Application of high-throughput sequencing to whole rabies viral genome characterisation and its use for phylogenetic re-evaluation of a raccoon strain incursion into the province of Ontario

Raccoon rabies remains a serious public health problem throughout much of the eastern seaboard of North America due to the urban nature of the reservoir host and the many challenges inherent in multi-jurisdictional efforts to administer co-ordinated and comprehensive wildlife rabies control programmes. Better understanding of the mechanisms of spread of rabies virus can play a significant role in guiding such control efforts. To facilitate a detailed molecular epidemiological study of raccoon rabies virus movements across eastern North America, we developed a methodology to efficiently determine whole genome sequences of hundreds of viral samples. The workflow combines the generation of a limited number of overlapping amplicons covering the complete viral genome and use of high throughput sequencing technology. The value of this approach is demonstrated through a retrospective phylogenetic analysis of an outbreak of raccoon rabies which occurred in the province of Ontario between 1999 and 2005. As demonstrated by the number of single nucleotide polymorphisms detected, whole genome sequence data were far more effective than single gene sequences in discriminating between samples and this facilitated the generation of more robust and informative phylogenies that yielded insights into the spatio-temporal pattern of viral spread. With minor modification this approach could be applied to other rabies virus variants thereby facilitating greatly improved phylogenetic inference and thus better understanding of the spread of this serious zoonotic disease. Such information will inform the most appropriate strategies for rabies control in wildlife reservoirs

Production of β-methylamino-L-alanine (BMAA) and its isomers by freshwater diatoms

© 2019 by the authors. β-methylamino-L-alanine (BMAA) is a non-protein amino acid that has been implicated as a risk factor for motor neurone disease (MND). BMAA is produced by a wide range of cyanobacteria globally and by a small number of marine diatoms. BMAA is commonly found with two of its constitutional isomers: 2,4-diaminobutyric acid (2,4-DAB), and N-(2-aminoethyl)glycine (AEG). The isomer 2,4-DAB, like BMAA, has neurotoxic properties. While many studies have shown BMAA production by cyanobacteria, few studies have looked at other algal groups. Several studies have shown BMAA production by marine diatoms; however, there are no studies examining freshwater diatoms. This study aimed to determine if some freshwater diatoms produced BMAA, and which diatom taxa are capable of BMAA, 2,4-DAB and AEG production. Five axenic diatom cultures were established from river and lake sites across eastern Australia. Cultures were harvested during the stationary growth phase and intracellular amino acids were extracted. Using liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS), diatom extracts were analysed for the presence of both free and protein-associated BMAA, 2,4-DAB and AEG. Of the five diatom cultures analysed, four were found to have detectable BMAA and AEG, while 2,4-DAB was found in all cultures. These results show that BMAA production by diatoms is not confined to marine genera and that the prevalence of these non-protein amino acids in Australian freshwater environments cannot be solely attributed to cyanobacteria

Effects of chlorpyrifos on macroinvertebrate communities in coastal stream mesocosms

This study measured the effects of a single pulse of chlorpyrifos at nominal concentrations of 1 and 10 μg/l on the macroinvertebrate community structure of a coastal stream mesocosm system. Analysis of data using Principal Response Curves (PRC) and Monte Carlo tests showed significant changes in the treated stream mesocosms relative to that of the controls. These changes in the macroinvertebrate assemblages occurred within 6 h, and persisted for at least 124 days after dosing. Significant community-level effects were detected at the lowest concentration on days 2 and 16 post-dosing, giving a no-observed effect concentration (NOECcommunity) of 1.2 μg/l (measured). The mayflies Atalophlebia sp. and Koorrnonga sp., Chironomidae and Acarina were all sensitive to chlorpyrifos and decreased in abundance in treated mesocosms after dosing. The fauna of these coastal stream mesocosms showed similar sensitivity to chlorpyrifos with that of other reported studies, but there was no evidence of recovery after 124 days. © 2007 Springer Science+Business Media, LLC

Toxicity and bioaccumulation of two non-protein amino acids synthesised by cyanobacteria, β-N-Methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), on a crop plant.

In order to study the toxicity of the cyanobacterial non-protein amino acids (NPAAs) L-β-N-methylamino-L-alanine (BMAA) and its structural isomer L-2,4-diaminobutyric acid (DAB) in the forage crop plant alfalfa (Medicago sativa), seedlings were exposed to NPAA-containing media for four days. Root growth was significantly inhibited by both treatments. The content of derivatised free and protein-bound BMAA and DAB in seedlings was then analysed by LC-MS/MS. Both NPAAs were detected in free and protein-bound fractions with higher levels detected in free fractions. Compared to shoots, there was approximately tenfold more BMAA and DAB in alfalfa roots. These results suggest that NPAAs might be taken up into crop plants from contaminated irrigation water and enter the food chain. This may present an exposure pathway for NPAAs in humans

Study protocol for a multicentre longitudinal mixed methods study to explore the Outcomes of ChildrEn and fAmilies in the first year after paediatric Intensive Care: the OCEANIC study.

INTRODUCTION: Annually in the UK, 20 000 children become very ill or injured and need specialist care within a paediatric intensive care unit (PICU). Most children survive. However, some children and their families may experience problems after they have left the PICU including physical, functional and/or emotional problems. It is unknown which children and families experience such problems, when these occur or what causes them. The aim of this mixed-method longitudinal cohort study is to understand the physical, functional, emotional and social impact of children surviving PICU (aged: 1 month-17 years), their parents and siblings, during the first year after a PICU admission. METHODS AND ANALYSIS: A quantitative study involving 300 child survivors of PICU; 300 parents; and 150-300 siblings will collect data (using self-completion questionnaires) at baseline, PICU discharge, 1, 3, 6 and 12 months post-PICU discharge. Questionnaires will comprise validated and reliable instruments. Demographic data, PICU admission and treatment data, health-related quality of life, functional status, strengths and difficulties behaviour and post-traumatic stress symptoms will be collected from the child. Parent and sibling data will be collected on the impact of paediatric health conditions on the family's functioning capabilities, levels of anxiety and social impact of the child's PICU admission. Data will be analysed using descriptive and inferential statistics. Concurrently, an embedded qualitative study involving semistructured interviews with 24 enrolled families at 3 months and 9 months post-PICU discharge will be undertaken. Framework analysis will be used to analyse the qualitative data. ETHICS AND DISSEMINATION: The study has received ethical approval from the National Health Services Research Ethics Committee (Ref: 19/WM/0290) and full governance clearance. This will be the first UK study to comprehensively investigate physical, functional, emotional and social consequences of PICU survival in the first-year postdischarge.Clinical Trials Registration Number: ISRCTN28072812 [Pre-results]

Assessing Doppler velocities of Rankin Inlet F-region echoes

Doppler velocities observed by the Rankin Inlet (RKN) PolarDARN radar are assessed with a focus on data in the beams oriented roughly along the magnetic meridian. Hourly scatter plots for every month are built. They are shown to vary widely, with median values showing very clear magnetic local time variation with maximum magnitude during pre-noon and pre-midnight hours. The histograms contain a significant amount of very small velocity data that dominates at farther ranges and during the daytime. Near noon data show generally antisunward flows but at large ranges/magnetic latitudes and very close to noon, sunward flows occur for periods of positive IMF Bz. The reverse flows are stronger during spring equinox. The velocity magnitude was found to depend linearly on the IMF Bz and interplanetary electric field. Velocities are often found to be smaller than those expected from the statistical convection model of Ruohoniemi and Greenwald –1996