Title : Development of a multiplex quantitative real-time PCR for the detection of Middelburg virus, Sindbis virus, and Ndumu virus

Abstract:

Background: Sindbis virus (SINV), Middelburg virus (MIDV), and Ndumu virus (NDUV) are mosquito-borne viruses belonging to the Alphavirus genus, family Togaviridae, and cause animal and human disease outbreaks in southern Africa after heavy rains. SINV and MIDV are associated with neurological disease in horses, livestock, and humans. In contrast, NDUV, although not yet linked to clinical disease, has zoonotic potential due to its close classification with SINV and MIDV. Despite detection in both human and animal cases, these viruses remain poorly characterised, and routine diagnostic tools are limited.

Aim: This study focused on developing and optimising a multiplex quantitative real-time PCR assay for the simultaneous detection of MIDV, SINV, and NDUV.

Methods: Virus-specific probes were designed and used with previously published primers for the detection of MIDV, SINV, and NDUV. These components were initially optimised, and their efficiency, limit of detection, and repeatability were determined in singleplex assays, after which the assays were combined and further optimised to develop a multiplex assay.

Results: All three singleplex assays demonstrated high amplification efficiencies (90.2–92.4%), strong linearity (R² > 0.95), and good repeatability across a range of target concentrations. The 95% limit of detection (LOD), determined by probit analysis, ranged from 4.83 to 5.92 copies/µL. The multiplex assay similarly achieved detection at low target concentrations, with LOD values ranging from 4.17 to 5.19 copies/µL.

Conclusions: The multiplex assay developed in this study represents a sensitive and reliable molecular tool for the detection of SINV, MIDV, and NDUV. Sequence mismatches between the MIDV, SINV, and NDUV probes and other alphaviruses are expected to minimise cross-reactivity. Future work will focus on validation using field-collected mosquito samples and expanding downstream applications to include genomic confirmation.

Keywords: Alphaviruses, Multiplex real-time PCR, Mosquito-borne viruses, Sindbis virus, Middelburg virus

Biography:

Elise Bonnet is a life scientist with a strong interest in molecular virology and the development of diagnostic tools for infectious diseases. Her work focuses on improving the detection and surveillance of mosquito-borne viruses, particularly alphaviruses of veterinary and public health importance in southern Africa. She is passionate about translating laboratory research into practical applications, especially in resource-limited and field settings. Elise values collaboration and interdisciplinary research and enjoys engaging with others who share an interest in infectious disease dynamics and molecular diagnostics.