Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface

Abstract

The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya’s Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies.

Composition and Genetic Diversity of Mosquitoes (Diptera: Culicidae) on Islands and Mainland Shores of Kenya’s Lakes Victoria and Baringo

“…Similarly, out of the 54 mosquito species identified in the LV region, 25 have been previously reported at LV in Kenya (Ochieng et al. 2013, Omondi et al. 2015) and Uganda (Kaddumukasa et al.…”

Arbovirus and insect‐specific virus discovery in Kenya by novel six genera multiplex high‐resolution melting analysis

“…; Omondi et al . ). Lambert & Lanciotti () employed triplex PCR for sequencing‐based identification of diverse arboviruses in the family Bunyaviridae ( Orthobunyavirus , Phlebovirus , Nairovirus ).…”

“…Nonetheless, high-throughput multiplex approaches, such as MassTag PCR (Briese et al 2005), TaqMan Arrays (Chao et al 2007;Liu et al 2016) and triplex RT-PCR enzyme hybridization assay (Dong et al 2013), have been used to screen for sets of specific arboviruses. Specific virus sequences can be differentiated among genusspecific PCR amplicons by electrospray ionization mass spectrometry (Eshoo et al 2007) and by their high-resolution melting (HRM) profiles (Naze et al 2009;Omondi et al 2015). Lambert & Lanciotti (2009) employed triplex PCR for sequencing-based identification of diverse arboviruses in the family Bunyaviridae (Orthobunyavirus, Phlebovirus, Nairovirus).…”

Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for forensic and biodiversity research pipelines

“…However, high-resolution melting (HRM) analysis has been used with a number of genes to identify species among diverse viruses 17 , bacteria 18 , malaria Plasmodium 19 , mosquitoes 20 and their bloodmeals 21 , plant products 22 , and animals within discrete families 23 , as well as human individuals 24 . HRM analysis is a fast, sensitive, and specific tool developed for genotyping PCR product sequence variations 25 that employs the use of intercalating fluorescent dyes, such as EvaGreen 26 .…”