Characterization of the Genome of Equine Herpesvirus 1 Subtype 2

Equine herpesvirus type-1 (EHV-1), a member of the Alphaherpesvirinae. is a major cause of abortion and respiratory disease in horses worldwide. It is also associated with a neurological syndrome, neonatal foal disease and more rarely, coital exanthema. Collectively, these diseases represent a significant economic loss to the thoroughbred industry each year. Two antigenically and genetically distinct subtypes of EHV-1 exist. They can be unequivocally differentiated by restriction endonuclease analysis of their DNAs. Molecular epizootiological studies in America and Australia indicate that both subtypes of EHV-1 are respiratory pathogens with the potential to cause abortion, but only subtype 1 has been associated with abortion storms and the neurological form of the disease. Most of the molecular data published concern the subtype 1 virus. At the onset of this project the genome of EHV-1 subtype 2 was totally uncharacterized. This was probably due in part to the original misconception that it was identical to EHV-1 subtype 1 and in part to the relative difficulty of growing this virus to a high titre in tissue culture. The purpose of this work was to determine whether the two subtypes of EHV-1 shared a common genome structure, to construct the first restriction endonuclease maps for EHV-1 subtype 2 and to investigate the homology between this virus and other members of the Alphaherpesvirinae by molecular hybridization and DNA sequence analysis. Electron microscopy of EHV-1 subtype 2 DNA which had been denatured and self-annealed indicated that a sequence of 11kbp approximately at one genome terminus is repeated in inverse orientation at one internal site. The inverted repeats were shown to be separated by a unique sequence of approximately 13kbp. The presence of repeated sequence within the EHV-1 subtype 2 genome was confirmed by hybridization studies using DNA probes isolated from virion DMA. A library of plasmid clones containing Ram HI fragments representing approximately 75% of the genome was prepared, and the clones were then used to derive Bam HI and Eco RI restriction endonuclease maps for EHV-1 subtype 2 DNA. The results show that the EHV-1 subtype 2 genome consists of two segments, L (111kbp) and S (35kbp). The S component consists of a unique sequence (Us; 9.6kpb -16kbp) flanked by inverted repeats (TRs and 9.5kbp - 12.7kbp). Published data indicate that the EHV-1 subtype 1 genome has a similar structure. However, the maps for the two subtypes are quite different. Eco RI and Bam HI cleave within the TRs/IRs , and so it was not possible to determine whether Us inverts relative to the L region, as it does in subtype 1. HSV-1 DNA fragments containing coding sequences for genes which have been shown previously to be well-conserved in the alphaherpesviruses were hybridized to EHV-1 subtype 2 DMA. Thus, the regions of the EHV-1 subtype 2 genome homologous to probes for the HSV-1 genes encoding the ribonucleotide reductase, the major capsid protein, the major DNA-binding protein and the immediate early protein VmwIE175 were identified. Cloned DNA fragments of EHV-1 subtype 2 were used in comparative hybridization experiments to further determine the extent and distribution of homologous sequences in the genomes of both subtypes of EHV-1 and HSV-1. Regions of detectable homology are arranged colinearly along the genomes suggesting that the three viruses share a common gene arrangement. These results imply that it should be possible to predict the locations of most EHV-1 subtype 2 genes on the basis of our existing knowledge of HSV-1 gene location and function. Published data indicate that the S segment is the least related region in the genomes of several members of the Alphaherpesvirinae and that the TRs /Us and IRs /Us junctions have altered in location, relative to adjacent genes, during evolution. To elucidate the nature of the genes near the EHV-1 subtype 2 TRs/Us junction, the DNA sequence of a 4.57kbp Bam HI fragment was determined using the Sanger chain terminating dideoxynucleotide method. The junction was located within a 100bp region by using several M13 clones in a hybridization study, indicating that TRs/IRs and Us are approximately 10.9kbp and 13.1kbp in size respectively. The G+C content of the TRs portion of the fragment is approximately 17% greater that that of the Us portion. An 8bp sequence is tandemly repeated within the TRs. Analysis of the sequence showed that Bam HI 1 contains two complete open reading frames and the parts of two others. The amino acid sequences of predicted EHV-1 subtype 2 proteins were compared with those coded by the S segments of VZV and HSV-1. Homologues of the four EHV-1 genes were detected in both HSV-1 and VZV. The EHV-1 genes and the TRs/Us junction have an arrangement intermediate between that of their HSV-1 and VZV counterparts. One of the EHV-1 subtype 2 genes apparently encodes a glycoprotein

Teachers' perceptions of Brandon's Matrix as a framework for the teaching and assessment of scientific methods in school science

This article utilizes a framework for classifying different scientific methods suggested by a philosopher of science (Brandon Synthese, 99, 59–73, 1994) called Brandon’s Matrix. It presents findings from teachers who took part in a funded project in England that looked at the nature of scientific methods in science investigations. Science investigations are an integral aspect of science education and, as such, are often included in high stakes examinations. Therefore, teachers need to have a good understanding of a range of scientific methods and their purposes in science investigations. The framework was used to ask teachers to classify science investigations based on how they teach them. It was also employed to devise assessments to measure students’ understanding of scientific methods. The teachers were introduced to the new approaches and their perceptions were gathered to understand if they supported this as a framework for their classroom practice. Evidence from the study suggested that Brandon’s Matrix appealed to teachers as a framework for practical science in schools, and they see potential benefits for its use in the teaching, learning, and assessment of science. Findings from the study showed it appealed to the teachers as a tool for classifying scientific methods, and how they also recognized the importance of assessing practical work and had an appreciation of the constraints and drivers in the current curriculum and assessment requirements in England. Implications for teachers’ professional development are discussed

A Game Approach to Teach Environmentally Benign Manufacturing in the Supply Chain

Multidisciplinary models of education are needed to prepare students for their role in a global work environment. Combined with this need is the reality of the new Millennial Generation entering the educational system with a different approach to learning. This paper introduces an interactive, educational engineering game designed to appeal to the Millennial Generation’s learning preferences. Shortfall is a prototype board game with a team approach and a trial and error methodology to introduce students to environmentally benign manufacturing in the supply chain using the automobile industry as a model. After playing the game, quantitative analysis showed that on average, students gained new knowledge and a changed perception of their confidence in their answers. Qualitative analysis of data demonstrated that students felt the game also helped them with the teamwork/communication aspects of supply chain. Future plans involve converting the game to a computer format to streamline its effectiveness for multi-institutional participation

Complete molecular genome analyses of equine rotavirus a strains from different continents reveal several novel genotypes and a largely conserved genotype constellation

In this study, the complete genome sequences of seven equine group A rotavirus (RVA) strains (RVA/Horse-tc/GBR/L338/1991/G13P[18], RVA/Horse-wt/IRL/03V04954/2003/G3P[12] and RVA/Horse-wt/IRL/04V2024/2004/G14P[12] from Europe; RVA/Horse-wt/ARG/E30/1993/ G3P[12], RVA/Horse-wt/ARG/E403/2006/G14P[12] and RVA/Horse-wt/ARG/E4040/2008/ G14P[12] from Argentina; and RVA/Horse-wt/ZAF/EqRV-SA1/2006/G14P[12] from South Africa) were determined. Multiple novel genotypes were identified and genotype numbers were assigned by the Rotavirus Classification Working Group: R9 (VP1), C9 (VP2), N9 (NSP2), T12 (NSP3), E14 (NSP4), and H7 and H11 (NSP5). The genotype constellation of L338 was unique: G13-P[18]-I6- R9-C9-M6-A6-N9-T12-E14-H11. The six remaining equine RVA strains showed a largely conserved genotype constellation: G3/G14-P[12]-I2/I6-R2-C2-M3-A10-N2-T3-E2/E12-H7, which is highly divergent from other known non-equine RVA genotype constellations. Phylogenetic analyses revealed that the sequences of these equine RVA strains are related distantly to nonequine RVA strains, and that at least three lineages exist within equine RVA strains. A small number of reassortment events were observed. Interestingly, the three RVA strains from Argentina possessed the E12 genotype, whereas the three RVA strains from Ireland and South Africa possessed the E2 genotype. The unusual E12 genotype has until now only been described in Argentina among RVA strains collected from guanaco, cattle and horses, suggesting geographical isolation of this NSP4 genotype. This conserved genetic configuration of equine RVA strains could be useful for future vaccine development or improvement of currently used equine RVA vaccines.Fil: Matthijnssens, Jelle. Katholikie Universiteit Leuven; BélgicaFil: Miño, Orlando Samuel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Papp, Hajnalka. Hungarian Academy of Sciences; HungríaFil: Potgieter, Christiaan. Ondersterpoort Veterinary Institute; SudáfricaFil: Novo, Luis. Katholikie Universiteit Leuven; BélgicaFil: Heylen, Elisabeth. Katholikie Universiteit Leuven; BélgicaFil: Zeller, Mark. Katholikie Universiteit Leuven; BélgicaFil: Garaicoechea, Lorena Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lengyel, György. Dr György Radó Military Medical Centre; HungríaFil: Kisfali, Péter. University Of Pécs; HungríaFil: Cullinane, Ann. Irish Equine Centre; IrlandaFil: Collins, P. J.. Cork Ins Of Technology; IrlandaFil: Ciarlet, Max. Novartis Vaccines and Diagnostics; Estados UnidosFil: O'Shea, Helen. Cork Ins Of Technology; IrlandaFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bányai, Krisztián. Hungarian Academy of Sciences; HungríaFil: Barrandeguy, María Edith. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Van Ranst, Marc. Katholikie Universiteit Leuven; Bélgic

The contribution of geogenic particulate matter to lung disease in indigenous children

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Indigenous children have much higher rates of ear and lung disease than non-Indigenous children, which may be related to exposure to high levels of geogenic (earth-derived) particulate matter (PM). The aim of this study was to assess the relationship between dust levels and health in Indigenous children in Western Australia (W.A.). Data were from a population-based sample of 1077 Indigenous children living in 66 remote communities of W.A. (>2,000,000 km2), with information on health outcomes derived from carer reports and hospitalisation records. Associations between dust levels and health outcomes were assessed by multivariate logistic regression in a multi-level framework. We assessed the effect of exposure to community sampled PM on epithelial cell (NuLi-1) responses to non-typeable Haemophilus influenzae (NTHi) in vitro. High dust levels were associated with increased odds of hospitalisation for upper (OR 1.77 95% CI [1.02–3.06]) and lower (OR 1.99 95% CI [1.08–3.68]) respiratory tract infections and ear disease (OR 3.06 95% CI [1.20–7.80]). Exposure to PM enhanced NTHi adhesion and invasion of epithelial cells and impaired IL-8 production. Exposure to geogenic PM may be contributing to the poor respiratory health of disadvantaged communities in arid environments where geogenic PM levels are high

Modeling Within-Host Dynamics of Influenza Virus Infection Including Immune Responses

Influenza virus infection remains a public health problem worldwide. The mechanisms underlying viral control during an uncomplicated influenza virus infection are not fully understood. Here, we developed a mathematical model including both innate and adaptive immune responses to study the within-host dynamics of equine influenza virus infection in horses. By comparing modeling predictions with both interferon and viral kinetic data, we examined the relative roles of target cell availability, and innate and adaptive immune responses in controlling the virus. Our results show that the rapid and substantial viral decline (about 2 to 4 logs within 1 day) after the peak can be explained by the killing of infected cells mediated by interferon activated cells, such as natural killer cells, during the innate immune response. After the viral load declines to a lower level, the loss of interferon-induced antiviral effect and an increased availability of target cells due to loss of the antiviral state can explain the observed short phase of viral plateau in which the viral level remains unchanged or even experiences a minor second peak in some animals. An adaptive immune response is needed in our model to explain the eventual viral clearance. This study provides a quantitative understanding of the biological factors that can explain the viral and interferon kinetics during a typical influenza virus infection

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Safety, immunogenicity, and reactogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines given as fourth-dose boosters following two doses of ChAdOx1 nCoV-19 or BNT162b2 and a third dose of BNT162b2 (COV-BOOST): a multicentre, blinded, phase 2, randomised trial

Background Some high-income countries have deployed fourth doses of COVID-19 vaccines, but the clinical need, effectiveness, timing, and dose of a fourth dose remain uncertain. We aimed to investigate the safety, reactogenicity, and immunogenicity of fourth-dose boosters against COVID-19.Methods The COV-BOOST trial is a multicentre, blinded, phase 2, randomised controlled trial of seven COVID-19 vaccines given as third-dose boosters at 18 sites in the UK. This sub-study enrolled participants who had received BNT162b2 (Pfizer-BioNTech) as their third dose in COV-BOOST and randomly assigned them (1:1) to receive a fourth dose of either BNT162b2 (30 µg in 0·30 mL; full dose) or mRNA-1273 (Moderna; 50 µg in 0·25 mL; half dose) via intramuscular injection into the upper arm. The computer-generated randomisation list was created by the study statisticians with random block sizes of two or four. Participants and all study staff not delivering the vaccines were masked to treatment allocation. The coprimary outcomes were safety and reactogenicity, and immunogenicity (antispike protein IgG titres by ELISA and cellular immune response by ELISpot). We compared immunogenicity at 28 days after the third dose versus 14 days after the fourth dose and at day 0 versus day 14 relative to the fourth dose. Safety and reactogenicity were assessed in the per-protocol population, which comprised all participants who received a fourth-dose booster regardless of their SARS-CoV-2 serostatus. Immunogenicity was primarily analysed in a modified intention-to-treat population comprising seronegative participants who had received a fourth-dose booster and had available endpoint data. This trial is registered with ISRCTN, 73765130, and is ongoing.Findings Between Jan 11 and Jan 25, 2022, 166 participants were screened, randomly assigned, and received either full-dose BNT162b2 (n=83) or half-dose mRNA-1273 (n=83) as a fourth dose. The median age of these participants was 70·1 years (IQR 51·6–77·5) and 86 (52%) of 166 participants were female and 80 (48%) were male. The median interval between the third and fourth doses was 208·5 days (IQR 203·3–214·8). Pain was the most common local solicited adverse event and fatigue was the most common systemic solicited adverse event after BNT162b2 or mRNA-1273 booster doses. None of three serious adverse events reported after a fourth dose with BNT162b2 were related to the study vaccine. In the BNT162b2 group, geometric mean anti-spike protein IgG concentration at day 28 after the third dose was 23 325 ELISA laboratory units (ELU)/mL (95% CI 20 030–27 162), which increased to 37 460 ELU/mL (31 996–43 857) at day 14 after the fourth dose, representing a significant fold change (geometric mean 1·59, 95% CI 1·41–1·78). There was a significant increase in geometric mean anti-spike protein IgG concentration from 28 days after the third dose (25 317 ELU/mL, 95% CI 20 996–30 528) to 14 days after a fourth dose of mRNA-1273 (54 936 ELU/mL, 46 826–64 452), with a geometric mean fold change of 2·19 (1·90–2·52). The fold changes in anti-spike protein IgG titres from before (day 0) to after (day 14) the fourth dose were 12·19 (95% CI 10·37–14·32) and 15·90 (12·92–19·58) in the BNT162b2 and mRNA-1273 groups, respectively. T-cell responses were also boosted after the fourth dose (eg, the fold changes for the wild-type variant from before to after the fourth dose were 7·32 [95% CI 3·24–16·54] in the BNT162b2 group and 6·22 [3·90–9·92] in the mRNA-1273 group).Interpretation Fourth-dose COVID-19 mRNA booster vaccines are well tolerated and boost cellular and humoral immunity. Peak responses after the fourth dose were similar to, and possibly better than, peak responses after the third dose