

RESUMO

Purpose: The aim of this study is to investigate the effects of wearing exoskeletons during welding on the quality of the weld seam. Material and methods: A total of n = 15 young healthy subjects with welding experience took part in the study. The study design defines a 1-hr workflow that abstracts welding and grinding tasks. The sequence is based on standard DIN EN ISO 9606-1 and reproduces authentic work sequences in the constrained body positions PF-workpiece in front of the body and PE-workpiece overhead. Each subject completed the entire workflow once with and once without passive shoulder exoskeleton in a randomized order. Results: The evaluation shows that the use of passive shoulder exoskeletons has a significant influence (p = .006 for Position PF; p = .029 for Position PE) on the welding parameter travel speed which significantly influences the quality of the weld seam. The quality scale (by the used augmented reality (AR) welding simulator) of the travel speed, which significantly determines the permissibility of the weld, increases by 5.80% in the constrained body position PF and by 28.87% in the constrained body position PE when using an exoskeleton. Discussion and conclusion: The score of the welding parameter travel speed, which is essential for the permissibility of the seam, shows a statistically significant increase when an assistance system is used. Further research during real welding with exoskeletons could be based on the setup and workflow of this study.

6.
Wearable Technol ; 3: e22, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-38486909

RESUMO

Industrial exoskeletons have recently gained importance as ergonomic interventions for physically demanding work activities. The growing demand for exoskeletons is leading to a need for new knowledge on the effectiveness of these systems. The Exoworkathlon, as a prospective study approach, aims to assess exoskeletons in realistic use cases and to evaluate them neutrally in their entirety. For this purpose, a first set of four realistic Parcours was developed with experts from relevant industries, the German Social Accident Insurance, and the Federal Institute for Occupational Safety and Health. In addition, a set of ratings was defined to assess subjective user feedback, work quality, and objective physiological parameters. Exoworkathlon aims to bring together developers, researchers, and end-users, strengthen collaborative exchanges, and promote a platform for the prospective holistic data collection for exoskeleton evaluation. In this article, the focus is on the background and methodology of Exoworkathlon.

7.
Artigo em Inglês | MEDLINE | ID: mdl-32708715

RESUMO

Occupational ergonomics in healthcare is an increasing challenge we have to handle in the near future. Physical assistive systems, so-called exoskeletons, are promising solutions to prevent work-related musculoskeletal disorders (WMSDs). Manual handling like pushing, pulling, holding and lifting during healthcare activities require practical and biomechanical effective assistive devices. In this article, a musculoskeletal-model-based development of an assistive exoskeleton is described for manual patient transfer in the surgery waiting room. For that purpose, kinematic data collected with an experimental set-up reproducing real patient transfer conditions are first used to define the kinetic boundary conditions for the model-based development approach. Model-based analysis reveals significant relief potential in the lower back and shoulder area of the musculoskeletal apparatus. This is corroborated by subjective feedback collected during measurements with real surgery assistants. A shoulder-arm exoskeleton design is then proposed, optimized and evaluated within the same simulation framework. The presented results illustrate the potential for the proposed design to reduce significantly joint compressions and muscle activities in the shoulder complex in the considered patient transfer scenarios.


Assuntos
Exoesqueleto Energizado , Ergonomia , Pessoal de Saúde , Humanos , Doenças Profissionais , Extremidade Superior
8.
Wearable Technol ; 1: e2, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-39050262

RESUMO

Although passive occupational exoskeletons alleviate worker physical stresses in demanding postures (e.g., overhead work), they are unsuitable in many other applications because of their lack of flexibility. Active exoskeletons that are able to dynamically adjust the delivered support are required. However, the automatic control of support provided by the exoskeleton is still a largely unsolved challenge in many applications, especially for upper limb occupational exoskeletons, where no practical and reliable approach exists. For this type of exoskeletons, a novel support control approach for lifting and carrying activities is presented here. As an initial step towards a full-fledged automatic support control (ASC), the present article focusses on the functionality of estimating the onset of user's demand for support. In this way, intuitive behavior should be made possible. The combination of movement and muscle activation signals of the upper limbs is expected to enable high reliability, cost efficiency, and compatibility for use in industrial applications. The functionality consists of two parts: a preprocessing-the motion interpretation-and the support detection itself. Both parts were trained with different subjects, who had to move objects. The functionality was validated both in the cases of (A) an unknown subject performing known tasks and (B) a known subject performing unknown tasks. The functionality showed sound results as it achieved a high accuracy () in training. In addition, the first validation results showed that this functionality is useful for integration in an appropriately adapted ASC and can then enable comfortable working.

9.
Top Spinal Cord Inj Rehabil ; 23(2): 120-130, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29339888

RESUMO

Advances in electric motors, electronics, and control systems have enhanced the capability and drivability of electric power mobility devices over the last 60 years. Yet, battery technologies used in powered mobility devices (PMDs) have not kept pace. Recent advances in pneumatic technology, primarily the high torque, low speed design of rotary piston air motors, directly align with the needs of PMD. Pneumatic technology has advantages over battery-powered technology, including lighter weight, lower operating costs, decreased environmental impact, better reliability, and increased safety. Two prototypes were created that incorporated rotary piston air motors, high-pressure air tanks, and air-pressure regulators. Prototype 1 was created by modifying an existing electric PMD. Range tests were performed to determine the feasibility of pneumatic technology and the optimal combination of components to allow the longest range possible at acceptable speeds over ideal conditions. Using a 1.44 L air tank for feasibility testing, prototype 1 was capable of traveling 800 m, which confirmed the feasibility of pneumatic technology usage in PMDs. Prototype 2 was designed based on the testing results from prototype 1. After further optimization of prototype 2, the average maximum range was 3,150 m. Prototype 2 is up to 28.3% lighter than an equivalent size electric PMD and can be fully recharged in approximately 2 minutes. It decreases the cost of PMDs by approximately $1,500, because batteries do not need to be replaced over the lifetime of the device. The results provide justification for the use of pneumatic technology in PMDs.


Assuntos
Fontes de Energia Elétrica , Desenho de Equipamento , Cadeiras de Rodas , Humanos
10.
Proc Inst Mech Eng H ; 227(12): 1255-64, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23966364

RESUMO

The shape of the acetabular cartilage follows the contact stress distribution across the joint. Accurate characterisation of this geometry may be useful for the development of acetabular cup devices that are more biomechanically compliant. In this study, the geometry of the acetabular cartilage was characterised by taking plaster moulds of the acetabulum from 24 dry bone human pelvises and digitising the mould shapes using a three-dimensional laser scanner. The articular bone surface geometry was analysed, and the shape of the acetabulum was approximated by fitting a best-fit sphere. To test the hypothesis that the acetabulum is non-spherical, a best-fit ellipsoid was also fitted to the geometry. In each case, points around the acetabular notch edge that disclosed the articular surface geometry were identified, and vectors were drawn between these and the best-fit sphere or ellipsoid centre. The significantly larger z radii (into the pole) of the ellipsoids indicated that the acetabulum was non-spherical and could imply that the kinematics of the hip joint is more complex than purely rotational motion, and the traditional ball-and-socket replacement may need to be updated to reflect this motion. The acetabular notch edges were observed to be curved, with males exhibiting deeper, wider and shorter notches than females, although the difference was not statistically significant (mean: p = 0.30) and supports the use of non-gender-specific models in anatomical studies.


Assuntos
Acetábulo/anatomia & histologia , Pontos de Referência Anatômicos/anatomia & histologia , Cartilagem Articular/anatomia & histologia , Articulação do Quadril/anatomia & histologia , Modelos Anatômicos , Pelve/anatomia & histologia , Simulação por Computador , Feminino , Humanos , Masculino
11.
Int Orthop ; 37(8): 1597-603, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23793460

RESUMO

PURPOSE: The purpose of this study was to compare bone healing of experimental osteotomies applying either piezosurgery or two different oscillating saw blades in a rabbit model. METHODS: The 16 rabbits were randomly assigned into four groups to comply with observation periods of one, two, three and five weeks. In all animals, four osteotomy lines were performed on the left and right nasal bone using a conventional saw blade, a novel saw blade and piezosurgery. RESULTS: All three osteotomy techniques revealed an advanced gap healing starting after one week. The most pronounced new bone formation took place between two and three weeks, whereby piezoelectric surgery revealed a tendency to faster bone formation and remodelling. Yet, there were no significant differences between the three modalities. CONCLUSIONS: The use of a novel as well as the piezoelectric bone-cutting instrument revealed advanced bone healing with a favourable surgical performance compared to a traditional saw.


Assuntos
Osso Nasal/cirurgia , Equipamentos Ortopédicos , Osteotomia/instrumentação , Osteotomia/métodos , Piezocirurgia/métodos , Cicatrização/fisiologia , Animais , Biópsia , Desenho de Equipamento , Modelos Animais , Osso Nasal/patologia , Osso Nasal/fisiologia , Osteogênese/fisiologia , Piezocirurgia/instrumentação , Projetos Piloto , Período Pós-Operatório , Coelhos , Instrumentos Cirúrgicos , Fatores de Tempo
12.
Arch Virol ; 158(10): 2209-26, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23636404

RESUMO

Nyamanini virus (NYMV) and Midway virus (MIDWV) are unclassified tick-borne agents that infect land birds and seabirds, respectively. The recent molecular characterization of both viruses confirmed their already known close serological relationship and revealed them to be nonsegmented, single- and negative-stranded RNA viruses that are clearly related to, but quite distinct from, members of the order Mononegavirales (bornaviruses, filoviruses, paramyxoviruses, and rhabdoviruses). A third agent, soybean cyst nematode virus 1 (SbCNV-1, previously named soybean cyst nematode nyavirus), was recently found to be an additional member of this new virus group. Here, we review the current knowledge about all three viruses and propose classifying them as members of a new mononegaviral family, Nyamiviridae.


Assuntos
Doenças das Aves/virologia , Nematoides/virologia , Vírus de RNA/classificação , Vírus de RNA/genética , Animais , Aves , Filogenia , Técnicas de Cultura de Tecidos , Cultura de Vírus , Replicação Viral
13.
J Virol ; 87(14): 8257-60, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23698291

RESUMO

We established a reverse genetics system for Nyamanini virus (NYMV) and recovered green fluorescent protein (GFP)-expressing virus from full-length cDNA. Using this technology, we assessed the functions of two poorly characterized viral genes. NYMV lacking open reading frame 2 (ORF2) could not be rescued, whereas virus lacking ORF4 was replication competent. ORF4-deficient NYMV readily established a persisting noncytolytic infection but failed to produce infectious viral particles, supporting the view that ORF4 represents an essential factor for NYMV particle assembly.


Assuntos
Mononegavirais/genética , Fases de Leitura Aberta/genética , Genética Reversa/métodos , Montagem de Vírus/genética , DNA Complementar/genética , Proteínas de Fluorescência Verde/metabolismo
14.
J Virol ; 86(19): 10739-47, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22837209

RESUMO

Tick-borne Nyamanini virus (NYMV) is the prototypic member of a recently discovered genus in the order Mononegavirales, designated Nyavirus. The NYMV genome codes for six distinct genes. Sequence similarity and structural properties suggest that genes 1, 5, and 6 encode the nucleoprotein (N), the glycoprotein (G), and the viral polymerase (L), respectively. The function of the other viral genes has been unknown to date. We found that the third NYMV gene codes for a protein which, when coexpressed with N and L, can reconstitute viral polymerase activity, suggesting that it represents a polymerase cofactor. The second viral gene codes for a small protein that inhibits viral polymerase activity and further strongly enhances the formation of virus-like particles when coexpressed with gene 4 and the viral glycoprotein G. This suggests that two distinct proteins serve a matrix protein function in NYMV as previously described for members of the family Filoviridae. We further found that NYMV replicates in the nucleus of infected cells like members of the family Bornaviridae. NYMV is a poor inducer of beta interferon, presumably because the viral genome is 5' monophosphorylated and has a protruding 3' terminus as observed for bornaviruses. Taken together, our results demonstrate that NYMV possesses biological properties previously regarded as typical for filoviruses and bornaviruses, respectively.


Assuntos
Mononegavirais/genética , Mononegavirais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Chlorocebus aethiops , Cães , Filoviridae/metabolismo , Genoma Viral , Glicoproteínas/química , Células HEK293 , Humanos , Nucleoproteínas/química , Fosforilação , Plasmídeos/metabolismo , Frações Subcelulares/metabolismo , Carrapatos , Células Vero , Proteínas da Matriz Viral/metabolismo
15.
Cell Host Microbe ; 11(5): 492-503, 2012 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-22607802

RESUMO

Bornaviruses are nonsegmented negative-strand RNA viruses that establish a persistent infection in the nucleus and occasionally integrate a DNA genome copy into the host chromosomal DNA. However, how these viruses achieve intranuclear infection remains unclear. We show that Borna disease virus (BDV), a mammalian bornavirus, closely associates with the cellular chromosome to ensure intranuclear infection. BDV generates viral factories within the nucleus using host chromatin as a scaffold. In addition, the viral ribonucleoprotein (RNP) interacts directly with the host chromosome throughout the cell cycle, using core histones as a docking platform. HMGB1, a host chromatin-remodeling DNA architectural protein, is required to stabilize RNP on chromosomes and for efficient BDV RNA transcription in the nucleus. During metaphase, the association of RNP with mitotic chromosomes allows the viral RNA to segregate into daughter cells and ensure persistent infection. Thus, bornaviruses likely evolved a chromosome-dependent life cycle to achieve stable intranuclear infection.


Assuntos
Vírus da Doença de Borna/fisiologia , Vírus da Doença de Borna/patogenicidade , Núcleo Celular/virologia , Replicação Viral , Ciclo Celular , Linhagem Celular , Segregação de Cromossomos , Proteína HMGB1/metabolismo , Histonas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Nucleoproteínas/metabolismo , Ligação Proteica , RNA Viral/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo
16.
Proc Natl Acad Sci U S A ; 108(17): 7206-11, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21482759

RESUMO

The terminal structures of the Borna disease virus (BDV) genome (vRNA) and antigenome (cRNA) differ from those of other negative strand RNA viruses, as both molecules possess four nucleotides at the 3' terminus without an apparent template at the 5' end of the opposite strand. Consequently, the v- and cRNA molecules are not perfect mirror images, a situation that is not compatible with conventional strategies to maintain genetic information. We show here that recombinant viruses recovered from cDNA lacking the nontemplated nucleotides efficiently reconstitute the 3' overhangs. Analyses of recombinant viruses encoding genetic markers in potential alternative template sequences demonstrated that the BDV v- and cRNA molecules are extended by a realign-and-elongation process on internal template motifs located in close proximity to the 3' ends of v- and cRNA, respectively. The data further suggest that cRNA elongation is restricted to a single template motif of the nascent strand, whereas elongation of vRNA might use multiple template motifs. We propose that the elongation of the 3' termini supports the terminal integrity of the genomic RNA molecules during BDV persistence, and furthermore provides an elegant strategy to eliminate the triphosphate groups from the 5' termini of the BDV v- and cRNA without compromising the genetic information of the virus.


Assuntos
Vírus da Doença de Borna/metabolismo , Genoma Viral/fisiologia , RNA Viral/biossíntese , Animais , Vírus da Doença de Borna/genética , Linhagem Celular , Marcadores Genéticos/fisiologia , RNA Viral/genética
17.
J Virol ; 84(10): 5438-42, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20219925

RESUMO

Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.


Assuntos
Doença de Borna/virologia , Vírus da Doença de Borna/patogenicidade , Sistema Nervoso/virologia , Animais , Vírus da Doença de Borna/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Coloração e Rotulagem/métodos
18.
J Gen Virol ; 90(Pt 8): 1932-1936, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19420156

RESUMO

The X protein of Borna disease virus (BDV) is an essential factor that regulates viral polymerase activity and inhibits apoptosis of persistently infected cells. We observed that a BDV mutant which carries an additional X gene replicated well in cell culture only after acquiring second-site mutations that selectively reduced expression of the endogenous X gene. In rat brains, the virus acquired additional mutations which inactivated the ectopic X gene or altered the sequence of X. These results demonstrate that BDV readily acquires mutations if strong selection pressure is applied. They further indicate that fine-tuning of X expression determines viral fitness.


Assuntos
Vírus da Doença de Borna/fisiologia , Dosagem de Genes , Regulação Viral da Expressão Gênica , Mutação de Sentido Incorreto , Proteínas Virais Reguladoras e Acessórias/biossíntese , Proteínas Virais Reguladoras e Acessórias/genética , Animais , Vírus da Doença de Borna/genética , Encéfalo/virologia , Análise Mutacional de DNA , Técnicas de Inativação de Genes , Ratos , Análise de Sequência de DNA , Replicação Viral
19.
J Virol ; 83(9): 4297-307, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19211764

RESUMO

Borna disease virus (BDV) is a neurotropic member of the order Mononegavirales with noncytolytic replication and obligatory persistence in cultured cells and animals. Here we show that the accessory protein X of BDV represents the first mitochondrion-localized protein of an RNA virus that inhibits rather than promotes apoptosis induction. Rat C6 astroglioma cells persistently infected with wild-type BDV were significantly more resistant to death receptor-dependent and -independent apoptotic stimuli than uninfected cells or cells infected with a BDV mutant expressing reduced amounts of X. Confocal microscopy demonstrated that X colocalizes with mitochondria and expression of X from plasmid DNA rendered human 293T and mouse L929 cells resistant to apoptosis induction. A recombinant virus encoding a mutant X protein unable to associate with mitochondria (BDV-X(A6A7)) failed to block apoptosis in C6 cells. Furthermore, Lewis rats neonatally infected with BDV-X(A6A7) developed severe neurological symptoms and died around day 30 postinfection, whereas all animals infected with wild-type BDV remained healthy and became persistently infected. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining revealed a significant increase in the number of apoptotic cells in the brain of BDV-X(A6A7)-infected animals, whereas the numbers of CD3(+) T lymphocytes were comparable to those detected in animals infected with wild-type BDV. Our data thus indicate that inhibition of apoptosis by X promotes noncytolytic viral persistence and is required for the survival of cells in the central nervous system of BDV-infected animals.


Assuntos
Apoptose , Doença de Borna/metabolismo , Doença de Borna/virologia , Vírus da Doença de Borna/metabolismo , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/virologia , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos/virologia , Doença de Borna/patologia , Vírus da Doença de Borna/genética , Linhagem Celular , Chlorocebus aethiops , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Ratos , Transativadores/química , Transativadores/genética
20.
J Virol ; 82(19): 9537-45, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18653450

RESUMO

An unusually long noncoding sequence is located between the N gene of Borna disease virus (BDV) and the genes for regulatory factor X and polymerase cofactor P. Most of these nucleotides are transcribed and seem to control translation of the bicistronic X/P mRNA. We report here that Vero cells persistently infected with mutant viruses containing minor alterations in this control region showed almost normal levels of N, X, and P proteins but exhibited greatly reduced levels of mRNAs coding for these viral gene products. Surprisingly, cells infected with these BDV mutants accumulated a viral transcript 1.9 kb in length that represents a capped and polyadenylated mRNA containing the coding regions of the N, X, and P genes. Cells infected with wild-type BDV also contained substantial amounts of this read-through mRNA, which yielded both N and P protein when translated in vitro. Viruses carrying mutations that promoted read-through transcription at the first gene junction failed to replicate in the brain of adult rats. In the brains of newborn rats, these mutant viruses were able to replicate after acquiring second-site mutations in or near the termination signal located downstream of the N gene. Thus, sequence elements adjacent to the core termination signal seem to regulate the frequency by which the polymerase terminates transcription after the N gene. We conclude from these observations that BDV uses read-through transcription for fine-tuning the expression of the N, X, and P genes which, in turn, influence viral polymerase activity.


Assuntos
Vírus da Doença de Borna/genética , Regulação Viral da Expressão Gênica , Mutação , Transcrição Gênica , Replicação Viral , Animais , Sequência de Bases , Vírus da Doença de Borna/metabolismo , Encéfalo/metabolismo , Chlorocebus aethiops , Modelos Genéticos , Dados de Sequência Molecular , Poliadenilação , RNA Mensageiro/metabolismo , Ratos , Células Vero
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