Development of a diagnostic method for the rocio virus and the use of new compounds as inhibitors of the flavivirus NS2B-NS3 protease-helicase

Abstract

Arboviruses (arthropod-borne viruses) are an ecological group of viruses transmitted mainly by hematophagous mosquitoes, involving complex cycles. Consisting of viruses of medical importance, their main representatives include mainly viruses belonging to the genus Flavivirus, Alphavirus and Orthobunyavirus. It is noteworthy that in several tropical and subtropical countries, including Brazil, these viruses have emerged and represent one of the main public health problems because they cause constant epidemics and outbreaks, such as the Dengue virus (DENV), Chikungunya virus (CHIKV), Zika virus (ZIKV) and Yellow Fever virus (YFV) recently. Additionally, constant epidemics increase the number of people seeking medical care and cause an overload in the public health network, resulting in high economic costs for the affected countries. The purpose of the present study is the development and standardization of an indirect peptide-ELISA test for the Rocio flavivirus (ROCV), in addition to the identification of isolated compounds as flavivirus NS2B-NS3 inhibitors; both initially selected through computational studies and molecular dynamics. The silent circulation of the Rocio virus was recently identified in the population, and the development of a cheap, sensitive and specific diagnostic method becomes extremely necessary in view of the scarcity of tests to identify this virus. Partial results of the study point to a unique peptide sequence of the Rocio virus, not present in other flaviviruses, obtained through molecular dynamics protein analysis of viral NS1. Antigenic prediction analyzes for the peptide were performed, showing a high in silico antigenicity score (these partial results were published in an international journal and are awaiting validation, to be carried out at BEPE). Partial results of the present study also demonstrate that the flavonoids isolated from natural products amentoflavone and quercetin are inhibitors of the NS2B-NS3 proteases of the flaviviruses West Nile, Rocio, Ilhéus, Yellow Fever, Saint-Louis Encephalitis and Dengue. The NS2B-NS3 protein complex is a serine protease of viruses of the genus Flavivirus and is considered a key target in the search for new antiviral drugs. Our docking studies propose a way of binding flavonoids to a specific allosteric binding site of the enzyme (the in silico results were also published in an indexed international journal). Furthermore, an initial in vitro study of the antiviral activity of quercetin hydrate recently published by us demonstrated in vitro inhibition against the Zika virus, corroborating our hypothesis. Our initial results help to understand the mechanism of serine protease inhibition of these viruses by flavonoids, essential for the development of improved inhibitors from these isolated molecules. The objective is to seek validation of the results of the present study obtained in silico/in vitro, along with appropriate training in a BSL-3 environment, as well as being instructed in animal models for Rocio virus and Ilhéus virus, mainly. A possible training in Arthropod Containment Level-3 (ACL3) is also foreseen, and the work could include assessment of competent vectors of Rocio (Culex ssp.) and vector transmission studies once the murine model is established. (AU)