Location: Foreign Arthropod Borne Animal Disease Research
Project Number: 3022-32000-024-010-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2022
End Date: Aug 31, 2025
Objective:
Mosquitoes transmit multiple zoonotic arboviruses including Rift Valley fever virus, yellow fever virus, dengue fever virus, Chikungunya virus among others. Many of these viruses are endemic in Kenya and are often associated with fatal haemorrhagic fever syndromes in humans. An outbreak of yellow fever was reported in Isiolo in February – March 2022 following intense rainfall that occurred earlier in the year. Multiple outbreaks of Rift VaIley fever (RVF) were also reported in Isiolo between December 2020 and January 2021. All these outbreaks affected low lying, flood-prone areas along Ewaso Nyiro river basin.
Approach:
Mosquito trapping: In each study area, mosquitoes will be collected for 3 consecutive days from 20 selected sites. Collection methods to be used are: (i) CDC light traps, (ii) CDC gravid traps, and (iii) BG sentinel traps. We intend to collect as many mosquitoes as possible because arbovirus transmission is usually maintained at a low level in a mosquito population. We will use the most effective collection combination with positioning and type of traps at each study site.
Storage of mosquitoes and identification: Mosquitoes will be collected alive in the evening and early morning, anaesthetized with ethyl acetate, sorted, and stored in cryovials in a nitrogen tank. They will be shipped to the Kenya Medical Research Institute (KEMRI) laboratories, where they will be morphologically identifiied. Mosquitoes will be grouped in pools of up to 25 mosquitoes per pool, according to the site, trap type, date of collection, sex, and species. Blood-fed mosquitoes will be placed individually in Eppendorf tubes for blood-meal determination.
Nucleic Acid extraction: Identified mosquito pools and individual mosquito abdomens will be processed for nucleic acid isolation. For blood-fed mosquitoes, the abdomens will be separated from the rest of the body before processing. After homogenization, the samples will be processed using the magnetic-based Viral DNA/RNA Kit. After nucleic acid extraction the total RNA will be subjected to cDNA synthesis using a High-Capacity cDNA Reverse Transcription Kit.
Molecular detection of viruses (RT-PCR): Mosquito pools will be transported to ILRI for molecular analyses. They will be screened for six genera (Flavivirus, Alphavirus, Phlebovirus, Orthobunyavirus, Nairovirus, and Thogotovirus) of arboviruses using a multiplex PCR that uses degenerate primers coupled with end-reaction high resolution melting analysis (PCR-HRM). This test has a high analytical sensitivity to detect viral nucleic acid for flaviviruses and thogotoviruses, alphaviruses and orthobunyaviruses. Positive samples from the multiplex PCR-HRM will be subjected to genus-specific amplification and representative positive samples will be selected and prepared for sequencing. The resulting sequence contigs will be used in nucleotide BLAST searches against the GenBank database.
Blood meal analyses: Analysis will be carried out on each individually extracted blood-fed mosquito to determine its vertebrate host. Primers for cytochrome b (cyt b) and 16S rRNA markers will be used to resolve the vertebrate host source of the blood-meals. DNA extracted from human, cattle, sheep, goat, pig, camel, and chicken samples will serve as positive controls in each of the runs. Directly purified amplicons will be Sanger-sequenced. The resulting cyt b and 16S rRNA sequences will be trimmed, edited, and analyzed and identities queried in GenBank using default BLAST parameters.
Nanopore sequencing: To metagenomically detect and characterize arboviruses virus from an infected mosquito, nanopore libraries will be prepared using the Oxford Nanopore Technologies sequencing protocols, and sequenced using a MinION Mk I. The resulting MinION files will be processed and analyzed.
