Análise in silico de transcrição reversa da reação em cadeia da polimerase em tempo real para detecção de arbovírus e padronização experimental para Orthobunyavirus oropoucheense e Alphavirus mayaro

Abstract

Arboviruses from the families Flaviviridae, Togaviridae, and more recently Peribunyaviridae, are responsible for outbreaks in several regions, representing a threat to public health. The genus Orthoflavivirus comprises important arboviruses such as Dengue virus, Zika virus, Yellow fever virus, West Nile virus, Ilhéus virus, Saint Louis encephalitis virus, and Rocio virus. Within the genus Alphavirus, notable viruses include Sindbis, Chikungunya, Ross River, Mayaro, and the Venezuelan, Eastern, and Western equine encephalitis viruses. Another arbovirus of major epidemiological importance in Brazil is Oropouche virus, which belongs to the genus Orthobunyavirus. Urbanization and globalization have contributed to the increased incidence of arboviral diseases, especially in tropical and subtropical regions. Therefore, effective diagnostic methods are required for the detection and identification of different arboviruses. Reverse Transcription Real-Time Polymerase Chain Reaction (RT-qPCR) is considered the gold standard technique for the diagnosis of viral diseases. Thus, the objective of this study was to analyze and standardize RT-qPCR assays for arbovirus detection through in silico analyses targeting Orthoflavivirus, Alphavirus, and Orthobunyavirus oropoucheense, in addition to performing experimental validation for Oropouche and Mayaro viruses. Oligonucleotides were selected through a literature review. In silico analyses were performed using different software tools, evaluating parameters such as similarity and alignment between viral consensus sequences and each oligonucleotide, primer length, melting temperature, guanine and cytosine content, primer dimer formation, and secondary structures. Based on these analyses, it was possible to establish the most suitable assays for each target virus and to select the best oligonucleotides for detection and identification of the different targets. Experimental validation was carried out using the selected oligonucleotides for Orthobunyavirus oropoucheense and Mayaro viruses in singleplex and multiplex assays. Regarding reaction specificity, no nonspecific amplification was observed, and there was little variation among quantification cycle values, indicating that the multiplex reaction did not compromise assay efficiency. To determine reaction efficiency through standard curves, Orthobunyavirus oropoucheense and Mayaro samples were subjected to serial dilutions. For statistical analysis of the standard curves, slope, intercept, and coefficient of determination were calculated using GraphPad Prism 8 software. The values obtained for the Orthobunyavirus oropoucheense virus assays were within the ideal range according to the literature. However, assays for Mayaro virus detection showed values outside the ideal ranges for slope (both multiplex and singleplex), efficiency (multiplex only), and coefficient of determination (singleplex only). Overall, the results demonstrate that multiplex assays provide results comparable to singleplex assays, in addition to offering advantages such as faster processing and lower cost by allowing the amplification of multiple targets in a single reaction.