New approaches to characterise viral pathogens in aquaculture

Aquaculture is the fastest growing food-producing sector in the world and of considerable economic, cultural and environmental relevance. This sector will be vital to achieving future food security demands, but its continued sustainable expansion is severely threatened by infectious diseases, with viral diseases amongst the most problematic to control. Unlike farmed livestock, fish are generally reared in open systems with constant circulation between farms and the natural aquatic environment. This routinely exposes the animals to naturally occurring viruses in the water, both pathogenic and non-pathogenic, which are generally uptaken through mucosal surfaces (i.e. gills and gut surfaces). However, with the increase in globalisation, aquatic species are frequently farmed in non-native habitats, thus exposing them not only to the pathogens present in wild relatives of the same species, but to pathogens of other species in their introduced habitat. Moreover, wild fish are threatened by viral disease outbreaks on fish farms due to the high density of individuals available to carry and transmit the pathogen. Characterising viral infections is therefore important to support the prevention and control of disease outbreaks, as understanding the disease agent enables both fish farmers and regulating agencies to tailor appropriate mitigation strategies. The routine use of whole genome sequencing to screen infected animals is not yet commonplace in the aquaculture industry, where genetic screening of viruses is largely done using PCR for 1 or 2 marker genes. However, the ‘genomic surveillance’ approach has been used to great effect in cases of disease outbreaks relevant to human health, and could be applied in aquaculture to enhance the resolution of molecular epidemiology investigations and diagnostic tests. Moreover, with the under-researched genetic diversity of aquatic viruses, significant advances in the understanding of host-pathogen interactions could be achieved with a denser and better curated genomic database of viruses. To address these knowledge gaps, I have developed and optimised several approaches to characterise aquatic viruses up-taking various sequencing methods depending on the resolution required for the specific study, using salmonid alphavirus (SAV) as a primary study system. To rapidly and accurately generate consensus-level genomes of specific pathogenic viruses, I developed a targeted PCR approach using overlapping long amplicons tiled across the SAV genome for full coverage. These amplicons are sequenced on the Oxford Nanopore Technologies MinION long-read platform. An analysis workflow was then optimised to generate consensus genomes while maintaining capability to discover SAV subtype-level co-infections by simultaneously mapping to multiple reference sequences. This approach can generate highly accurate consensus sequences (as judged by independent Sanger sequencing) and detect co-infections of strains with ≥ 95% pairwise identity over a 2kb region, even when minor infecting strains are present at just 5% frequency. This approach was used to investigate the population dynamics and phylogeography of the SAV3 epidemic in Norwegian aquaculture, revealing repeated seedings of SAV3 from ‘source’ to ‘sink’ counties. To characterise viral genetic diversity within a host, I applied a targeted sequence capture strategy to obtain SAV genomes at high coverage (using Illumina technology) from infected fish using both pooled and individual tissue samples. This approach utilises RNA baits to capture and enrich for specific DNA (or cDNA) strands in a sample, and allows for greater sequencing efficiency. These baits, while designed from specific templates, are less specific than PCR primers and can tolerate a certain amount of template mismatches, thus capturing all genetic variation of a specific viral species within a sample. This approach was used to compare the genetic diversity of SAV in farmed Atlantic salmon and rainbow trout, in addition to two wild flatfish species, sampled from multiple regions in Scottish and Irish waters. In the same study, I provided evidence of complex infections on single fish farms, and for co-infections within single wild fish. Finally, I developed a pipeline to detect viral infections in metagenomics samples, which can be applied even when the infectious agent is unknown. This involves an optional step of mapping to the host reference genome to increase efficiency of later steps, assembly of the remaining reads with a transcriptome assembler, and identifying viral transcripts using homology-based tools. Before implementation, this pipeline was benchmarked against several datasets, including a simulated virome and a simulated co-infection of two strains of the same virus. It was also tested against datasets with known pathogens, resulting in similar efficiencies of detection as a mapping-based approach. Finally the pipeline was used on datasets with unknown viromes to demonstrate its applicability to detect novel viral species. Overall, my research has led to the development of several cutting-edge approaches for the genomic analysis of aquatic viruses and other pathogens, and helps clarify which approach is most useful in different epidemiological settings. I also demonstrate that genome-wide analyses of viral pathogens impacting salmonid aquaculture generates valuable additional information on viral diversity compared to standard surveillance methods using particular marker genes, advocating for route use of genomic approaches in this sector

The X-ray Spectrum and Spectral Energy Distribution of FIRST J155633.8+351758: a LoBAL Quasar with a Probable Polar Outflow

We report the results of a new 60 ks Chandra X-ray Observatory Advanced CCD Imaging Spectrometer S-array (ACIS-S) observation of the reddened, radio-selected, highly polarized `FeLoBAL' quasar FIRST J1556+3517. We investigated a number of models of varied sophistication to fit the 531-photon spectrum. These models ranged from simple power laws to power laws absorbed by hydrogen gas in differing ionization states and degrees of partial covering. Preferred fits indicate that the intrinsic X-ray flux is consistent with that expected for quasars of similarly high luminosity, i.e., an intrinsic, dereddened and unabsorbed optical to X-ray spectral index of -1.7. We cannot tightly constrain the intrinsic X-ray power-law slope, but find indications that it is flat (photon index Gamma = 1.7 or flatter at a >99% confidence for a neutral hydrogen absorber model). Absorption is present, with a column density a few times 10^23 cm^-2, with both partially ionized models and partially covering neutral hydrogen models providing good fits. We present several lines of argument that suggest the fraction of X-ray emissions associated with the radio jet is not large. We combine our Chandra data with observations from the literature to construct the spectral energy distribution of FIRST J1556+3517 from radio to X-ray energies. We make corrections for Doppler beaming for the pole-on radio jet, optical dust reddening, and X-ray absorption, in order to recover a probable intrinsic spectrum. The quasar FIRST J1556+3517 seems to be an intrinsically normal radio-quiet quasar with a reddened optical/UV spectrum, a Doppler-boosted but intrinsically weak radio jet, and an X-ray absorber not dissimilar from that of other broad absorption line quasars.Comment: to be published in MNRA

There is a low rate of major adverse cardiovascular events in chest pain patients with a moderate risk heart score referred from urgent care for expedited outpatient cardiology evaluation: a multi-center study

Background The HEART score is an effective method of risk stratifying emergency department (ED) patients with chest pain. The rate of major adverse cardiovascular events (MACE) in patients with moderate HEART score referred from an urgent care (UC) for an expedited outpatient cardiology evaluation for 11 months was described in 133 patients in a previous study. This is a follow-up study with 18 months of data and 206 patients.Aim. The primary outcome was to examine the rate of MACE when patients with moderate HEART score were referred for an expedited outpatient cardiology follow-up after evaluation in urgent care. The secondary outcome was to determine if there is a decrease in rate of ED transfer after this protocol was introduced.Methods. A cross-sectional study was conducted by a multispecialty group in Las Vegas, Nevada, which included 206 patients with a HEART score of 4 to 6 (i.e.: moderate risk) who presented to one of five UC centers with chest pain or an anginal equivalent. A streamlined evaluation protocol to assess each HEART score component was adopted by all UC providers to facilitate an expedited outpatient cardiology follow-up, as an alternative to referral to the emergency department. Data was collected from February 14, 2019 through August 13, 2020. The population was followed for 6 weeks with a primary endpoint of MACE determined by electronic medical record review and direct phone contact with patients. Outcomes were confirmed in 98% of patients. Chest pain transfer data was compared between 12 months prior to implementing HEART protocol and 18 months of data analysis while using the new protocol.Results. Over the course of 18 months, 206 patients with a moderate risk HEART score were referred to outpatient cardiology in an expedited manner. The average age was 65 with 53% female and 47% male patients. 150 patients (73% of the 206) were seen within 3 days, 114 (55%) underwent stress testing, 6 (3%) had coronary computed tomography angiogram, and 6 (3%) received an invasive coronary angiogram. Five patients were found to have MACE: one patient who had a non-ST-elevation myocardial infarction and subsequent coronary stent, two patients were found to have obstructive disease after coronary angiography with subsequent coronary artery bypass graft, one patient had an abnormal stress test and subsequent coronary stent, and one patient had critical mitral stenosis, multi-vessel coronary artery disease and underwent coronary artery bypass graft with mitral valve replacement with complications of renal failure and COVID-19 and expired. The emergency department referral rate declined by 21%.Conclusion. Patients with a moderate risk HEART score referred from UC for an expedited outpatient cardiology evaluation had a low rate of MACE and no deaths due to delay of care. There was also a significant decrease in the rate of ED referrals.Background. The HEART score is an effective method of risk stratifying emergency department (ED) patients with chest pain. The rate of major adverse cardiovascular events (MACE) in patients with moderate HEART score referred from an urgent care (UC) for an expedited outpatient cardiology evaluation for 11 months was described in 133 patients in a previous study. This is a follow-up study with 18 months of data and 206 patients.Aim. The primary outcome was to examine the rate of MACE when patients with moderate HEART score were referred for an expedited outpatient cardiology follow-up after evaluation in urgent care. The secondary outcome was to determine if there is a decrease in rate of ED transfer after this protocol was introduced.Methods. A cross-sectional study was conducted by a multispecialty group in Las Vegas, Nevada, which included 206 patients with a HEART score of 4 to 6 (i.e.: moderate risk) who presented to one of five UC centers with chest pain or an anginal equivalent. A streamlined evaluation protocol to assess each HEART score component was adopted by all UC providers to facilitate an expedited outpatient cardiology follow-up, as an alternative to referral to the emergency department. Data was collected from February 14, 2019 through August 13, 2020. The population was followed for 6 weeks with a primary endpoint of MACE determined by electronic medical record review and direct phone contact with patients. Outcomes were confirmed in 98% of patients. Chest pain transfer data was compared between 12 months prior to implementing HEART protocol and 18 months of data analysis while using the new protocol.Results. Over the course of 18 months, 206 patients with a moderate risk HEART score were referred to outpatient cardiology in an expedited manner. The average age was 65 with 53% female and 47% male patients. 150 patients (73% of the 206) were seen within 3 days, 114 (55%) underwent stress testing, 6 (3%) had coronary computed tomography angiogram, and 6 (3%) received an invasive coronary angiogram. Five patients were found to have MACE: one patient who had a non-ST-elevation myocardial infarction and subsequent coronary stent, two patients were found to have obstructive disease after coronary angiography with subsequent coronary artery bypass graft, one patient had an abnormal stress test and subsequent coronary stent, and one patient had critical mitral stenosis, multi-vessel coronary artery disease and underwent coronary artery bypass graft with mitral valve replacement with complications of renal failure and COVID-19 and expired. The emergency department referral rate declined by 21%.Conclusion. Patients with a moderate risk HEART score referred from UC for an expedited outpatient cardiology evaluation had a low rate of MACE and no deaths due to delay of care. There was also a significant decrease in the rate of ED referrals

Evolution and Expression of Tissue Globins in Ray-Finned Fishes

We thank Professor Ian A. Johnston FRSE and Dr Daniel Garcia de la Serrana (School of Biology, University of St. Andrews) for providing tissues samples for African butterflyfish and spotted gar. We are grateful to Professor Peter W.H. Holland FRS (Department of Zoology, University of Oxford) for sharing sequence databases for Osteoglossiformes. We thank Professor Christopher J. Secombes (Institute of Biological and Environmental Sciences, University of Aberdeen) for gifting rainbow trout used in the study. Mr Ronald McKay contributed towards Pantodon molecular work during his undergraduate research. MDG is a PhD student funded by the BBSRC EASTBIO Doctoral Training Partnership (DTP) (BB/J01446X/1). The study received support from institutional funds within the University of Aberdeen and from an undergraduate Research Experience Placement scheme granted by the BBSRC EASTBIO DTP scheme.Peer reviewedPublisher PD

HexPak and GradPak: variable-pitch dual-head IFUs for the WIYN 3.5m Telescope Bench Spectrograph

We describe the design, construction, and expected performance of two new fiber integral field units (IFUs) --- HexPak and GradPak --- for the WIYN 3.5m Telescope Nasmyth focus and Bench Spectrograph. These are the first IFUs to provide formatted fiber integral field spectroscopy with simultaneous sampling of varying angular scales. HexPak and GradPak are in a single cable with a dual-head design, permitting easy switching between the two different IFU heads on the telescope without changing the spectrograph feed: the two heads feed a variable-width double-slit. Each IFU head is comprised of a fixed arrangement of fibers with a range of fiber diameters. The layout and diameters of the fibers within each array are scientifically-driven for observations of galaxies: HexPak is designed to observe face-on spiral or spheroidal galaxies while GradPak is optimized for edge-on studies of galaxy disks. HexPak is a hexagonal array of 2.9 arcsec fibers subtending a 40.9 arcsec diameter, with a high-resolution circular core of 0.94 arcsec fibers subtending 6 arcsec diameter. GradPak is a 39 by 55 arcsec rectangular array with rows of fibers of increasing diameter from angular scales of 1.9 arcsec to 5.6 arcsec across the array. The variable pitch of these IFU heads allows for adequate sampling of light profile gradients while maintaining the photon limit at different scales.Comment: 10 pages, 4 figures, presented at SPIE, Astronomical Telescopes and Instrumentation, 1 - 6 July 2012, Amsterdam, Netherland

High intensity exercise as a dishabituating stimulus restores counterregulatory responses in recurrently hypoglycemic rodents

Hypoglycemia is a major adverse effect of insulin therapy for people with type 1 diabetes (T1D). Profound defects in the normal counterregulatory response to hypoglycemia explain the frequency of hypoglycemia occurrence in T1D. Defective counterregulation results to a large extent from prior exposure to hypoglycemia per se, leading to a condition called impaired awareness of hypoglycemia (IAH), the cause of which is unknown. In the current study, we investigate the hypothesis that IAH develops through a special type of adaptive memory referred to as habituation. To test this hypothesis, we used a novel intense stimulus (high-intensity exercise) to demonstrate two classic features of a habituated response, namely dishabituation and response recovery. We demonstrate that after recurrent hypoglycemia the introduction of a novel dishabituating stimulus (a single burst of high-intensity exercise) in male Sprague-Dawley rats restores the defective hypoglycemia counterregulatory response. In addition, the rats showed an enhanced response to the novel stimulus (response recovery). We make the further observation using proteomic analysis of hypothalamic extracts that high-intensity exercise in recurrently hypoglycemic rats increases levels of a number of proteins linked with brain-derived neurotrophic factor signaling. These findings may lead to novel therapeutic approaches for individuals with T1D and IAH.</jats:p