A Fatal Case of Louping-ill in a Dog: Immunolocalization and Full Genome Sequencing of the Virus.

Louping-ill (LI), caused by louping-ill virus (LIV), results in a frequently fatal encephalitis primarily affecting sheep and red grouse (Lagopus lagopus scotica), but it does occur in other species. An adult male Border collie dog was definitively diagnosed with fatal LI and the lesion profile, LIV antigen distribution and full genome sequence of the LIV responsible were investigated to determine if this differed significantly from sheep-derived LIV. No gross lesions were present. The histological lesions were confined to the central nervous system and comprised of lymphocytic perivascular cuffs, glial foci, neuronal necrosis and neuronophagia. Immunolocalization of viral antigen showed small amounts present in neurons only. These histological and immunohistochemical findings were similar to those reported in affected sheep. Compared with published full genome sequences of sheep-derived LIV, only very minor differences were present and phylogenetically the virus clustered individually between a subclade containing Scottish strains, LIV 369/T2 and G and another subclade containing an English isolate LIV A. The LIV isolated from the dog shares a common progenitor with LIV A. These findings suggest there is no canine-specific LIV strain, dogs are susceptible to sheep-associated strains of LI and with the increase in tick prevalence, and therefore exposure to LIV, a safe, effective vaccine for dogs may be required.

Identifying genotype specific elevated-risk areas and associated herd risk factors for bovine tuberculosis spread in British cattle.

Bovine tuberculosis (bTB) is a chronic zoonosis with major health and economic impact on the cattle industry. Despite extensive control measures in cattle and culling trials in wildlife, the reasons behind the expansion of areas with high incidence of bTB breakdowns in Great Britain remain unexplained. By balancing the importance of cattle movements and local transmission on the observed pattern of cattle outbreaks, we identify areas at elevated risk of infection from specific Mycobacterium bovis genotypes. We show that elevated-risk areas (ERAs) were historically more extensive than previously understood, and that cattle movements alone are insufficient for ERA spread, suggesting the involvement of other factors. For all genotypes, we find that, while the absolute risk of infection is higher in ERAs compared to areas with intermittent risk, the statistically significant risk factors are remarkably similar in both, suggesting that these risk factors can be used to identify incipient ERAs before this is indicated by elevated incidence alone. Our findings identify research priorities for understanding bTB dynamics, improving surveillance and guiding management to prevent further ERA expansion.

Novel hemotropic mycoplasmas are widespread and genetically diverse in vampire bats.

Bats (Order: Chiroptera) have been widely studied as reservoir hosts for viruses of concern for human and animal health. However, whether bats are equally competent hosts of non-viral pathogens such as bacteria remains an important open question. Here, we surveyed blood and saliva samples of vampire bats from Peru and Belize for hemotropic Mycoplasma spp. (hemoplasmas), bacteria that can cause inapparent infection or anemia in hosts. 16S rRNA gene amplification of blood showed 67% (150/223) of common vampire bats (Desmodus rotundus) were infected by hemoplasmas. Sequencing of the 16S rRNA gene amplicons revealed three novel genotypes that were phylogenetically related but not identical to hemoplasmas described from other (non-vampire) bat species, rodents, humans, and non-human primates. Hemoplasma prevalence in vampire bats was highest in non-reproductive and young individuals, did not differ by country, and was relatively stable over time (i.e., endemic). Metagenomics from pooled D. rotundus saliva from Peru detected non-hemotropic Mycoplasma species and hemoplasma genotypes phylogenetically similar to those identified in blood, providing indirect evidence for potential direct transmission of hemoplasmas through biting or social contacts. This study demonstrates vampire bats host several novel hemoplasmas and sheds light on risk factors for infection and basic transmission routes. Given the high frequency of direct contacts that arise when vampire bats feed on humans, domestic animals, and wildlife, the potential of these bacteria to be transmitted between species should be investigated in future work.

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.

Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model.

West Nile virus (WNV) occurs as a population of genetic variants (quasispecies) infecting a single animal. Previous low-resolution viral genetic diversity estimates in sampled wild birds and mosquitoes, and in multiple-passage adaptation studies in vivo or in cell culture, suggest that WNV genetic diversification is mostly limited to the mosquito vector. This study investigated genetic diversification of WNV in avian hosts during a single passage using next-generation sequencing. Wild-captured carrion crows were subcutaneously infected using a clonal Middle-East WNV. Blood samples were collected 2 and 4 days post-infection. A reverse-transcription (RT)-PCR approach was used to amplify the WNV genome directly from serum samples prior to next-generation sequencing resulting in an average depth of at least 700 × in each sample. Appropriate controls were sequenced to discriminate biologically relevant low-frequency variants from experimentally introduced errors. The WNV populations in the wild crows showed significant diversification away from the inoculum virus quasispecies structure. By contrast, WNV populations in intracerebrally infected day-old chickens did not diversify from that of the inoculum. Where previous studies concluded that WNV genetic diversification is only experimentally demonstrated in its permissive insect vector species, we have experimentally shown significant diversification of WNV populations in a wild bird reservoir species.

Estimating epidemiological parameters for bovine tuberculosis in British cattle using a Bayesian partial-likelihood approach.

Fitting models with Bayesian likelihood-based parameter inference is becoming increasingly important in infectious disease epidemiology. Detailed datasets present the opportunity to identify subsets of these data that capture important characteristics of the underlying epidemiology. One such dataset describes the epidemic of bovine tuberculosis (bTB) in British cattle, which is also an important exemplar of a disease with a wildlife reservoir (the Eurasian badger). Here, we evaluate a set of nested dynamic models of bTB transmission, including individual- and herd-level transmission heterogeneity and assuming minimal prior knowledge of the transmission and diagnostic test parameters. We performed a likelihood-based bootstrapping operation on the model to infer parameters based only on the recorded numbers of cattle testing positive for bTB at the start of each herd outbreak considering high- and low-risk areas separately. Models without herd heterogeneity are preferred in both areas though there is some evidence for super-spreading cattle. Similar to previous studies, we found low test sensitivities and high within-herd basic reproduction numbers (R0), suggesting that there may be many unobserved infections in cattle, even though the current testing regime is sufficient to control within-herd epidemics in most cases. Compared with other, more data-heavy approaches, the summary data used in our approach are easily collected, making our approach attractive for other systems.

Developing a framework for risk-based surveillance of tuberculosis in cattle: a case study of its application in Scotland.

Due to its substantially lower prevalence of bovine tuberculosis (bTB) relative to other areas of Great Britain, Scotland was designated as an officially (bovine) TB-free region in 2009. This paper investigates resultant possibilities for reducing surveillance by developing risk-based alternatives to current 4-year testing of eligible herds. A model of freedom of infection was used to develop strategies that specifically tested herds that are at risk of infection but would probably not be identified by slaughterhouse meat inspection. The performance of current testing is mimicked by testing all herds that slaughter fewer than 25% of their total stock per year and regularly import animals from high-incidence areas of England and Wales or from Ireland. This system offers a cost reduction by requiring 25% fewer herd and animal tests and 25% fewer false positives.

Computational modelling reveals feedback redundancy within the epidermal growth factor receptor/extracellular-signal regulated kinase signalling pathway.

The epidermal growth factor receptor (EGFR) activated extracellular-signal regulated kinase (ERK) pathway is a central cell signalling pathway that mediates many biological responses including cell proliferation, transformation, survival and motility. Deregulation of the pathway either through mutation of components or overexpression of EGFRs is associated with several forms of cancer. Under normal conditions, EGF stimulates a rapid but transient activation of ERK as the signal is rapidly shutdown, whereas under cancerous conditions, the ERK signal cannot be shutdown and is sustained. Computational modelling techniques have been used to investigate the signalling dynamics of the EGFR/ERK pathway, focusing on identifying the key processes involved in signal termination and what role the ERK to son of sevenless (SOS) negative feedback loop plays in generating a transient response. This model predicts that this negative feedback loop is not needed to achieve a transient activation of ERK as the process of receptor degradation alone is enough to terminate the signal. Importantly, the behaviour and predictions of this model are verified with laboratory data, as is essential for modern systems biology approaches. Further analysis showed that the feedback loop and receptor degradation were both redundant processes, as each could compensate for the absence of the other. This led to the prediction that in the case of a receptor which is not degraded, such as the insulin receptor, the negative feedback loop to SOS will actually be essential for a transient response to be achieved. Overall, the results shed new light on the role of negative feedback in EGF receptor signalling and suggest that different receptors are dependent on different features within the ERK pathway when relaying their signals.

A novel starch for the treatment of glycogen storage diseases.

OBJECTIVE: To determine whether a new starch offers better short-term metabolic control than uncooked cornstarch in patients with glycogen storage diseases (GSDs). STUDY DESIGN: A short-term double-blind cross-over pilot study comparing uncooked physically modified cornstarch (WMHM20) with uncooked cornstarch in patients with GSD types Ia, Ib and III. Twenty-one patients (ages 3-47, 9 female) were given 2 g/kg cornstarch or WMHM20 mixed in water. Blood glucose, lactate and insulin, and breath hydrogen and (13)CO2 enrichment were measured, at baseline and after each load. The hourly biochemical evaluations terminated when blood glucose was < or = 3.0 mmol/L, when the study period had lasted 10 h or when the patient wished to end the test. The alternative starch was administered under similar trial conditions a median of 10 days later. RESULTS: The median starch load duration was 9 h for WMHM20 versus 7 h for cornstarch. Glucose decreased more slowly (p = 0.05) and lactate was suppressed faster (p = 0.17) for WMHM20 compared with cornstarch. Peak hydrogen excretion was increased (p = 0.05) when cornstarch was taken. CONCLUSION: These data indicate longer duration of euglycaemia and better short-term metabolic control in the majority of GSD patients with WMHM20 compared to cornstarch.

YETI: Yeast Exploration Tool Integrator.

UNLABELLED: Yeast Exploration Tool Integrator (YETI) is a novel bioinformatics tool for the integrated visualization and analysis of functional genomic data sets from the budding yeast Saccharomyces cerevisiae. AVAILABILITY: YETI is freely available for use over the WWW, or download under license, at http://www.bru.ed.ac.uk/~orton/yeti.html

Iris lymphoma in a pediatric cardiac transplant recipient: clinicopathologic findings.

PURPOSE: To report the unusual presentation of a confirmed iris lymphoma in the setting of posttransplant lymphoproliferative disorder. DESIGN: Case report with clinicopathologic findings. METHODS: The medical record was reviewed for clinical manifestations, course of disease, and pathologic and radiologic findings. RESULTS: A 4-year-old girl who had undergone a cardiac transplant at 3 months of age was found to have unilateral iris lesions associated with granulomatous uveitis. Her condition was unresponsive to local and systemic steroids, so she underwent an iris biopsy that was found to contain a B cell-derived monoclonal lymphoproliferation. Treatment with systemic chemotherapy including cyclophosphamide and prednisone resulted in a transient, partial response. Localized radiotherapy induced total regression of the iris lesions. She continues to be observed and has been free of recurrence or metastasis for the past 12 months. CONCLUSIONS: Because of the increased use of organ transplantation, eye care specialists should consider the diagnosis of intraocular lymphoma when confronted with uveitis-like syndrome unresponsive to topical steroid therapy. Although there are numerous treatment options for intraocular lymphoma, radiation therapy was found to be effective in the treatment of this case.

Ocular alkali burn associated with automobile air-bag activation.

Alkali burns of the eye can result in permanent visual impairment and are therefore potentially devastating. Immedicate diagnosis and treatment are essential to a good prognosis. The authors report the case of a 52-year-old woman who suffered alkali keratitis as the result of the activation of an automobile air bag. This type of injury will be seen more frequently as more cars are equipped with air bags and should be suspected in drivers and passengers involved in accidents in which air bags have been activated.

The 20-prism-dioptre base-out test: an indicator of peripheral binocularity.

OBJECTIVE: To assess the ability of the 20-prism-dioptre base-out test to detect the presence of peripheral fusion in children too young to undergo formal sensory testing. DESIGN: Case series. SETTING: Pediatric ophthalmology service at the Children's Hospital of Western Ontario, London. PATIENTS: Eighty orthophoric patients (mean age at presentation 19 months [range 4 to 50 months]) with a family history of strabismus or with a diagnosis of pseudostrabismus, blocked tear duct or chalazion, followed for an average of 36 (range 6 to 129) months, and 41 patients with surgically corrected infantile esotropia (mean age at testing with the 20-PD base-out test 21 months [range 9 to 50 months]), followed for an average of 53 (range 14 to 117) months. INTERVENTIONS: Twenty-PD base-out test and red-green Worth four-dot test at near (0.3 m). OUTCOME MEASURE: Presence of peripheral fusion. RESULTS: The 20-PD base-out test had a positive predictive value of 100% in detecting peripheral fusion in the orthophoric patients and 93% (95% confidence interval [CI] 82% to 100%) in the patients with surgically corrected infantile esotropia. CONCLUSIONS: Although the 20-PD base-out test had a negative predictive value of 21% (95% CI 0% to 43%), a positive response remains a useful indicator of peripheral binocular single vision in children too young to undergo more formal sensory testing.

Natural history of presumed congenital Brown syndrome.

OBJECTIVE: To evaluate the stability of the ocular alignment in patients with presumed congenital Brown syndrome. DESIGN: A retrospective review of patients with Brown syndrome with an emphasis on nonsurgical cases. Follow-up of at least 1 year was required for inclusion in the study. SETTING: Patients were selected for this study from the pediatric ophthalmology services at the Ivey Institute of Ophthalmology, London, Ontario, and the Children's Hospital of Eastern Ontario, Ottawa. PATIENTS: A cohort of 71 patients with presumed congenital Brown syndrome. Two cases were bilateral. Eleven cases were excluded because of insufficient length of follow-up, leaving 60 patients with an average follow-up of 46 months. All patients were assessed and followed up by a pediatric ophthalmologist. RESULTS: Of 38 patients who had no hypotropia in primary position at presentation, only two (5%) patients experienced a worsening with the development of a small vertical strabismus during the follow-up period. Six (10%) of the entire group of 60 patients experienced a complete spontaneous resolution of the deficiency in elevation at 4, 6, 8, 10, 12, and 15 years of age. CONCLUSION: Among patients with congenital Brown syndrome, those who are orthotropic in the primary position tend to remain stable or improve over time without surgical intervention.

Serum angiotensin converting enzyme activity in chronic beryllium disease.

Serum angiotensin converting enzyme (SACE) activity is used as a marker of sarcoidosis activity and severity, but in chronic beryllium disease (CBD) the studies of SACE give conflicting results. We examined SACE activity in 23 CBD patients, five patients with beryllium sensitization, and 25 beryllium-exposed control subjects. CBD patients underwent complete clinical evaluation, including physical examination, pulmonary function testing, exercise physiology testing, chest radiography, and bronchoscopy with bronchoalveolar lavage and biopsy. CBD SACE activity was systematically compared with these clinical markers of disease severity. Of CBD patients, 22% had elevated SACE activity. The test did not discriminate CBD patients from those in the beryllium-sensitized or beryllium-exposed groups. However, SACE activity in CBD correlated with the extent of pulmonary granulomatous inflammation as reflected by the symptom of breathlessness, the number of white cells in bronchoalveolar lavage (r = 0.44), the number of lavage lymphocytes (r = 0.58), the lavage lymphocyte percentage (r = 0.55), and the profusion of small opacities on chest radiograph (r = 0.41). The test-retest reliability of the assay was high (r = 0.84), as was the agreement between fresh and -70 degrees C frozen sera (r = 0.93). We conclude that SACE activity levels may reflect the extent of pulmonary granulomatous inflammation in CBD but that the test does not help discriminate disease from nondisease.

Changes in astigmatism after surgery for congenital ptosis.

To determine the effect of ptosis surgery on the astigmatic refractive error in pediatric patients, we assessed 88 eyes postoperatively at 3, 6, and 12 months. Thirty-six eyes of unilateral cases were used as controls. There was an overall increase in average astigmatic refractive error of the study group by 0.30 diopters, while the control group decreased 0.15 D. Thirty-six percent of study eyes changed by more than 0.75 D. Results were similar for both fascia lata slings and levator resections. Those patients older than 4 years showed an increase in average cylinder (0.50 D), while those younger than 4 years decreased their average cylinder (0.20 D). Careful monitoring with postoperative refractions is required in pediatric ptosis patients.

Treatment of blepharospasm and hemifacial spasm with botulinum A toxin: a Canadian multicentre study.

Botulinum A exotoxin was recently approved for use in Canada. We describe the efficacy of botulinum toxin in the management of 235 patients with blepharospasm (mean age 64.3 years) and 130 patients with hemifacial spasm (mean age 60.4 years) treated at three Canadian ophthalmologic centres between 1984 and 1989. A total of 98% of the patients with blepharospasm and 100% of the patients with hemifacial spasm had significant relief of their symptoms; however, 11% of the former and 2% of the latter did not respond to the usual starting concentrations of the drug and needed stronger dosages for relief. The duration of relief varied widely in both groups. Up to 7% of patients had ineffective treatments but responded to subsequent injections. Analysis of variance and linear trend statistics showed that there were no changes in the mean duration of relief over the first several treatments for individual patients in either group. Side effects were transient and included ptosis, exposure keratitis, epiphora and strabismus.

Factors associated with binocular single vision in microtropia/monofixation syndrome.

We reviewed the charts of 398 patients with microtropia/monofixation syndrome to determine what factors influence the level of binocularity attained. Most patients (82%) exhibited some degree of stereoacuity. Patients with a later age at presentation of the initial deviation were more likely to exhibit stereoacuity (p less than 0.001). Patients with smaller initial deviations were also more likely to exhibit stereoacuity (p = 0.016), as were those with smaller manifest deviations on final testing (p less than 0.001). Patients with amblyopia on final testing were less likely to demonstrate stereoacuity (p = 0.0001). Generally, the more intervention required in the form of optical or surgical correction, the poorer the level of binocularity.

Topical 2.5% and 5% phenylephrine: comparison of effects on heart rate and blood pressure.

We compared the effects of 2.5% and 5% concentrations of topical ocular phenylephrine hydrochloride on blood pressure and heart rate in 200 adult patients undergoing extracapsular cataract surgery under either general or neuroleptic anesthesia. Although there was little initial change in mean systolic or diastolic blood pressure in either group, once anesthesia was induced there was a rise of 1 to 5 mm Hg in the lower-dose group, compared with 17 to 26 mm Hg in the higher-dose group. A slight decrease in mean heart rate of 5 to 6 beats/min was noted immediately after the instillation of either the 2.5% or 5% solution. The results suggest that lower doses of topical phenylephrine are safer than higher doses when used preoperatively. This may be particularly significant in hypertensive patients, who have higher baseline blood pressure.