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BackgroundRapid identification of individuals with acute respiratory infections is crucial for preventing nosocomial infections. For rapid diagnosis, especially in EDs, lateral flow devices (LFDs) are a convenient, inexpensive option with a rapid turnaround. Several ‘multiplex’ LFDs (M-LFDs) now exist, testing for multiple pathogens from a single swab sample. We evaluated the real-world performance of M-LFD versus PCR testing in detecting influenza A, B and SARS-CoV-2) in the ED setting.MethodsAfter preliminary evaluation of an M-LFD (SureScreen) with laboratory-grown virus and PCR-negative clinical samples, it was evaluated in a real-world setting at the ED of St Thomas’ Hospital (London, UK) from 1 December 2022 to 21 April 2023. Eligible participants were ≥18 years of age, admitted with respiratory symptoms and received concurrent M-LFD and PCR tests. Main endpoints were sensitivity to detect influenza A/B (primary) and SARS-CoV-2 (secondary) versus PCR. The probability of a true positive in relation to viral concentration (expressed as PCR cycle threshold (Ct)) was analysed using logistic regression.ResultsIn total, 808 symptomatic participants were included (49.8% female; mean age 46.9 years). Test sensitivity (95% CI) was 67.0% (56.9% to 76.1%) for influenza A (n=100), 94.1% (71.3% to 99.9%) for influenza B (n=17) and 48.2% (39.7% to 56.8%) for SARS-CoV-2 (n=141). Sensitivity for SARS-CoV-2 was significantly lower than that for influenza A and B (p=0.0057 and p=0.00088, respectively). The probability of a true positive was 98% for Ct<25 for influenza A and SARS-CoV-2 (influenza B non-evaluable). No co-infections were identified by PCR or M-LFD.ConclusionThe real-world performance of SureScreen M-LFD was consistent with laboratory evaluation and achieved a high sensitivity for individuals with high viral concentration, most likely to be infectious. Given the representative UK population sample, results could be generalised for use in other settings.

To detect SARS-CoV-2 amongst asymptomatic care home staff in England, a dual-technology weekly testing regime was introduced on 23 December 2020. A lateral flow device (LFD) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) test were taken on the same day (day 0) and a midweek LFD test was taken three to four days later. We evaluated the effectiveness of using dual-technology to detect SARS-CoV-2 between December 2020 to April 2021. Viral concentrations derived from qRT-PCR were used to determine the probable stage of infection and likely level of infectiousness. Day 0 PCR detected 1,493 cases of COVID-19, of which 53% were in the early stages of infection with little to no risk of transmission. Day 0 LFD detected 83% of cases that were highly likely to be infectious. On average, LFD results were received 46.3 h earlier than PCR, enabling removal of likely infectious staff from the workplace quicker than by weekly PCR alone. Demonstrating the rapidity of LFDs to detect highly infectious cases could be combined with the ability of PCR to detect cases in the very early stages of infection. In practice, asymptomatic care home staff were removed from the workplace earlier, breaking potential chains of transmission.

HP1 proteins bind methylated histone H3 Lys9, a hallmark of heterochromatin, and mediate heterochromatin spreading by recruiting histone methyltransferase activities. New studies have now identified a long noncoding RNA called BORDERLINE that prevents spreading of the HP1 protein Swi6 and histone H3 Lys9 methylation beyond the pericentromeric repeat region of fission yeast chromosome 1. Transcription of eukaryotic genomes is more widespread than was previously anticipated and results in the production of many non–protein-coding RNAs (ncRNAs) whose functional relevance is poorly understood. Here we demonstrate that ncRNAs can counteract the encroachment of heterochromatin into neighboring euchromatin. We have identified a long ncRNA (termed BORDERLINE) that prevents spreading of the HP1 protein Swi6 and histone H3 Lys9 methylation beyond the pericentromeric repeat region of Schizosaccharomyces pombe chromosome 1. BORDERLINE RNAs act in a sequence-independent but locus-dependent manner and are processed by Dicer into short RNAs referred to as brdrRNAs. In contrast to canonical centromeric short interfering RNAs, brdrRNAs are rarely loaded onto Argonaute. Our analyses reveal an unexpected regulatory activity of ncRNAs in demarcating an epigenetically distinct chromosomal domain that could also be operational in other eukaryotes.

BackgroundSARS-CoV-2 lineage B.1.1.7 has been associated with an increased rate of transmission and disease severity among subjects testing positive in the community. Its impact on hospitalised patients is less well documented.MethodsWe collected viral sequences and clinical data of patients admitted with SARS-CoV-2 and hospital-onset COVID-19 infections (HOCIs), sampled 16 November 2020 to 10 January 2021, from eight hospitals participating in the COG-UK-HOCI study. Associations between the variant and the outcomes of all-cause mortality and intensive therapy unit (ITU) admission were evaluated using mixed effects Cox models adjusted by age, sex, comorbidities, care home residence, pregnancy and ethnicity.FindingsSequences were obtained from 2341 inpatients (HOCI cases=786) and analysis of clinical outcomes was carried out in 2147 inpatients with all data available. The HR for mortality of B.1.1.7 compared with other lineages was 1.01 (95% CI 0.79 to 1.28, p=0.94) and for ITU admission was 1.01 (95% CI 0.75 to 1.37, p=0.96). Analysis of sex-specific effects of B.1.1.7 identified increased risk of mortality (HR 1.30, 95% CI 0.95 to 1.78, p=0.096) and ITU admission (HR 1.82, 95% CI 1.15 to 2.90, p=0.011) in females infected with the variant but not males (mortality HR 0.82, 95% CI 0.61 to 1.10, p=0.177; ITU HR 0.74, 95% CI 0.52 to 1.04, p=0.086).InterpretationIn common with smaller studies of patients hospitalised with SARS-CoV-2, we did not find an overall increase in mortality or ITU admission associated with B.1.1.7 compared with other lineages. However, women with B.1.1.7 may be at an increased risk of admission to intensive care and at modestly increased risk of mortality.

RNA structural elements play critical roles in several viral processes. An attempt to elucidate the role of one such RNA structural element encoded by the 5’ Untranslated Region (5’UTR), in regulating virus replication and attenuation of Venezuelan equine encephalitis virus (VEEV) is described. VEEV is one of the pathogenic members of the Alphavirus genus in the Togaviridae family. VEEV infection causes debilitating illness complicated by neurological manifestations. The only available vaccine for VEE infection, the attenuated strain VEEV TC-83 provides minimal protection against virulent strains, but the molecular basis for its attenuation remains poorly understood. Interestingly however, the attenuation of TC-83 was shown to strongly depend on two point mutations, one of which, the G3A mutation, was found in the 5’UTR of the viral genome. Results from my biochemical and biophysical studies demonstrate that the G3A mutation strongly affects the structure of the VEEV 5’UTR. Further functional analysis revealed that this change in 5’UTR RNA structure affects various processes in virus replication. The G3A mutation moderately enhanced translation of the downstream polyprotein, and strongly increased replication of the viral genome, but led to a significant decrease in the synthesis of subgenomic RNA (sgRNA). Based on my findings and those of others, I propose a model for attenuation of the vaccine strain TC-83. The enhanced functionality of the TC-83 5’UTR in viral processes prompted further investigation into the structural requirements within the VEEV 5’UTR for efficient virus replication. Results from these studies revealed that the sequence, secondary structure and stability of the stem-loop in this region are critical for virus replication. Mutations affecting any of the above resulted in pseudorevertants that either acquired compensatory AU or AUG repeat sequences in the 5’UTR, or accumulated mutations in the VEEV non-structural proteins. Results from my mutational analyses thus provide evidence that during the replication of the viral genome, the ends of the dsRNA replication intermediate stay single stranded and fold into individual stem-loops that are critical for virus replication, and the sequence and folding determines the efficiency of the promoter in this region for genomic RNA synthesis.

The underlying hallmark of centromeres is the presence of specialized nucleosomes in which histone H3 is replaced by CENP-A. The events that mediate the installation of CENP-A in place of H3 remain poorly characterized. H2A.Z is linked to transcriptional competence and associates with mammalian centromeres. We find that H2A.ZPht1 and the Swr1 complex are enriched in fission yeast CENP-ACnp1 chromatin. Our analysis shows that Swr1, Msc1 and H2A.ZPht1 are required to maintain CENP-ACnp1 chromatin integrity. Cell cycle analyses demonstrate that H2A.ZPht1 is deposited in S phase, coincident with the deposition of placeholder H3, and prior to CENP-ACnp1 replenishment in G2. Establishment assays reveal that H2A.ZPht1 and Swr1 are required for de novo assembly of CENP-ACnp1 onto naïve centromere DNA. We propose that features akin to promoters within centromere DNA program the incorporation of H2A.ZPht1 via Swr1, and mediate the replacement of resident H3 nucleosomes with CENP-A nucleosomes thereby defining centromeres.

The underlying hallmark of centromeres is the presence of specialized nucleosomes in which histone H3 is replaced by CENP-A. The events that mediate the installation of CENP-A in place of H3 remain poorly characterized. H2A.Z is linked to transcriptional competence and associates with mammalian centromeres. We find that H2A.Z/Pht1 and the Swr1 complex are enriched in fission yeast CENP-A/Cnp1 chromatin. Our analysis shows that H2A.Z/Pht1, Swr1 and Msc1 are required to maintain CENP-A/Cnp1 chromatin integrity. Cell cycle analyses demonstrate that H2A.Z/Pht1 is deposited in S phase, coincident with the deposition of placeholder H3, and prior to CENP-A/Cnp1 replenishment in G2. Establishment assays reveal that H2A.Z/Pht1 and Swr1 are required for de novo assembly of CENP-A/Cnp1 onto naive centromere DNA. We propose that features akin to promoters within centromere DNA program the incorporation of H2A.Z/Pht1 via Swr1, and mediate the replacement of resident H3 nucleosomes with CENP-A nucleosomes thereby defining centromeres.