Single base mutations in the nucleocapsid gene of SARS-CoV-2 affects amplification efficiency of sequence variants and may lead to assay failure

Reverse transcriptase quantitative PCR (RT-qPCR) is the main diagnostic assay used to detect SARS-CoV-2 RNA in respiratory samples. RT-qPCR is performed by specifically targeting the viral genome using complementary oligonucleotides called primers and probes. This approach relies on prior knowledge of the genetic sequence of the target. Viral genetic variants with changes to the primer/probe binding region may reduce the performance of PCR assays and have the potential to cause assay failure. In this work we demonstrate how two single nucleotide variants (SNVs) altered the amplification curve of a diagnostic PCR targeting the Nucleocapsid (N) gene and illustrate how threshold setting can lead to false-negative results even where the variant sequence is amplified. We also describe how in silico analysis of SARS-CoV-2 genome sequences available in the COVID-19 Genomics UK Consortium (COG-UK) and GISAID databases was performed to predict the impact of sequence variation on the performance of 22 published PCR assays. The vast majority of published primer and probe sequences contain sequence mismatches with at least one SARS-CoV-2 lineage. We recommend that visual observation of amplification curves is included as part of laboratory quality procedures, even in high throughput settings where thresholds are set automatically and that in silico analysis is used to monitor the potential impact of new variants on established assays. Ideally comprehensive in silico analysis should be applied to guide selection of highly conserved genomic regions to target with future SARS-CoV-2 PCR assays

Untargeted metagenomics protocol for the diagnosis of infection from CSF and tissue from sterile sites

Metagenomic next-generation sequencing (mNGS) is an untargeted technique capable of detecting all microbial nucleic acid within a sample. This protocol outlines our wet laboratory method for mNGS of cerebrospinal fluid (CSF) specimens and tissues from sterile sites. We use this method routinely in our clinical service, processing 178 specimens over the past 2.5 years in a laboratory that adheres to ISO:15189 standards. We have successfully used this protocol to diagnose multiple cases of encephalitis and hepatitis

Islands of linkage in an ocean of pervasive recombination reveals two-speed evolution of human cytomegalovirus genomes

Human cytomegalovirus (HCMV) infects most of the population worldwide, persisting throughout the host's life in a latent state with periodic episodes of reactivation. While typically asymptomatic, HCMV can cause fatal disease among congenitally infected infants and immunocompromised patients. These clinical issues are compounded by the emergence of antiviral resistance and the absence of an effective vaccine, the development of which is likely complicated by the numerous immune evasins encoded by HCMV to counter the host's adaptive immune responses, a feature that facilitates frequent super-infections. Understanding the evolutionary dynamics of HCMV is essential for the development of effective new drugs and vaccines. By comparing viral genomes from uncultivated or low-passaged clinical samples of diverse origins, we observe evidence of frequent homologous recombination events, both recent and ancient, and no structure of HCMV genetic diversity at the whole-genome scale. Analysis of individual gene-scale loci reveals a striking dichotomy: while most of the genome is highly conserved, recombines essentially freely and has evolved under purifying selection, 21 genes display extreme diversity, structured into distinct genotypes that do not recombine with each other. Most of these hyper-variable genes encode glycoproteins involved in cell entry or escape of host immunity. Evidence that half of them have diverged through episodes of intense positive selection suggests that rapid evolution of hyper-variable loci is likely driven by interactions with host immunity. It appears that this process is enabled by recombination unlinking hyper-variable loci from strongly constrained neighboring sites. It is conceivable that viral mechanisms facilitating super-infection have evolved to promote recombination between diverged genotypes, allowing the virus to continuously diversify at key loci to escape immune detection, while maintaining a genome optimally adapted to its asymptomatic infectious lifecycle

The Panchromatic Hubble Andromeda Treasury II. Tracing the Inner M31 Halo with Blue Horizontal Branch Stars

We attempt to constrain the shape of M31's inner stellar halo by tracing the surface density of blue horizontal branch (BHB) stars at galactocentric distances ranging from 2 kpc to 35 kpc. Our measurements make use of resolved stellar photometry from a section of the Panchromatic Hubble Andromeda Treasury (PHAT) survey, supplemented by several archival Hubble Space Telescope observations. We find that the ratio of BHB to red giant stars is relatively constant outside of 10 kpc, suggesting that the BHB is as reliable a tracer of the halo population as the red giant branch. In the inner halo, we do not expect BHB stars to be produced by the high metallicity bulge and disk, making BHB stars a good candidate to be a reliable tracer of the stellar halo to much smaller galactocentric distances. If we assume a power-law profile r^(-\alpha) for the 2-D projected surface density BHB distribution, we obtain a high-quality fit with a 2-D power-law index of \alpha=2.6^{+0.3}_{-0.2} outside of 3 kpc, which flattens to \alpha<1.2 inside of 3 kpc. This slope is consistent with previous measurements but is anchored to a radial baseline that extends much farther inward. Finally, assuming azimuthal symmetry and a constant mass-to-light ratio, the best-fitting profile yields a total halo stellar mass of 2.1^{+1.7}_{-0.4} x 10^9 M_sun. These properties are comparable with both simulations of stellar halo formation formed by satellite disruption alone, and with simulations that include some in situ formation of halo stars.Comment: 15 pages, 1 table, 5 figures, accepted for publication in Ap

Evaluating 'Prefer not to say' Around Sensitive Disclosures

As people's offline and online lives become increasingly entwined, the sensitivity of personal information disclosed online is increasing. Disclosures often occur through structured disclosure fields (e.g., drop-down lists). Prior research suggests these fields may limit privacy, with non-disclosing users being presumed to be hiding undesirable information. We investigated this around HIV status disclosure in online dating apps used by men who have sex with men. Our online study asked participants (N=183) to rate profiles where HIV status was either disclosed or undisclosed. We tested three designs for displaying undisclosed fields. Visibility of undisclosed fields had a significant effect on the way profiles were rated, and other profile information (e.g., ethnicity) could affect inferences that develop around undisclosed information. Our research highlights complexities around designing for non-disclosure and questions the voluntary nature of these fields. Further work is outlined to ensure disclosure control is appropriately implemented around online sensitive information disclosures

Signal Appropriation of Explicit HIV Status Disclosure Fields in Sex-Social Apps used by Gay and Bisexual Men

HIV status disclosure fields in online sex-social applications ("apps") are designed to help increase awareness, reduce stigma, and promote sexual health. Public disclosure could also help those diagnosed relate to others with similar statuses to feel less isolated. However, in our interview study (n=28) with HIV positive and negative men who have sex with men (MSM), we found some users preferred to keep their status private, especially when disclosure could stigmatise and disadvantage them, or risk revealing their status to someone they knew offline in a different context. How do users manage these tensions between health, stigma, and privacy? We analysed our interview data using signalling theory as a conceptual framework and identify participants developing 'signal appropriation' strategies, helping them manage the disclosure of their HIV status. Additionally, we propose a set of design considerations that explore the use of signals in the design of sensitive disclosure fields

High Viral Diversity and Mixed Infections in Cerebral Spinal Fluid From Cases of Varicella Zoster Virus Encephalitis.

BACKGROUND: Varicella zoster virus (VZV) may cause encephalitis, both with and without rash. Here we investigate whether viruses recovered from the central nervous system (CNS; encephalitis or meningitis) differ genetically from those recovered from non-CNS samples. METHODS: Enrichment-based deep sequencing of 45 VZV genomes from cerebral spinal fluid (CSF), plasma, bronchoalveolar lavage (BAL), and vesicles was carried out with samples collected from 34 patients with and without VZV infection of the CNS. RESULTS: Viral sequences from multiple sites in the same patient were identical at the consensus level. Virus from vesicle fluid and CSF in cases of meningitis showed low-level diversity. By contrast, plasma, BAL, and encephalitis had higher numbers of variant alleles. Two CSF-encephalitis samples had high genetic diversity, with variant frequency patterns typical of mixed infections with different clades. CONCLUSIONS: Low viral genetic diversity in vesicle fluid is compatible with previous observations that VZV skin lesions arise from single or low numbers of virions. A similar result was observed in VZV from cases of VZV meningitis, a generally self-limiting infection. CSF from cases of encephalitis had higher diversity with evidence for mixed clade infections in 2 cases. We hypothesize that reactivation from multiple neurons may contribute to the pathogenesis of VZV encephalitis.Action Medical research GN2424 This work was supported by a UK MRC New Investigator Award to D. P. D; UCL/UCLH BRC (J. B.); Action Medical Research (grant number GN2424 to C. J. H); Swedish Research Council (P. N. and T. B.). The work was also support by an NIHR Fellowship (grant number DRF-2013-06-168 to F. M.), the Meningitis Research Foundation (grant number 0904.0), an NIHR Programme Grant in Applied Research (grant number RP-PG-0108-10048 to T. S.), and the NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool

Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses

: Background : Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. We investigated whether we could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore Technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing of known pathogens and could improve sensitivity. Methods: We evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers. Results: Capture with the Twist CVRP increased sensitivity by at least 10–100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600–6000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT. Conclusions: Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context

Disequilibrium Carbon, Oxygen, and Nitrogen Chemistry in the Atmospheres of HD 189733b and HD 209458b

We have developed 1-D photochemical and thermochemical kinetics and diffusion models for the transiting exoplanets HD 189733b and HD 209458b to study the effects of disequilibrium chemistry on the atmospheric composition of "hot Jupiters." Here we investigate the coupled chemistry of neutral carbon, hydrogen, oxygen, and nitrogen species, and we compare the model results with existing transit and eclipse observations. We find that the vertical profiles of molecular constituents are significantly affected by transport-induced quenching and photochemistry, particularly on cooler HD 189733b; however, the warmer stratospheric temperatures on HD 209458b can help maintain thermochemical equilibrium and reduce the effects of disequilibrium chemistry. For both planets, the methane and ammonia mole fractions are found to be enhanced over their equilibrium values at pressures of a few bar to less than a mbar due to transport-induced quenching, but CH4 and NH3 are photochemically removed at higher altitudes. Atomic species, unsaturated hydrocarbons (particularly C2H2), some nitriles (particularly HCN), and radicals like OH, CH3, and NH2 are enhanced overequilibrium predictions because of quenching and photochemistry. In contrast, CO, H2O, N2, and CO2 more closely follow their equilibrium profiles, except at pressures < 1 microbar, where CO, H2O, and N2 are photochemically destroyed and CO2 is produced before its eventual high-altitude destruction. The enhanced abundances of HCN, CH4, and NH3 in particular are expected to affect the spectral signatures and thermal profiles HD 189733b and other, relatively cool, close-in transiting exoplanets. We examine the sensitivity of our results to the assumed temperature structure and eddy diffusion coefficientss and discuss further observational consequences of these models.Comment: 40 pages, 16 figures, accepted for publication in Astrophysical Journa

A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin.L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV.VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts