Assignment of epidemiological lineages in an emerging pandemic using the pangolin tool.

Funder: Oxford Martin School, University of OxfordThe response of the global virus genomics community to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been unprecedented, with significant advances made towards the 'real-time' generation and sharing of SARS-CoV-2 genomic data. The rapid growth in virus genome data production has necessitated the development of new analytical methods that can deal with orders of magnitude of more genomes than previously available. Here, we present and describe Phylogenetic Assignment of Named Global Outbreak Lineages (pangolin), a computational tool that has been developed to assign the most likely lineage to a given SARS-CoV-2 genome sequence according to the Pango dynamic lineage nomenclature scheme. To date, nearly two million virus genomes have been submitted to the web-application implementation of pangolin, which has facilitated the SARS-CoV-2 genomic epidemiology and provided researchers with access to actionable information about the pandemic's transmission lineages

Isolation of a natural DNA virus of <i>Drosophila melanogaster</i>, and characterisation of host resistance and immune responses

<div><p><i>Drosophila melanogaster</i> has played a key role in our understanding of invertebrate immunity. However, both functional and evolutionary studies of host-virus interaction in <i>Drosophila</i> have been limited by a dearth of native virus isolates. In particular, despite a long history of virus research, DNA viruses of <i>D</i>. <i>melanogaster</i> have only recently been described, and none have been available for experimental study. Here we report the isolation and comprehensive characterisation of Kallithea virus, a large double-stranded DNA virus, and the first DNA virus to have been reported from wild populations of <i>D</i>. <i>melanogaster</i>. We find that Kallithea virus infection is costly for adult flies, reaching high titres in both sexes and disproportionately reducing survival in males, and movement and late fecundity in females. Using the <i>Drosophila</i> Genetic Reference Panel, we quantify host genetic variance for virus-induced mortality and viral titre and identify candidate host genes that may underlie this variation, including <i>Cdc42-interacting protein 4</i>. Using full transcriptome sequencing of infected males and females, we examine the transcriptional response of flies to Kallithea virus infection and describe differential regulation of virus-responsive genes. This work establishes Kallithea virus as a new tractable model to study the natural interaction between <i>D</i>. <i>melanogaster</i> and DNA viruses, and we hope it will serve as a basis for future studies of immune responses to DNA viruses in insects.</p></div

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

Trait means, genetic variance (V_G), total phenotypic variance (V_P), heritability (H²), and coefficient of genetic variation (CV_G) in titre and mortality following KV infection in the DGRP.

<p>Trait means, genetic variance (V_G), total phenotypic variance (V_P), heritability (H²), and coefficient of genetic variation (CV_G) in titre and mortality following KV infection in the DGRP.</p

Genetic variation in resistance to KV.

<p>(A) We measured LT50 in both sexes, and titre in females, following KV injection in the DGRP. For titre, each bar represents the mean (and standard error) titre relative to fly genome copy-number, as assessed by qPCR for 5 vials of 10 flies for each of 125 DGRP lines. For LT50, each bar represents the mean time until half the flies (in a vial of 10) were dead, for three vials per line, per sex. (B, C) We used a multi-response linear mixed model to calculate genetic correlation between the traits. Shown are the raw data (left), and the estimated line effects (right) after accounting for any injection date and qPCR plate effects, and for the estimated variance among lines. Each point is a DGRP line measured for both phenotypes. We find a strong positive correlation between male and female LT50 values (B). We also observe a weak positive correlation between titre and LT50 (C).</p

KV induces differential regulation of chorion, virus defense, and serine endopeptidase genes.

<p>(A) Volcano plots showing fold changes and p-values from Wald tests for differential expression of <i>D</i>. <i>melanogaster</i> genes following KV infection for females (left), males (center), those different between the sexes (right), with DAV read count fit as a covariate and nominal significance threshold of p < 0.001. In each panel, the genes with the smallest p-values are labelled. All of the genes that were significantly differentially regulated between the sexes are highly significant in females. (B) Highly induced genes are mostly functionally unannotated, but include some with known roles in viral pathogenesis. (C) The male response is correlated with females, but muted, with few genes identified as significantly differentially expressed in males. Genes with weak evidence of differential expression in either sex (p < 0.05) are plotted, where the dotted line represents a perfect correlation, and red points are genes identified as significantly differentially expressed. (D) The top GO enrichment terms for each GO class (Molecular Function, Biological Process, Cellular Component) were genes involved the chorion, virus defense, and serine peptidase activity. For each plot, estimated fold changes and their associated standard errors are plotted for every gene matching the GO term, regardless of the significance of the Wald test. Generally, chorion genes were downregulated, virus defense genes were upregulated, and serine peptidases were downregulated.</p

KV causes male-biased mortality, increased lethargy, and decreased fecundity.

<p>(A) Injection of KV virus into <i>OreR</i> flies led to sex-specific mortality. Infected females (red dotted line) experienced a small but significant increase in mortality, but males (blue dotted line) experienced a significantly larger rate of mortality after day 10. Flies injected with control gradient solution were unaffected (solid lines). Each point is the mean and standard error for the proportion of flies alive in each vial (10 vials of 10 flies). (B) Although females remained alive for longer, they were more lethargic. We assessed daily movement of flies injected with either chloroform-inactivated KV (green) or active KV (purple). KV-infected flies moved less from days 3–7 post-infection. (C) Females also displayed altered egg laying behaviour. Thirty pairs of flies were injected with inactive chloroform treated KV (green) or active KV (purple). KV-infected flies laid a slightly, but not significantly, higher number of eggs during early infection (1 and 2 DPI) but laid significantly fewer eggs in late infection (7 and 8 DPI). This reduction in egg laying is due to a shutdown of oogenesis before vitellogenesis (D, E), and ovaries from KV-infected flies house a lower proportion of ovarioles that include late-stage and mature egg chambers (F) and a higher proportion which contain apoptotic nurse cells (G). Ovaries were analysed 10 DPI, and error bars (F,G) show the standard error.</p

Confirmation of antiviral genes identified in GWAS.

<p>KV titre was measured in flies expressing a foldback hairpin targeting 18 genes identified in the GWAS, using GAL4 lines that knock each down in either the whole fly or specifically in the gut. (A) The data were used to estimate random effects associated with each gene knock down, plotted with 95% highest posterior density intervals. (B) Knock-down of the most confident association in the GWAS, <i>Cip4</i>, caused reduced <i>Cip4</i> RNA levels and (C) increased viral titre. (D) The associated variant (3L_4363810_SNP), was polymorphic (G/A), representing a nonsynonymous polymorphism in some splice variants, and survival following KV infection was significantly increased in fly lines with the “A” genotype, especially in females. Each point in comparison of survival in the two genotypes is a line mean. (*MCMCp < 0.05).</p

Genome-wide association of polymorphism in the DGRP with KV-induced titre and mortality.

<p>Manhattan plots showing the p-value for the effect of each polymorphism on viral titre (purple) and mortality (green). The top SNPs for each phenotype are shown in expanded inset panels, including surrounding genes. For clarity “CG” is omitted from gene identifiers. Horizontal lines show significance thresholds obtained through randomisation (p_rand = 0.05 in blue; p_rand = 0.01 in red).</p