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.

A New Clade of Insect-Specific Flaviviruses from Australian Anopheles Mosquitoes Displays Species-Specific Host Restriction

“…Coevolution of the Anopheles ISFs with their host species provides an explanation for the narrow host restriction of these viruses and is, to some degree, corroborated by our data, as each virus was detected in a single Anopheles species. This does not preclude the possibility of several viruses being found in the same species in other mosquito populations; two different ISF sequences were detected in A. gambiae from Kenya and Senegal, approximately 8,000 km apart (11, 12). …”

“…So far, cISFs have been isolated predominantly from Aedes or Culex mosquitoes and separate into two phylogenetic clusters according to the genus of their host. Only limited sequencing data have been reported for Anopheles -associated ISFs—one virus from Liberia with two full genome sequences ( Anopheles flavivirus [AnFV]) and four partial sequences from Senegal and Kenya ( Anopheles squamosus flavivirus [AnsFV] and Anopheles gambiae flavivirus [AngFV])—and none have been isolated or characterized (11, 12). …”

Tsetse blood-meal sources, endosymbionts, and trypanosome infections provide insight into African trypanosomiasis transmission in the Maasai Mara National Reserve, a wildlife-human-livestock interface

“…We were able to identify blood-meals from wild-caught non-engorged flies. Unlike serological and other PCR-based techniques for blood-meal identification [57,70,71], the use of HRM to detect sequence variants is fast, cost-effective, accurate, easy-to-use, and sensitive, making it a more economical tool for blood-meal analysis [30,72] and pathogen detection/identification [71,73,74]. Sequencing of representative samples with combined human-hippopotamus and human-African buffalo peaks confirmed their mixed blood-meal status (S2 Fig.…”

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

“…Though some studies have only considered one 31 or two 32 different DNA markers, this study demonstrates increased robustness of species identification by PCR-HRM when using combined analysis of three mtDNA markers. While the reliability of sequencing, as widely applied in species identification, is undoubted, the high costs associated with it may not be sustainable in some instances, especially where large sample sizes are analyzed 17 . PCR coupled to HRM offers a quicker, cheaper, and relatively easy-to-work-with real-time PCR alternative 25 .…”