Novel strains of Culex flavivirus and Hubei chryso-like virus 1 from the Anopheles mosquito in western Kenya

• Published in: Virus research 2024-01, Vol.339, p.199266-199266, Article 199266
• Main Authors: Lwande, Olivia Wesula, Näslund, Jonas, Sjödin, Andreas, Lantto, Rebecca, Luande, Verah Nafula, Bucht, Göran, Ahlm, Clas, Agwanda, Bernard, Obanda, Vincent, Evander, Magnus
• Format: Article
• Language: English
• Subjects: Animals; Anopheles - genetics; Anopheles spp; Culex; Culex flavivirus; Flavivirus; Hubei chryso-like virus 1; Insect Viruses - genetics; Kenya; mosquito-borne viruses; Next generation target enrichment protocol; Phylogeny; RNA; Western Kenya
• ISSN: 0168-1702; 1872-7492; 1872-7492
• DOI: 10.1016/j.virusres.2023.199266

•Complete sequences of Hubei chryso-like virus segments and the genome of Culex flavivirus in Anopheles spp.•Culex flavivirus and Hubei chryso-like virus in a single Anopheles spp. indicates coinfection.•Insect specific viruses may not be mosquito-species specific.•First report of utilisation of Twist CVRP to detect viruses from mosquito samples. Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogenates were sequenced. Genome sequences of two viruses were completed. One was identified as the single-stranded RNA Culex flavivirus and the other as th