INVESTIGATION OF CELL SIGNALING PATHWAYS INVOLVED IN INFECTION BY NEUROTROPIC ARBOVIRUSES

• Grant number: 24/03757-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: August 01, 2024
• End date: July 31, 2027
• Field of knowledge: Biological Sciences - Immunology
• Principal Investigator: Rafael Freitas de Oliveira Franca
• Grantee: Bruna Manuella Souza Silva
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Arboviruses represent a serious public health problem which has worsened in recent years due to the insertion of new years due to the introduction of new viruses into highly susceptible populations. The concomitant circulation of different viruses results in a high burden on health systems, which are unprepared to deal with the different clinical forms of these infections. It is known that arbovirus infection generally results in an acute febrile condition, but a significant proportion of infections develop into severe neurological conditions. In this context, the molecular mechanisms involved in controlling the infection versus the evolution of the different clinical forms remain unknown. This proposal aims to evaluate the applicability of a CRISPRCas9 library as a tool for identifying critical factors for infection and cell damage caused by different arboviruses. by different arboviruses. This library consists of a grouping of 77,441 guides directed at the deletion of approximately 19,000 human genes. In this way, we will be able to (screening of the human genome for factors related to the host response to experimental arbovirus infection. experimental arbovirus infection through CRISPR/Cas9-mediated gene deletion in permissive cells. cells. After constructing cell clones constitutively expressing the Cas9 enzyme and transduction of the library by lentivirus, we will subject the knockout cell populations to lethal infection with isolates of the Chikungunya, Mayaro and Oropouche viruses. Finally, we will determine genes and pathways involved in replication and cell damage by sequencing the surviving cells and identifying the identification of the guides present in that population. The genes identified will be ranked and grouped according to the pathway or cellular process in which they participate using gene annotation tools (Gene Ontology). Functional assays for the identified genes will subsequently be carried out, which may include, but are not limited to: i) viral load assessment assays for genes coding for receptors and/or surface proteins ii) cell death assessment assays for genes involved in signaling for apoptosis or other forms of cell death iii) inflammatory cytokine and chemokine profiles for genes involved in activating inflammation. In this way, this proposal may contribute to the identification of molecular pathways involved in arbovirus infection, and may add new knowledge about the processes of virus-host interaction, which in turn may lead to the discovery of potential new therapeutic targets.