Desenvolvimento e validação de construções do vírus Mayaro com genes de luciferase como ferramentas para ensaios antivirais

Abstract

The Mayaro virus (MAYV), an arbovirus from the Alphavirus genus and Togaviridae family, is the etiologic agent of Mayaro fever. Endemic in South and Central Americas, it is maintained in animal reservoirs within a sylvatic cycle. However, the virus exhibits high adaptability to new hosts and vectors, establishing an urban cycle. Mayaro fever presents symptoms similar to other arboviruses, potentially resulting in long-term polyarthritis and polyarthralgia. Currently, there is no licensed vaccine or antiviral treatment against MAYV, which contributes to virus dissemination. In this context, this work aimed to develop viral constructs for use in high-throughput antiviral research. The plasmids were produced by reverse genetics, based on the MAYV BeAr 20290 strain, containing human cytomegalovirus promoter and nanoluciferase (CMV-MAYV-nanoluc) and firefly luciferase (CMV-MAYV-firefly) reporter genes. The luminescence genes were inserted into the viral genome between the end of nsP4 and the 5' terminus of the structural genes. Subsequently, this viral genome segment was combined with a CMV promoter obtained through polymerase chain reaction (PCR). After sequencing, the plasmids were amplified using competent DH5-α bacteria. Subsequently, CMV-MAYV-nanoluc and CMV-MAYV-firefly were transfected into Vero E6 cells, generating infectious MAYV-nanoluc and MAYV-firefly particles, respectively. The viruses replicated efficiently with properties similar to the parental wild-type MAYV, and the expression of the reporter genes reflected viral replication rates. Additionally, using MAYV-nanoluc, we analyzed the repositioning potential of rimantadine hydrochloride (rtdH), licensed drug for the treatment of Influenza, as an anti-MAYV agent. The drug inhibited MAYV with an SI of 2.3 and also showed activity against Zika virus (ZIKV) with an SI of 2.4. Additionally, the compound EIDD-2749, described as an inhibitor of RNA-dependent RNA polymerase with potent antiviral activity against alphavirus, was used to validate the MAYV-nanoluc tool for antiviral trials, with an SI of 1.8 x 107. Our data demonstrated that the MAYV-nanoluc clone is a valuable tool for antiviral assays, enhancing the screening of new anti-MAYV agents. Furthermore, rtdH showed a broad-spectrum antiviral activity against MAYV and ZIKV.