Venezuelan equine encephalitis complex, Madariaga and Eastern equine encephalitis viruses genome detection in human and mosquito populations

Abstract

Background. Venezuelan equine encephalitis (VEE) complex, eastern equine encephalitis virus (EEEV), and Maradiaga virus (MADV), are associated with severe neurological disease in human and equine hosts. We aimed to overcome the lack of available molecular diagnostics for these pathogens by designing and clinically evaluating real-time reverse transcription PCRs (rRT-PCRs) for 1) VEE complex and 2) MADV/EEEV. Methodology/Principal Findings. Primers and probes were designed from alignments of all publicly available complete genome sequences for VEE complex, EEEV and MADV. Linear range for the VEE complex and MADV/EEEV rRT-PCRs extended from 2.0 to 8.0 log10 copies/uL with exclusive detection of the respective viruses. Fifteen serum samples from febrile patients collected from 2015 and 2017 outbreaks in Panama were tested to evaluate the new assays. Ten samples (66.7%) samples were positive for VEEV, and in one sample, rRT-PCR detected both VEEV and MADV. Early acute samples (≤ 3 days since onset) with coincidental anti-VEEV IgM and IgG antibodies were also found positive for VEEV viral RNA. Of 150 mosquito pools, 3 tested positive for VEEV by rRT-PCR. Two of these also yielded VEEV isolates. Conclusions. The VEE complex and MADV/EEEV rRT-PCRs provide accurate detection while yielding significant benefits over available molecular methods. Data from the clinical evaluation provide further evidence for VEE complex and MADV co-infections and suggest that re-infection with VEE complex viruses is possible.