RT-RPA as a dual tool for detection and phylogenetic analysis of epidemic arthritogenic alphaviruses

Chikungunya (CHIKV), o'nyong-nyong (ONNV), and Mayaro (MAYV) viruses are transmitted by mosquitoes and known to cause a debilitating arthritogenic syndrome. These alphaviruses have emerged and re-emerged, leading to outbreaks in tropical and subtropical regions of Asia, South America, and Africa. Despite their prevalence, there persists a critical gap in the availability of sensitive and virus-specific point-of-care (POC) diagnostics. Traditional immunoglobulin-based tests such as enzyme-linked immunosorbent assay (ELISA) often yield cross-reactive results due to the close genetic relationship between these viruses. Molecular diagnostics such as quantitative polymerase chain reaction (qPCR) offer high sensitivity but are limited by the need for specialized laboratory equipment. Recombinase polymerase amplification (RPA), an isothermal amplification method, is a promising alternative to qPCR, providing rapid results with minimal equipment requirements. Here, we report the development and validation of three virus-specific RT-RPA-based rapid tests for CHIKV, ONNV, and MAYV. These tests demonstrated both speed and sensitivity, capable of detecting 10-100 viral copies within 20 min of amplification, without exhibiting cross-reactivity. Furthermore, we evaluated the clinical potential of these tests using serum and tissue samples from CHIKV, ONNV, and MAYV-infected mice, as well as CHIKV-infected human patients. We demonstrate that the RPA amplicons derived from the patient samples can be sequenced, enabling cost-effective molecular epidemiological studies. Our findings highlight the significance of these rapid and specific diagnostics in improving the early detection and management of these arboviral infections, particularly in resource-limited settings.