Isolation and genetic analysis of the chikungunya virus from Aedes aegypti and Aedes albopictus mosquitoes captured in Central America

Introduction. The habitat of mosquitoes belonging to the genera Aedes spp., Culex spp., Culiseta spp. is in South and Central America, including Nicaragua. Monitoring of the spread of mosquito vectors and assessment of the infection with arboviruses can provide information on possible occurrence of new diseases or an increase in the reported cases, changes in the infectivity of viruses for humans due to changes in pathogen transmitters. The purpose of this study was isolation and identification of arboviruses belonging to the Flavivirus and Alphavirus genera from A. albopictus, A. aegypti, Culiseta spp., Culex spp. mosquitoes captured in forests of Nicaragua. Materials and methods. A. albopictus, A. aegypti, Culiseta spp., Culex spp. mosquitoes were captured during the dry season in 2021 in forested areas of Nicaragua in four different locations. Mosquitoes were sorted into pools, each containing 5-8 mosquitoes (236 pools in total). Using the reverse transcription polymerase chain reaction, the pools were tested for the presence of chikungunya (CHIKV), dengue, Zika, and yellow fever viruses. Positive pools were inoculated into the C6/36 cell culture to obtain isolates and for their further sequencing. Results. The dengue virus was detected only in Aedes spp. mosquitoes: in 7 pools — A. aegypti, in 1 — A. albopictus. CHIKV was also detected only in Aedes spp. mosquitoes: in 3 pools — A. aegypti, in 1 — A. albopictus. The sequencing of nucleotide sequences of 6К, Е1, Е2, and NS1 genes of CHIKV isolated from A. albopictus mosquitoes showed that compared to the similar gene sequences from CHIKV isolates recovered from A. aegypti mosquitoes, the 6K gene region contained 4 nucleotide and 4 amino acid substitutions, while the E1 region contained 16 nucleotide substitutions, 10 of them led to amino acid substitutions; the E2 region contained 14 nucleotide and 11 amino acid substitutions; the NS1 region contained 33 nucleotide and 19 amino acid substitutions

Indication and Identification of Dengue and Chikungunya Viruses in Aedes spp. Mosquitoes Captured in Central America

The purpose of study was to isolate arboviruses from mosquitoes of different species in the cell culture and to identify them by using molecular and immunochemical techniques.Materials and methods. Viruses were isolated in C6/36 cell cultures. The pathogens were identified by using enzyme-linked immunosorbent assay (ELISA) kits for detection of antigens of dengue, Chikungunya, West Nile and Sindbis viruses as well as the reverse transcription polymerase chain reaction (RT-PCR) with specific primers and Sanger sequencing.Results. A total of 102 mosquitoes belonging to three genera, Culex spp, Culiseta spp., Aedes spp., were studied. Mosquitoes of each species or genus were divided into pools, each containing 4–5 mosquitoes. The study of suspensions of only 2 mosquito pools obtained from Aedes aegypti and Aedes albopictus, starting from the 3rd passage, showed changes in the C6/36 cell monolayer. Starting from the 4th passage, an antigen of Chikungunya virus was detected using ELISA test in the suspension obtained from the Aedes albopictus pool. Dengue virus was detected in the 5th passage from the materials obtained from the Aedes aegypti pool. Thus, antigens of the Chikungunya and dengue viruses were detected only in 2 of 23 examined pools of mosquitoes of different genera. Materials of the 5th passage were analyzed by RT-PCR with specific primers for dengue and Chikungunya viruses. It was confirmed that the isolate obtained from Aedes albopictus mosquitoes contained RNA of the Chikungunya virus and corresponded to the East/Central/South African genotype, while the isolate obtained from Aedes aegypti mosquitoes contained RNA of the dengue type 2 virus.Conclusion. The obtained nucleotide sequences of the Chikungunya virus were deposited in the GenBank international database under accession numbers MN271691 and MN271692