Aim of the study: The Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a viral zoonosis that progresses without symptoms in animals, but gives rise to a lethal threat for humans. The CCHFV is a group IV biological hazard and, for this reason, it requires a BSL-4 containment facility to be manipulated, which is a great dampening for conducting any research on it. Anyway, there are different approaches that permit to bypass the BSL-4 restrictions, like using a nonpathogenic pseudotyped virus, where the envelope protein of the heterologous virus (i.e. glycoprotein Gc and Gn of CCHFV) are assembled into the membrane of the nonpathogenic virus. Since it presents only the glycoproteins of CCHFV, it acquires the immunogenicity and the tropism of CCHFV but can be manipulated in a BSL-2 containment facility. Our work focuses on the development of a BSL-2 executable tool for serological studies and the identification of entry inhibitors of the CCHFV. Methods: As viral model we selected a recombinant Vesicular Stomatitis Virus (rVSV- ΔG-GFP), in which the glycoprotein gene was replaced with a reporter gene, i.e. in our case this gene was the GFP. The rVSV-ΔG-GFP is characterize by an high profile of biosafety because it is single cycle replication virus. Results and conclusion: First, we have optimized a protocol for the preparation of the rVSV-ΔG-GFP pseudotyped with the CCHFV’s glycoprotein (rVSV-CCHFVG). Human cells HEK293T previously transfected with pC-G, a pCAGGS/MCS plasmid containing the CCHFV-M segment’s cDNA, were infected with the rVSV-ΔG-GFP containing the native G glycoprotein. The pseudotyped viral particles released in the cell culture medium are collected and ultracentrifugated in a sucrose cushion to concentrate and clean the viral stock. Then, the viral stock is titrated by serial dilution and evaluation of the GFP positive cells. Second, the rVSV-CCHFVG has been used in a serum neutralization assay using a panel of sera (collected by people from a non-endemic area) and the serum of a Bulgarian laboratory worker vaccinated for the CCHF, as positive control. The results show that the rVSV-CCHFVG is correctly neutralized only by the serum of the vaccinated worker and the neutralization titer correlate with data published evaluating the efficacy of the Bulgarian vaccine against the CCHFV. Overall, the results support the feasibility of the rVSV-CCHFVG based assay for serological studies.

DEVELOPMENT OF A BSL-2 TOOL BASED ON THE PSEUDOTYPED VESICULAR STOMATITIS VIRUS TO STUDY CCHFV SERO-EPIDEMIOLOGY AND FOR ANTIVIRAL DISCOVERY

SALVATI, MARIA VITTORIA;PAROLIN, MARIA CRISTINA;CALISTRI, ARIANNA;PALU', GIORGIO;SALATA, CRISTIANO
2017

Abstract

Aim of the study: The Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a viral zoonosis that progresses without symptoms in animals, but gives rise to a lethal threat for humans. The CCHFV is a group IV biological hazard and, for this reason, it requires a BSL-4 containment facility to be manipulated, which is a great dampening for conducting any research on it. Anyway, there are different approaches that permit to bypass the BSL-4 restrictions, like using a nonpathogenic pseudotyped virus, where the envelope protein of the heterologous virus (i.e. glycoprotein Gc and Gn of CCHFV) are assembled into the membrane of the nonpathogenic virus. Since it presents only the glycoproteins of CCHFV, it acquires the immunogenicity and the tropism of CCHFV but can be manipulated in a BSL-2 containment facility. Our work focuses on the development of a BSL-2 executable tool for serological studies and the identification of entry inhibitors of the CCHFV. Methods: As viral model we selected a recombinant Vesicular Stomatitis Virus (rVSV- ΔG-GFP), in which the glycoprotein gene was replaced with a reporter gene, i.e. in our case this gene was the GFP. The rVSV-ΔG-GFP is characterize by an high profile of biosafety because it is single cycle replication virus. Results and conclusion: First, we have optimized a protocol for the preparation of the rVSV-ΔG-GFP pseudotyped with the CCHFV’s glycoprotein (rVSV-CCHFVG). Human cells HEK293T previously transfected with pC-G, a pCAGGS/MCS plasmid containing the CCHFV-M segment’s cDNA, were infected with the rVSV-ΔG-GFP containing the native G glycoprotein. The pseudotyped viral particles released in the cell culture medium are collected and ultracentrifugated in a sucrose cushion to concentrate and clean the viral stock. Then, the viral stock is titrated by serial dilution and evaluation of the GFP positive cells. Second, the rVSV-CCHFVG has been used in a serum neutralization assay using a panel of sera (collected by people from a non-endemic area) and the serum of a Bulgarian laboratory worker vaccinated for the CCHF, as positive control. The results show that the rVSV-CCHFVG is correctly neutralized only by the serum of the vaccinated worker and the neutralization titer correlate with data published evaluating the efficacy of the Bulgarian vaccine against the CCHFV. Overall, the results support the feasibility of the rVSV-CCHFVG based assay for serological studies.
2017
Atti del congresso
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3240129
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