IBV : potential as a vaccine vector and identification of a novel subgenomic mRNA

Using an IBV reverse genetics system a series of recombinant viruses were generated to investigate the potential for utilizing IBV as a vaccine vector. Through the replacement of non-essential regions of the IBV genome, with eGFP or hRluc, factors influencing the stability of recombinant viruses expressing heterologous genes were determined. Expression of heterologous proteins was possible from a variety of virus constructs. The stability of recombinant viruses varied depending on the genome location of the heterologous gene, with replacement of Gene 5 proving to be most stable following passage in cell culture. Stability was strongly influenced by the MOI at which viruses were passaged, with low MOIs resulting in increased stability. The replacement of Gene 5 with a heterologous virus gene may be a suitable target for development of a bivalent vaccine capable of protecting against IBV and a second avian viral disease. Analysis of recombinant IBV mRNA expression profiles led to an investigation into an uncharacterized RNA species, and its link to the IBV intergenic region. A novel subgenomic mRNA was identified associated with the intergenic region that was shown to be transcribed via a non-canonical transcription regulatory sequence. In contradiction to the current model of coronavirus transcription this mRNA has a transcription regulatory sequence derived mainly from the leader, and not the body, transcription regulatory sequence. The non-canonical sequence was shown to be responsible for reduced transcription levels of the intergenic region mRNA. This project proposes the presence of an additional IBV subgenomic mRNA, transcribed via a non-canonical mechanism, and encoding a novel 5th accessory protein of IBV and closely related gammacoronaviruses

Постспленектомічний спленоз, як предиктор розвитку рецидиву хвороби Верльгофа

Спленектомія (СЕ) у хворих на хворобу Верльгофа (ХВ), виконується у випадку неефективності консервативного лікування цього тяжкого захворювання. Не зважаючи на високий ризик оперативного втручання, пов'язаний з притаманним для цього захворювання порушеннями в системі згортання крові, СЕ продовжує активно використовуватися , так як є найбільш ефективним методом лікування ХВ

Hydroxypropyl methylcellulose-based nasal sprays effectively inhibit in vitro SARS-CoV-2 infection and spread

The ongoing coronavirus disease (COVID-19) pandemic has required a variety of non-medical interventions to limit the transmission of the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One such option is over-the-counter nasal sprays that aim to block virus entry and transmission within the nasal cavity. In this study, we assessed the ability of three hydroxypropyl methylcellulose (HPMC)-based powder nasal sprays, produced by Nasaleze, to inhibit SARS-CoV-2 infection and release in vitro. Upon application, the HPMC powder forms a gel-like matrix within the nasal cavity—a process we recapitulated in cell culture. We found that virus release from cells previously infected with SARS-CoV-2 was inhibited by the gel matrix product in a dose-dependent manner, with virus levels reduced by >99.99% over a 72 h period at a dose of 6.4 mg/3.5 cm2. We also show that the pre-treatment of cells with product inhibited SARS-CoV-2 infection, independent of the virus variant. The primary mechanism of action appears to be via the formation of a physical, passive barrier. However, the addition of wild garlic provided additional direct antiviral properties in some formulations. We conclude that HPMC-based nasal sprays may offer an additional component to strategies to limit the spread of respiratory viruses, including SARS-CoV-

Imprecise recombinant viruses evolve via a fitness-driven, iterative process of polymerase template-switching events

Funding: This work was supported by the Biotechnology and Biological Sciences Research Council - https://bbsrc.ukri.org/ - (BB/M009343/1 to D.J.E) and an ISSF award from The Wellcome Trust - https://wellcome.org/ - to the BSRC, University of St Andrews. F.G.A was supported by a PhD studentship from the Ministry of Education, Government of Saudi Arabia. S.J was supported by a Microbiology Society - https://microbiologysociety.org/ - Harry Smith Vacation Studentship awarded to K.B.Viruses with positive-sense RNA genomes, such as poliovirus, have several mechanisms by which they evolve. One of these is the process of recombination involving the large-scale exchange of genetic information. Recombination occurs during replication when the viral polymerase, bound to the nascent RNA chain, switches from copying one genome to another. However, the polymerase does not always accurately switch between the two, resulting in sequence duplications or deletions, and genomes that are referred to as imprecise. Over multiple rounds of replication sequence duplications are lost and genomes are resolved to wild type length, but it is unclear how this occurs. Here we used synthetic polioviruses containing defined sequence duplications to determine that the genome population undergoes repeated rounds of recombination until sequence duplications are lost and viruses with precise, wild type length genomes are selected for. This selection is based on the overall fitness of the virus population, with less fit imprecise viruses evolving more quickly. Our study suggests that recombination is a continual process where virus fitness drives the selection of a small subset of recombinant variants. These data are important for understanding how novel viruses evolve via recombination and how this process can be blocked to prevent novel and dangerous pathogens from arising.Publisher PDFPeer reviewe

Generated randomly and selected functionally? The nature of enterovirus recombination

FA was supported by a PhD studentship from the Ministry of Education, Government of Saudi Arabia and KB was supported by Biological Sciences Research Council award BB/M009343/1to D.J.E and an ISSF award from The Wellcome Trust to the BSRC, University of St Andrews.Genetic recombination in RNA viruses is an important evolutionary mechanism. It con-tributes to population diversity, host/tissue adaptation and compromises vaccine efficacy. Both the molecular mechanism and initial products of recombination are relatively poorly understood. We used an established poliovirus-based in vitro recombination assay to investigate the roles of se-quence identity and RNA structure, both implicated or inferred from analysis of circulating recom-binant viruses, in the process. In addition, we used next generation sequencing to investigate the early products of recombination after cellular co-infection with different poliovirus serotypes. In independent studies we find no evidence for a role for RNA identity or structure in determining recombination junctions location. Instead, genome function and fitness are of greater importance in determining the identity of recombinant progeny. These studies provide further insights into this important evolutionary mechanism and emphasise the critical nature of the selection process on a mixed virus population.Publisher PDFPeer reviewe

Community Co-Design: From Magic Squares to Magic Dresses

This project joined elementary, secondary, and postsecondary students with a textile museum, community cultural centre, and provincial science centre to collaborate on textile design and printing, as well as garment design, creation, and exhibition. While the design process centered on the concept of the magic square, each contribution was independent and progressive, and handed off to the next group like a baton in a relay race

Systematic review of the psychometric performance of generic childhood multi-attribute utility instruments

Background Childhood multi-attribute utility instruments (MAUIs) can be used to measure health utilities in children (aged ≤ 18 years) for economic evaluation. Systematic review methods can generate a psychometric evidence base that informs their selection for application. Previous reviews focused on limited sets of MAUIs and psychometric properties, and only on evidence from studies that directly aimed to conduct psychometric assessments. Objective This study aimed to conduct a systematic review of psychometric evidence for generic childhood MAUIs and to meet three objectives: (1) create a comprehensive catalogue of evaluated psychometric evidence; (2) identify psychometric evidence gaps; and (3) summarise the psychometric assessment methods and performance by property. Methods A review protocol was registered with the Prospective Register of Systematic Reviews (PROSPERO; CRD42021295959); reporting followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guideline. The searches covered seven academic databases, and included studies that provided psychometric evidence for one or more of the following generic childhood MAUIs designed to be accompanied by a preference-based value set (any language version): 16D, 17D, AHUM, AQoL-6D, CH-6D, CHSCS-PS, CHU9D, EQ-5D-Y-3L, EQ-5D-Y-5L, HUI2, HUI3, IQI, QWB, and TANDI; used data derived from general and/or clinical childhood populations and from children and/or proxy respondents; and were published in English. The review included ‘direct studies’ that aimed to assess psychometric properties and ‘indirect studies’ that generated psychometric evidence without this explicit aim. Eighteen properties were evaluated using a four-part criteria rating developed from established standards in the literature. Data syntheses identified psychometric evidence gaps and summarised the psychometric assessment methods/results by property. Results Overall, 372 studies were included, generating a catalogue of 2153 criteria rating outputs across 14 instruments covering all properties except predictive validity. The number of outputs varied markedly by instrument and property, ranging from 1 for IQI to 623 for HUI3, and from zero for predictive validity to 500 for known-group validity. The more recently developed instruments targeting preschool children (CHSCS-PS, IQI, TANDI) have greater evidence gaps (lack of any evidence) than longer established instruments such as EQ-5D-Y, HUI2/3, and CHU9D. The gaps were prominent for reliability (test–retest, inter-proxy-rater, inter-modal, internal consistency) and proxy-child agreement. The inclusion of indirect studies (n = 209 studies; n = 900 outputs) increased the number of properties with at least one output of acceptable performance. Common methodological issues in psychometric assessment were identified, e.g., lack of reference measures to help interpret associations and changes. No instrument consistently outperformed others across all properties. Conclusion This review provides comprehensive evidence on the psychometric performance of generic childhood MAUIs. It assists analysts involved in cost-effectiveness-based evaluation to select instruments based on the application-specific minimum standards of scientific rigour. The identified evidence gaps and methodological issues also motivate and inform future psychometric studies and their methods, particularly those assessing reliability, proxy-child agreement, and MAUIs targeting preschool children

Multiple novel non-canonically transcribed sub-genomic mRNAs produced by avian coronavirus infectious bronchitis virus

Funding: This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/L003988/1 and 1645891, and strategic funding to The Pirbright Institute, BBS/E/I/00007035, BBS/E/I/00007034, BBS/E/I/00007037 and BBS/E/I/00007039.Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.Publisher PDFPeer reviewe

Small vessel disease and dietary salt intake: cross sectional study and systematic review

Background: Higher dietary salt intake increases the risk of stroke and may increase white matter hyperintensity (WMH) volume. We hypothesized that a long-term higher salt intake may be associated with other features of small vessel disease (SVD). Methods: We recruited consecutive patients with mild stroke presenting to the Lothian regional stroke service. We performed brain magnetic resonance imaging, obtained a basic dietary salt history, and measured the urinary sodium/creatinine ratio. We also carried out a systematic review to put the study in the context of other studies in the field. Results: We recruited 250 patients, 112 with lacunar stroke and 138 with cortical stroke, with a median age of 67.5 years. After adjustment for risk factors, including age and hypertension, patients who had not reduced their salt intake in the long term were more likely to have lacunar stroke (odds ratio [OR], 1.90; 95% confidence interval [CI], 1.10-3.29), lacune(s) (OR, 2.06; 95% CI, 1.09-3.99), microbleed(s) (OR, 3.4; 95% CI, 1.54, 8.21), severe WMHs (OR, 2.45; 95% CI 1.34-4.57), and worse SVD scores (OR, 2.17; 95% CI, 1.22-3.9). There was limited association between SVD and current salt intake or urinary sodium/creatinine ratio. Our systematic review found no previously published studies of dietary salt and SVD. Conclusion: The association between dietary salt and background SVD is a promising indication of a potential neglected contributory factor for SVD. These results should be replicated in larger, long-term studies using the recognized gold-standard measures of dietary sodium

Mechanically ventilated patients shed high titre live SARS-CoV2 for extended periods from both the upper and lower respiratory tract

Background SARS-CoV-2 infection can lead to severe acute respiratory distress syndrome needing intensive care admission and may lead to death. As a virus that transmits by respiratory droplets and aerosols, determining the duration of viable virus shedding from the respiratory tract is critical for patient prognosis, and informs infection control measures both within healthcare settings and the public domain. Methods We prospectively examined upper and lower airway respiratory secretions for both viral RNA and infectious virions in mechanically ventilated patients admitted to the intensive care unit of the University Hospital of Wales. Samples were taken from the oral cavity (saliva), oropharynx (sub-glottic aspirate), or lower respiratory tract (non-directed bronchoalveolar lavage (NBL) or bronchoalveolar lavage (BAL)) and analyzed by both qPCR and plaque assay. Results 117 samples were obtained from 25 patients. qPCR showed extremely high rates of positivity across all sample types, however live virus was far more common in saliva (68%) than in BAL/NBAL (32%). Average titres of live virus were higher in subglottic aspirates (4.5x10 7) than in saliva (2.2x10 6) or BAL/NBAL (8.5x10 6) and reached >10 8 PFU/ml in some samples. The longest duration of shedding was 98 days, while most patients (14/25) shed live virus for 20 days or longer. Conclusions Intensive care unit patients infected with SARS-CoV-2 can shed high titres of virus both in the upper and lower respiratory tract and tend to be prolonged shedders. This information is important for decision making around cohorting patients, de-escalation of PPE, and undertaking potential aerosol generating procedures