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

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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement JPC is funded by the Clarendon Scholarship from University of Oxford and Lincoln-Kingsgate Scholarship from Lincoln College, University of Oxford (grant number SFF1920\_CB2\_MPLS_1293647). This work was supported by SENACYT, through the grants number FID-16-201 and FID-2021-96 grant to JPC; the National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant K08AI110528 to JJW) and the National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant K08AI110528 to JJW) and Centers for Research in Emerging Infectious Diseases (CREID) Coordinating Research on Emerging Arboviral Threats Encompassing the Neotropics (CREATE-NEO) 1U01AI151807 grant awarded to NV/KAH by the National Institutes of Health (NIH); and by the Medical Research Council‐Sao Paulo Research Foundation CADDE partnership award (MR/S0195/1 and FAPESP18/14389‐0 to NRF) (https://caddecentre.org). CAD was supported by the NIHR HPRU in Emerging and Zoonotic Infections, a partnership between PHE, University of Oxford, University of Liverpool and Liverpool School of Tropical Medicine (grant no. NIHR200907). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Gorgas Memorial Institute gave ethical approval for this work Ethics committee/IRB of Emory gave ethical approval for this work Ethics committee/IRB of Universidad Nacional de Asuncion gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript