Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga, and Eastern equine encephalitis viruses: application in human and mosquito public health surveillance in Panama

Abstract

Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by mosquitoes. They cause febrile and sometimes severe neurological diseases in human and equine hosts. Detecting them during the acute phase is hindered by non-specific symptoms and limited diagnostic tools. We designed and clinically assessed real-time reverse transcription polymerase chain reaction assays (rRT-PCRs) for VEEV complex, MADV, and EEEV using whole-genome sequences. Validation involved 15 retrospective serum samples from 2015 to 2017 outbreaks, 150 mosquito pools from 2015, and 118 prospective samples from 2021 to 2022 surveillance in Panama. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from outbreaks, with one having both VEEV complex and MADV RNAs. VEEV complex RNA was found in five suspected dengue cases from disease surveillance. The rRT-PCR assays identified VEEV complex RNA in three Culex (Melanoconion) vomerifer pools, leading to VEEV isolates in two. Phylogenetic analysis revealed the VEEV ID subtype in positive samples. Notably, 11.9% of dengue-like disease patients showed VEEV infections. Together, our rRT-PCR validation in human and mosquito samples suggests that this method can be incorporated into mosquito and human encephalitic alphavirus surveillance programs in endemic regions.

Keywords: Eastern equine encephalitis virus; Madariaga virus; Venezuelan equine encephalitis; alphavirus; rRT-PCR.

Fig 1

VEE amplification curves across a range of concentrations. Amplification curves are shown across a range of concentrations for the VEE complex rRT-PCR with ssDNA (gray curves, subtype IAB) and RNA (pink dotted curves, subtype IC). ssDNA was tested in quadruplicate at 8.0, 6.0, 4.0, 2.0, and 1.0 log₁₀ copies/µL (labeled a–e, respectively). 10-fold dilutions of VEEV subtype IC RNA were tested in duplicate starting at the highest concentration available (5.0 log₁₀ copies/µL).

Fig 2

Map with the distribution of VEEV human cases in Darien province in 2015 and 2017, and health centers in Panama and Darien provinces. (A) Distribution of VEEV cases used for protocol validation. Red dots represent the number of cases reported by locality. (B) Distribution of health centers used for prospective febrile surveillance in Panama and Darien provinces. The map was created with ArcGIS Desktop 10.6 using shapefiles from Esri [World Countries Generalized (2021); https://www.arcgis.com/home/item.html?id=2b93b06dc0dc4e809d3c8db5cb96ba69]. Data sources for the shapefiles include Esri, Garmin International Inc., U.S. Central Intelligence Agency, and National Geographic Society (24).

Fig 3

Flowchart of patient recruitment, characteristics, and RT-PCR results of febrile patients detected throughout disease surveillance. Febrile patients were recruited from 16 November 2021 to 1 December 2022, in 10 health care centers in Panama and Darien provinces.

Fig 4

VEEV complex maximum likelihood phylogenetic tree. Maximum likelihood phylogeny was estimated using 139 complete or near complete VEEV genomes. Publicly available Panamanian VEEV ID subtype strains are highlighted in gray (n = 15), and genomes generated in this study (n = 7) are highlighted in red. Bootstrap statistical support is shown for selected nodes. NCBI GenBank accession numbers for the new VEEV genomes are: OR644785, OR644786, OR644788, OR644801, OR644802, OR644803.