Construction and characterization of a ZIKV sub-genomic replicon system for the discovery of antiviral agents.

• Grant number: 18/05130-3
• Support Opportunities: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): June 01, 2018
• Effective date (End): February 28, 2023
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Glaucius Oliva
• Grantee: Rafaela Sachetto Fernandes
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 13/07600-3 - CIBFar - Center for Innovation in Biodiversity and Drug Discovery, AP.CEPID

Abstract

An outbreak of zika fever hit the Americas in 2015 and became a major epidemy in 2016, prompting the World Health Organization (WHO) to declare a state of international emergency. In Brazil, the Zika virus (ZIKV) has been associated with congenital malformations and serious neurological diseases such as microcephaly and Guillain-Barré syndrome. Considering the risk of another ZIKV epidemy and the absence of effective and prophylactic treatment against the virus, the development of rapid and reliable antiviral assays that allow the analysis of large compound libraries is a major challenge for the discovery of anti-ZIKV drugs. The replicon is a self-replicating sub-genomic system in which the genes encoding the virus structural proteins are replaced by a reporter gene, such as a luciferase or a fluorescent protein. The inhibitory effects of the compounds on viral RNA replication can be easily assessed by measuring the reduction of luminescent or fluorescent signals. The objective of the proposed project is the construction and characterization of a ZIKV replicon expressing the Renilla luciferase reporter gene (Rluc). We will use standard molecular biology techniques, such as PCR and cloning, to assemble the replicon expression cassette containing the Rluc gene and insert it into the plasmid pUC57 for propagation in E. coli along with a T7 promoter. Once the plasmid is obtained containing the replicon construct, we will transfect mammalian cells (BHK) with the mRNA transcribed from that strain. Expression of the viral proteins will be assessed by indirect immunofluorescence and the replicon competence will be analyzed by the measurement of luciferase activity. Once standardized, this cell line will be the basis for testing new antiviral agents in our laboratory. This project is linked to the CEPID CIBFar project of FAPESP and also aims to establish a basis for studies of several viruses in our laboratory.

Scientific publications

(4)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

NOSKE, GABRIELA DIAS; GAWRILJUK, VICTOR OLIVEIRA; FERNANDES, RAFAELA SACHETTO; FURTADO, NATHALIA DIAS; BONALDO, MYRNA CRISTINA; OLIVA, GLAUCIUS; GODOY, ANDRE SCHUTZER. Structural characterization and polymorphism analysis of the NS2B-NS3 protease from the 2017 Brazilian circulating strain of Yellow Fever virus. BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, v. 1864, n. 4, APR 2020. (16/19712-9, 18/05130-3, 13/07600-3)

DE GODOY, ANDRE SCHUTZER; FERNANDES, RAFAELA SACHETTO; CAMPOS AGUIAR, ANNA CAROLINE; BUENO, RENATA VIEIRA; DE MORAES ROSO MESQUITA, NATHALYA CRISTINA; CARVALHO GUIDO, RAFAEL VICTORIO; OLIVA, GLAUCIUS. Structural and mechanistic insight from antiviral and antiparasitic enzyme drug targets for tropical infectious diseases. CURRENT OPINION IN STRUCTURAL BIOLOGY, v. 59, p. 65-72, DEC 2019. (16/19712-9, 18/05130-3, 13/07600-3, 15/18192-9)

FERNANDES, RAFAELA S.; FREIRE, MARJORIE C. L. C.; BUENO, V, RENATA; GODOY, ANDRE S.; GIL, LAURA H. V. G.; OLIVA, GLAUCIUS. Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses. Viruses-Basel, v. 12, n. 6, JUN 2020. (16/19712-9, 18/05130-3, 13/07600-3, 18/17095-8)

FERNANDES, RAFAELA SACHETTO; DE GODOY, ANDRE SCHUTZER; SANTOS, IGOR ANDRADE; NOSKE, GABRIELA DIAS; DE OLIVEIRA, KETLLYN IRENE ZAGATO; GAWRILJUK, VICTOR OLIVEIRA; JARDIM, ANA CAROLINA GOMES; OLIVA, GLAUCIUS. Discovery of an imidazonaphthyridine and a riminophenazine as potent anti-Zika virus agents through a replicon-based high-throughput screening. VIRUS RESEARCH, v. 299, JUL 2 2021. (18/05130-3, 16/19712-9, 13/07600-3)