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

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.