Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga and Eastern equine encephalitis viruses: application in clinical diagnostic and mosquito surveillance

Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV) and Venezuelan equine encephalitis virus complex (VEEV) are New World mosquito-borne alphaviruses and cause severe neurological disease in human and equine hosts. However, their detection during the acute phase is complicated by non-specific clinical manifestations and lack of available diagnostic tools. To develop and clinically evaluate rRT-PCRs for VEEV complex, MADV and EEEV, primers and probes were designed from publicly available whole-genome sequences. The rRT-PCRs were validated using 15 retrospective serum samples from febrile patients collected during the 2015 and 2017 alphavirus outbreaks in Panama. In addition, the protocol was validated with 150 mosquito pools from 2015, and with 118 samples from prospective disease surveillance from 2021 and 2022. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from the 2015 and 2017 outbreaks, and in one of these ten samples, both VEEV complex and MADV RNAs were detected. Additionally, VEEV complex RNA was detected in 5 suspected dengue from prospective disease surveillance. The rRT-PCR assays detected VEEV complex RNA in 3 from Culex (Melanoconion) vomerifer pools, 2 of which yielded VEEV isolates. Untargeted sequencing and phylogenetic analysis identified VEEV ID subtype in seven VEEV complex RNA positive sample. The VEEV complex, MADV and EEEV rRT-PCRs provide accurate detection while yielding significant benefits over currently available molecular methods. Our results suggest that 11.9% of suspected dengue cases in Panama are VEEV infections.