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.

IDENTIFICATION OF AN IMPORTANT FACTOR INVOLVED IN CCHFV INFECTION

Cristiano Salata;
2023

Abstract

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.
2023
Abstract Book
3rd International Conference on Crimean - Congo Hemorrhagic Fever
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3495407
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