IDENTIFICATION OF AN IMPORTANT FACTOR INVOLVED IN CCHFV INFECTION

Despite intensive research, much of the molecular pathogenesis of CCHFV is still unknown. Genome-wide screening methods (particularly CRISPR/Cas9-based screens and insertional mutagenesis in haploid cell systems) have facilitated and accelerated the identification and characterization of host genes involved in infectious diseases. Combining haploid cells with genome saturating chemical mutagenesis using N-Ethyl-N-nitrosourea, we have developed an unbiased screening system that interrogates single nucleotide variants for their relevance in viral infections. To identify host factors involved in CCHFV infections, we performed resistant screens with a viral RNA replication competent vesicular stomatitis virus, pseudotyped with the glycoproteins of the CCHFV (VSV-CCHF_G). Resistant clones were individually selected, expanded and rescreened using the infectious CCHFV IbAr10200 laboratory strain. Subsequently, whole exome sequencing was conducted on the resistant clones. Three clones showing nearly 100% resistance to CCHFV displayed mutations in the gene encoding for protein we named X. Through the use of knocked out haploid and diploid cells as well as soluble form of this protein on VSV-CCHF_G, CCHFV IbAr 10200 and CCHFV isolate, we showed that this protein is important for CCHFV infection. These data were then confirmed in vivo, in a mice model. By using an unbiaised screening system, our study identified an important factor involved for CCHFV cell entry and infection

Crimean-Congo haemorrhagic fever virus uses LDLR to bind and enter host cells

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV. Laboratory and clinical strains of Crimean-Congo haemorrhagic fever virus use LDLR to bind and enter host cells in blood vessel organoids and mice. Infection can also occur through ApoE, possibly present on virus particles

A Highly Efficient TiO2-XCx Nano-Heterojunction Photocatalyst for Visible-Light Induced Antibacterial Applications

Visible-light-induced antibacterial activity of carbon-doped anatase-brookite titania nano-heterojunction photocatalysts are reported for the first time. These heterostructures were prepared using a novel low temperature (100 °C) non-hydrothermal low power microwave (300 W) assisted method. Formation of interband C 2p states was found to be responsible for the band gap narrowing of the carbon doped heterojunctions. The most active photocatalyst obtained after 60 minutes of microwave irradiation exhibits a 2-fold higher visible-light induced photocatalytic activity in contrast to the standard commercial photocatalyst Evonik-Degussa P-25. Staphylococcus aureus inactivation rate constant for carbon-doped nano-heterojunctions and the standard photocatalyst was 0.0023 and -0.0081 min-1 respectively. It is proposed that the photo-excited electrons (from the C 2p level) are effectively transferred from the conduction band of brookite to that of anatase causing efficient electron-hole separation, which is found to be responsible for the superior visible-light induced photocatalytic and antibacterial activities of carbon-doped anatase-brookite nano-heterojunctions. KEYWORDS: Titanium dioxide; Microwave synthesis; Carbon-doping; Visible-light induced photocatalysis; Antibacterial activity; Heterojunction

CRISPR-UMI : single-cell lineage tracing of pooled CRISPR-Cas9 screens

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens