Analytical and clinical evaluation of a duplex RT-qPCR assay for the detection and identification of o’nyong-nyong and chikungunya virus

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The mosquito-borne alphavirus o’nyong-nyong virus (ONNV) has proven its potential to cause major human outbreaks. On the African continent, ONNV causes unspecific febrile illness and co-circulates with the close relative chikungunya virus (CHIKV). The true scale of ONNV burden is poorly understood in Africa, because of the scarce availability of molecular in-house and commercial assays, strong cross-reactivity between ONNV and CHIKV in serological assays and a lack of surveillance. We designed a new RT-qPCR assay targeting the E1 gene for the detection of ONNV that can be used in monoplex or in duplex format, combined with a previously published CHIKV monoplex assay targeting the nsp1. The lower limit of detection with 95% positivity rate was determined to be <10 RNA copies/µL in monoplex and duplex format for both ONNV and CHIKV. Both monoplex assays and the duplex assay proved to be linear within the tested range of approximately 108 to 102 RNA copies/µl, and showed 100% specificity against a wide panel of arbovirus supernatants as well as several other pathogens in clinical samples. Testing of CHIKV positive serum and ONNV spiked plasma samples confirmed the suitability of the assays in a clinical setting. The new assays provide a robust tool for molecular ONNV as well as ONNV/CHIKV simultaneous detection and may contribute to clarify the true burden of the two viruses, to improve arbovirus surveillance and to strengthen epidemics preparedness in Africa.