title: Arbovirus and insect-specific virus discovery in Kenya by novel six genera multiplex high-resolution melting analysis.
abstract: A broad diversity of arthropod-borne viruses (arboviruses) of global health concern are endemic to East Africa, yet most surveillance efforts are limited to just a few key viral pathogens. Additionally, estimates of arbovirus diversity in the tropics are likely to be underestimated as their discovery has lagged significantly over past decades due to limitations in fast and sensitive arbovirus identification methods. Here, we developed a nearly pan-arbovirus detection assay that uses high-resolution melting (HRM) analysis of RT-PCR products from highly multiplexed assays to differentiate broad diversities of arboviruses. We differentiated 15 viral culture controls and seven additional synthetic viral DNA sequence controls, within Flavivirus, Alphavirus, Nairovirus, Phlebovirus, Orthobunyavirus and Thogotovirus genera. Among Bunyamwera, sindbis, dengue and Thogoto virus serial dilutions, detection by multiplex RT-PCR-HRM was comparable to the gold standard Vero cell plaque assays. We applied our low-cost method for enhanced broad-range pathogen surveillance from mosquito samples collected in Kenya and identified diverse insect-specific viruses, including a new clade in anopheline mosquitoes, and Wesselsbron virus, an arbovirus that can cause viral haemorrhagic fever in humans and has not previously been isolated in Kenya, in Culex spp. and Anopheles coustani mosquitoes. Our findings demonstrate how multiplex RT-PCR-HRM can identify novel viral diversities and potential disease threats that may not be included in pathogen detection panels of routine surveillance efforts. This approach can be adapted to other pathogens to enhance disease surveillance and pathogen discovery efforts, as well as the study of pathogen diversity and viral evolutionary ecology.
authors.affiliation: International Centre of Insect Physiology and Ecology (icipe), P.O. Box 30772, Nairobi, 00100, Kenya.
authors.forename: Jandouwe
authors.identifier: http://orcid.org/0000-0002-5097-6605
authors.initials: J
authors.lastname: Villinger
authors.affiliation: International Centre of Insect Physiology and Ecology (icipe), P.O. Box 30772, Nairobi, 00100, Kenya.|Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya.
authors.forename: Martin K
authors.initials: MK
authors.lastname: Mbaya
authors.affiliation: International Centre of Insect Physiology and Ecology (icipe), P.O. Box 30772, Nairobi, 00100, Kenya.|Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ouso
authors.affiliation: International Centre of Insect Physiology and Ecology (icipe), P.O. Box 30772, Nairobi, 00100, Kenya.|Zoological Institute, Katholieke Universiteit, Naamsestraat 59, P.O. Box 3000, Leuven, Belgium.
authors.forename: Purity N
authors.initials: PN
authors.lastname: Kipanga
authors.affiliation: Kenya Medical Research Institute (KEMRI), Nairobi, Kenya.
authors.forename: Joel
authors.initials: J
authors.lastname: Lutomiah
authors.affiliation: International Centre of Insect Physiology and Ecology (icipe), P.O. Box 30772, Nairobi, 00100, Kenya.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Masiga
mesh_terms: D000351:Africa, Eastern; D000818:Animals; D001103:Arboviruses / Q000302:isolation & purification*; D009033:Culicidae / Q000821:virology*; D005416:Flavivirus / Q000302:isolation & purification*; D006801:Humans; D007304:Insect Viruses / Q000302:isolation & purification*; D007630:Kenya; D020133:Reverse Transcriptase Polymerase Chain Reaction
keywords: arboviruses; emerging communicable diseases; high-resolution melting analysis; insect-specific flaviviruses; multiplex polymerase chain reaction; pathogen discovery