Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks.

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Bioinformatics tools for analysing viral genomic data.

The field of viral genomics and bioinformatics is experiencing a strong resurgence due to high-throughput sequencing (HTS) technology, which enables the rapid and cost-effective sequencing and subsequent assembly of large numbers of viral genomes. In addition, the unprecedented power of HTS technologies has enabled the analysis of intra-host viral diversity and quasispecies dynamics in relation to important biological questions on viral transmission, vaccine resistance and host jumping. HTS also enables the rapid identification of both known and potentially new viruses from field and clinical samples, thus adding new tools to the fields of viral discovery and metagenomics. Bioinformatics has been central to the rise of HTS applications because new algorithms and software tools are continually needed to process and analyse the large, complex datasets generated in this rapidly evolving area. In this paper, the authors give a brief overview of the main bioinformatics tools available for viral genomic research, with a particular emphasis on HTS technologies and their main applications. They summarise the major steps in various HTS analyses, starting with quality control of raw reads and encompassing activities ranging from consensus and de novo genome assembly to variant calling and metagenomics, as well as RNA sequencing.

Le champ de la génomique virale et de la bio-informatique connaît actuellement un nouvel essor grâce à la technologie du séquençage à haut débit (SHD), qui permet de séquencer puis d'assembler rapidement un très grand nombre de génomes viraux, à un coût abordable. De surcroît, grâce à la puissance sans précédent des technologies du SHD, il est désormais possible d'analyser la diversité des virus au sein d'un hôte ainsi que la dynamique des quasi-espèces afin d'élucider d'importantes questions biologiques ayant trait à la transmission virale, à la résistance vis-à-vis des vaccins et au passage d'un hôte à l'autre. Le SHD permet également d'identifier rapidement des virus connus ou potentiellement nouveaux dans des échantillons de terrain ou cliniques, ce qui apporte de nouveaux outils pour la découverte des virus et la métagénomique. La bio-informatique joue un rôle central dans le développement des applications du SHD car ce domaine en constante évolution génère des séries de données aussi nombreuses que complexes dont le traitement et l'analyse requièrent en permanence de nouveaux algorithmes et logiciels. Les auteurs font rapidement le point sur les principaux outils de la bio-informatique utilisés dans la recherche sur les génomes viraux, en mettant particulièrement l'accent sur les technologies du SHD et sur leurs applications les plus importantes. Ils décrivent schématiquement les grandes étapes de différents types d'analyse recourant au SHD, depuis le contrôle qualité des lectures brutes jusqu'aux activités telles que l'assemblage de séquences consensus et de novo du génome, l'appel de variants et la métagénomique, et enfin le séquençage d'ARN.

El campo de la genómica vírica y la bioinformática conoce hoy un renovado dinamismo gracias a las técnicas de secuenciación de alto rendimiento, que permiten secuenciar con rapidez y rentabilidad, y a continuación ensamblar, un gran número de genomas víricos. Además, la potencia sin precedentes que ofrecen estas técnicas ha hecho posible analizar la diversidad vírica dentro de los anfitriones y la dinámica de cuasiespecies en relación con importantes interrogantes biológicos tocantes a la transmisión de virus, la resistencia a las vacunas o el salto de un anfitrión a otro. Con la secuenciación de alto rendimiento también es posible identificar con celeridad los virus tanto conocidos como eventualmente nuevos que estén presentes en muestras clínicas u obtenidas sobre el terreno, lo que aporta nuevas herramientas al arsenal disponible en los campos del descubrimiento de virus y la metagenómica. La bioinformática ha sido un factor capital en el auge de las aplicaciones de técnicas de secuenciación de alto rendimiento, pues continuamente se necesitan nuevos algoritmos y programas informáticos para procesar y analizar los vastos y complejos conjuntos de datos que se generan en un ámbito sujeto a tan rápida evolución. Los autores repasan brevemente las principales herramientas bioinformáticas que existen para la investigación en genómica vírica, prestando especial atención a las técnicas de secuenciación de alto rendimiento y sus principales aplicaciones. Asimismo, resumen las etapas básicas de diversos procedimientos de análisis por secuenciación de alto rendimiento, empezando por el control de calidad de las lecturas brutas y pasando por labores que van desde el ensamblaje del genoma con creación de secuencia consenso o ensamblaje de novo hasta la asignación de variantes (variant calling) o la metagenómica, sin olvidar la secuenciación de ARN.

Drosophila wingless and pair-rule transcripts localize apically by dynein-mediated transport of RNA particles.

Asymmetric mRNA localization targets proteins to their cytoplasmic site of function. We have elucidated the mechanism of apical localization of wingless and pair-rule transcripts in the Drosophila blastoderm embryo by directly visualizing intermediates along the entire path of transcript movement. After release from their site of transcription, mRNAs diffuse within the nucleus and are exported to all parts of the cytoplasm, regardless of their cytoplasmic destinations. Endogenous and injected apical RNAs assemble selectively into cytoplasmic particles that are transported apically along microtubules. Cytoplasmic dynein is required for correct localization of endogenous transcripts and apical movement of injected RNA particles. We propose that dynein-dependent movement of RNA particles is a widely deployed mechanism for mRNA localization.

Merlin, the Drosophila homologue of neurofibromatosis-2, is specifically required in posterior follicle cells for axis formation in the oocyte.

In Drosophila, the formation of the embryonic axes is initiated by Gurken, a transforming growth factor alpha signal from the oocyte to the posterior follicle cells, and an unknown polarising signal back to the oocyte. We report that Drosophila Merlin is specifically required only within the posterior follicle cells to initiate axis formation. Merlin mutants show defects in nuclear migration and mRNA localisation in the oocyte. Merlin is not required to specify posterior follicle cell identity in response to the Gurken signal from the oocyte, but is required for the unknown polarising signal back to the oocyte. Merlin is also required non-autonomously, only in follicle cells that have received the Gurken signal, to maintain cell polarity and limit proliferation, but is not required in embryos and larvae. These results are consistent with the fact that human Merlin is encoded by the gene for the tumour suppressor neurofibromatosis-2 and is a member of the Ezrin-Radixin-Moesin family of proteins that link actin to transmembrane proteins. We propose that Merlin acts in response to the Gurken signal by apically targeting the signal that initiates axis specification in the oocyte.

Small bristles, the Drosophila ortholog of NXF-1, is essential for mRNA export throughout development.

We identified a temperature-sensitive allele of small bristles (sbr), the Drosophila ortholog of human TAP/NXF-1 and yeast Mex67, in a screen for mutants defective in mRNA export. We show that sbr is essential for the nuclear export of all mRNAs tested in a wide range of tissues and times in development. High resolution and sensitive in situ hybridization detect the rapid accumulation of individual mRNA species in sbr mutant nuclei in particles that are distinct from nascent transcript foci and resemble wild-type export intermediates. The particles become more numerous and intense with increasing time at the restrictive temperature and are exported very rapidly after shifting back to the permissive temperature. The mRNA export block is not due indirectly to a defect in splicing, nuclear protein import, or aberrant nuclear ultrastructure, suggesting that in sbr mutants, mRNA is competent for export but fails to dock or translocate through NPCs. We conclude that NXF-1 is an essential ubiquitous export factor for all mRNAs throughout development in higher eukaryotes.

Perturbing nuclear transport in Drosophila eye imaginal discs causes specific cell adhesion and axon guidance defects.

To study nucleocytoplasmic transport during multicellular development, we developed a sensitive nuclear protein import assay in living blastoderm embryos. We show that dominant negative truncations of the human nuclear transport receptor karyopherinbeta/Importinbeta (DNImpbeta) disrupt mRNA export and protein import in Drosophila. To test the sensitivity of different developmental processes to nuclear trafficking perturbations, we expressed DNImpbeta behind the morphogenetic furrow of the eye disc, at a time when photoreceptors are patterned and project their axons to the brain. DNImpbeta expression does not disrupt the correct specification of different photoreceptors, but causes a defect in cell adhesion that leads to some photoreceptors descending below the layer of ommatidia. The photoreceptors initially project their axons correctly to the posterior, but later their axons are unable to enter the optic stalk en route to the brain and continue to project an extensive network of misguided axons. The axon guidance and cell adhesion defects are both due to a disruption in the function of Ketel, the Drosophila ortholog of Importinbeta. We conclude that cell adhesion and axon guidance in the eye have specific requirements for nucleocytoplasmic transport, despite involving processes that occur primarily at the cell surface.

Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei.

When some genes are silenced, their positions within the nucleus can change dramatically [1] [2]. It is unclear, however, whether genes move to new positions when they are activated [3]. The chromosomes within the polarized nuclei of the fruit fly Drosophila have a well-characterized apical-basal orientation (the Rabl configuration [4]). Using a high-resolution in situ hybridization method [5], we found that each of 15 transcribed genes was localized as predicted by their chromosomal position and by the known polarized organization of the chromosomes. We also found that, within their specific apical-basal plane, most nascent transcript foci could occupy any radial position. There was no correlation between the apical-basal position of the transcribed locus and the final cytoplasmic site of localization of the RNA along the apical-basal axis of the cell. There was also no relationship between the distance of loci from the nuclear periphery and the amount of nascent mRNA decorating the gene. Our results are consistent with the view that effective transcription can occur without major re-localization of the genes themselves.

In vivo and in vitro evidence of altered nitric oxide metabolism in the spontaneously diabetic, insulin-dependent BB/Edinburgh rat.

1. Altered vasoreactivity may contribute significantly to the pathogenesis of diabetic vascular complications. This study investigated the effect of (a) insulin-related diabetes, and (b) chronic in vivo administration of N(omega)-nitro-L-arginine ester (L-NAME), a nitric oxide (NO) synthase inhibitor, on mean arterial pressure and in vitro vascular reactivity to noradrenaline in mesenteric arterial bed preparations from spontaneously diabetic, insulin-dependent and treated BB rats, the best animal model of insulin-dependent mellitus (IDDM) currently available. Four groups of animals from the Edinburgh colony (BB/E) of spontaneous diabetic BB rats were studied: age-matched (mean +/- s.e. mean = 156 +/- 2d) non-diabetic (glycated haemoglobin = 3.8 +/- 0.1%) and insulin-treated diabetic (glycated haemoglobin = 6.2 +/- 0.5%; duration of diabetes = 56 +/- 4 d) groups were either L-NAME treated (oral dose = 27 +/- 1 mg kg-1 d-1; duration of treatment from 30 until 153 days of age) or untreated. Although our diabetic BB/E rats do not achieve overall normoglycaemia, individual adjustment of the daily insulin dose administered to every diabetic rat achieves better glycaemic control than previous groups studying altered vascular reactivity and endothelial dysfunction in this animal model of diabetes. 2. Mean arterial pressure (measured directly via indwelling carotid arterial cannulae) was not significantly different between non-diabetic (116 +/- 3 mmHg; n = 10) and diabetic (122 +/- 2 mmHg; n = 12) BB/E rats. L-NAME treatment significantly (P < 0.001) increased mean arterial pressure in both groups (165 +/- 6 mmHg; n = 9 and 142 +/- 4 mmHg; n = 6 respectively) but the degree of hypertension observed in L-NAME-treated diabetic rats was significantly (P < 0.01) attenuated compared to non-diabetic rats treated with L-NAME. 3. Mesenteric arterial bed preparations were cannulated under anesthesia, excised and intralumenally perfused ex vivo with noradrenaline (0.2-20 microM). Basal perfusion pressures were not significantly different in mesentery preparations from non-diabetic (27.0 +/- 2.6 mmHg) and diabetic (27.1 +/- 3.2 mmHg) BB/E rats. There was no significant difference in maximal response above basal perfusion pressure (MAX) or pEC50, defined as the negative log of the agonist concentration required to give 50% of the maximal response above basal perfusion pressure, to noradrenaline in untreated non-diabetic (166 +/- 7 mmHg and 5.74 +/- 0.05 respectively) and diabetic (170 +/- 11 mmHg and 5.59 +/- 0.05) BB/E rats. 4. In vivo treatment of non-diabetic and diabetic BB/E rats with L-NAME had no significant effect on basal perfusion pressure (25.9 +/- 4.3 mmHg and 28.5 +/- 3.9 mmHg respectively). L-NAME treatment in vivo increased (P < 0.001) MAX to noradrenaline of non-diabetic rats (224 +/- 8 mmHg) but did not affect the value for diabetic rats (178 +/- 14 mmHg). L-NAME treatment did not alter after the pEC50 values in either group (5.71 +/- 0.05 and 5.65 +/- 0.05). 5. Consistent with previous studies using vascular preparations from spontaneously diabetic BB rats, mesentery preparations from diabetic BB/E rats (n = 12) exhibited a significantly reduced vasodilator response to acetylcholine (F value = 4.4, P < 0.05) across the concentration range studied compared to non-diabetic BB/E rats (n = 12) although there was no significant difference in maximal relaxation (diabetic 53.1 +/- 4.3% vs non-diabetic 55.7 +/- 5.5%) or pEC50, (diabetic 6.92 +/- 0.25 vs non-diabetic 7.49 +/- 0.22). There was no significant (F value = 0.8, P > 0.1) difference in the response to GTN between preparations from non-diabetic and diabetic rats (maximal relaxation: 49.6 +/- 3.7% vs 48.5 +/- 4.3%; pEC50: 7.84 +/- 0.12 vs 7.89 +/- 0.22 respectively). 6. In conclusion, vascular responsiveness to noradrenaline is not impaired in spontaneously diabetic BB/E rats with significantly better glycaemic control than those used in previous studies. However, following chronic L-NAME treatment, diabetic BB/E rats exhibit attenuated hypertension and an absence of enhanced vascular responsiveness to noradrenaline in vitro compared to similarly treated non-diabetic rats. These results, together with the significantly impaired endothelium-dependent vasodilatation and unchanged endothelium-independent vasodilatation in vitro of preparations from diabetic BB/E rats, are consistent with the hypothesis that functional changes in the synthesis and metabolism of NO (rather than altered vascular responsiveness to NO) occur in diabetes. Our results indicate that good glycaemic control alone is insufficient to prevent these abnormalities in NO availability and further studies to characterize the origin of these changes are necessary.