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

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Introduction

Mosquitoes of different genera transmit a large number of disease-causing viruses, primarily belonging to families Flaviviridae (genus Flavivirus) and Togaviridae (genus Alphavirus) [1, 2]. The habitat of Aedes spp., Culexspp., Culisetaspp. mosquitoes is not limited to tropical and subtropical countries. These species of mosquitoes are widespread in countries with moderate climate in the southern and northern hemispheres [2-4]. Therefore, there is a high probability that transmitters can interchange, thus causing changes in virus infectivity for humans [5].

Three genotypes have been described for the Chikungunya virus: East/Central/South African, West Afri­can and Asian [6]. The improved detection of the Chi- kungunya virus in collected mosquitoes and in diseased people made it possible to thoroughly study nucleotide sequences of its isolates and changed the beliefs about the genotypes of this pathogen. For example, some re­searchers speak about genotypes of the Indian Ocean and the Caribbean lineage [6-8]. Only 4 serotypes have been described for the dengue virus [5, 9]. Consider­ing the increasing detectability of viruses transmitted by mosquitoes and the fact that they are pathogenic for humans, development of prevention methods and treatment of diseases caused by alphaviruses and flaviviruses is highly significant. Therefore, the isolation of viruses directly from the transmitters captured in their natural habitats and the study of the isolated strains are constituent parts of this process.

The purpose of this study was isolation and iden­tification of arboviruses belonging to the Flavivirus and Alphavirus genera from mosquitoes of Aedes albopic­tus, Aedes aegypti, Culiseta spp., Culex spp. species.

Materials and methods

Mosquitoes were captured during the dry season in the forested areas of Nicaragua (Tipitapa municipa­lity) having 12.325527N 85.974662W and 12.323326N 85.974275W coordinates. Among the captured mosqui­toes there were representatives of Aedes genera (Aedes albopictus, Aedes aegypti species), Culiseta spp. and Culex spp. genera. After their species had been identi­fied, the mosquitoes were separated into pools of 4-5 insects of the same species. Each pool was homoge­nized in 0.4 ml of phosphate-buffered saline, pH 7.4, to obtain the suspension.

Isolation of viruses. The obtained mosquito sus­pension was applied to the monolayer of C6/36 mosqui­to cells, which was grown in cell culture flasks (Corn­ing, USA) having a 25 cm² surface area. After one-hour adsorption, DMEM cell medium (Chumakov Federal Scientific Center for Research and Development of Im­mune and Biological Products) was added to the flasks, together with 2% fetal serum (Gibco). The flasks were placed into an incubator and remained there at 32°C and 5% CO₂. The monolayer was monitored daily un­der a microscope until a cytopathic effect.

Identification of virus antigens. Reagent kits from Bioservice, namely BioScreen-Dengue (AG), BioScreen-Chikungunya (AG), BioScreen-WNV (AG), BioScreen-Sindbis (AG), were used to detect an­tigens of Chikungunya, dengue, Sindbis and West Nile in samples of the cell culture fluid. The reaction was performed and the results were recorded in accordance with the instructions to the reagent kits.

Polymerase chain reaction (PCR). The Ampli- Sens® Magno-Sorb (InterLabService) kit was used to isolate RNA from 100 μ! of the virus-containing culture fluid in accordance with the manufacturer’s protocol.

The reverse primers pE2CHVrev1, pE2CH- Vrev2, pElCHVrevl, pE1CHVrev2, pNSlCHVrevl, pNS1CHVrev2, pNS1CHVrev3 for the Chikungunya virus (Table 1), pDV2rt (5-CAGCCATGGCAGCG-GTAGGTC-3’) primers for the dengue virus, and the reagent kit for the reverse transcription (RT) (Syntol) were used to perform the RT reaction on the virus RNA matrix and to receive cDNA. During the first stage, 2 μ] of the reverse primer (10 pmol/μl) were mixed with 6 μl of the isolated RNA, and then the mixture was heated during 5 minutes at 95°C. Then the tubes were cooled down during 2 minutes at the indoor temperature and were filled with another 22 μl of the mixture for RT (9 μl of deionized water, 12 μl of 2.5-fold buffer for RT, 2.5 μl of MMLV-reverse transcriptase and were incubated during 30 minutes at 42°C. The mixture was heated during 5 minutes at 95°C to inactive the reverse transcriptase.

 

Oligonucleotides shown in Table 1 were used for obtaining E1, E2 and NS1 gene fragments of the Chi­kungunya virus and their sequencing.

The PCR on the cDNA obtained after RT was per­formed by using the following combinations of primers for the Chikungunya virus:

• gene E1 — pElCHVforl and pElCHVrevl, pE1CHVfor2 and pE1CHVrev2;
• gene E2 — pE2CHVfor1 and pE2CHVrev1. pE2CHVfor2 and pE2CHVrev2;
• gene NS1 — pNSlCHVforl and pNSlCHVrevl, pNS1CHVfor2 and pNS1CHVrev2, pNS 1 CHVfor3 and pNS1CHVrev3.

For the dengue virus, the PCR was performed as described previously [10]: pDV2for (5’-CCAAA- CAACCCGCCACTCTAAGG-3’) and pDV2rev (5’-GTTATCACGACAGTGTATTCCA-3’). The PCR mixture was received by mixing of 1 μl of the for­ward and 1 μl of the reverse primer at 10 pmol/pl 10 μl of the multipurpose 2.5-forld reaction mixture for PCR (Syntol), 8 μl of deionized water, and 5 μl cDNA added later. The amplification was conducted by using the XP Cycler instrument (Hangzhou Bioer Techonology) according to the following protocol: 95°C — 1 min 30 sec; 30 cycles: 95°C — 20 sec, 55°C — 15 sec, 72°C — 30 sec; the final elongation at 72°C — 10 min. The protocol for amplification of the dengue virus: 95°C — 1 min 30 sec; 40 cycles: 95°C — 20 sec, 57°C — 15 sec, 72°C — 30 sec; the final elongation at 72°C — 10 min.

The measuring of the minimum infection rate (MIR) of the studied mosquitoes was conducted as de­scribed previously [11, 12].

Results

A total of 102 mosquitoes of three genera were studied (Table 2). The largest number of mosquitoes belonged to the Aedes genus represented by Aedes ae­gypti and Aedes albopictus species. Each sample of sus­pension went through 4 successive “blind” passages on C6/36 cells. The condition of the cell monolayer was monitored under the microscope during 5 days after the passage.

 

As a result, it was confirmed that the isolate ob­tained from Aedes albopictus mosquitoes contained the RNA of the Chikungunya virus and did not contain the RNA of the dengue virus. The isolate obtained from Aedes aegypti mosquitoes contained the RNA of the dengue virus and did not contain the RNA of the Chikungunya virus. Based on the results of sequencing, the isolate of the dengue virus was classified as type 2. The PCR products of E1, E2 and NS1 genes, which were obtained by the amplification of the isolate of the Chikungunya virus, were also sequenced.

The obtained sequences are deposited in the Gen- Bank under numbers MN271691 and MN271692. The BLAST computer program was used for classification of the nucleotide sequences of the isolate of the Chikungunya virus, which were classified as the East/Central/ South African genotype.

Changes in the cell monolayer were detected only in 2 pools of Aedes aegypti and Aedes albopictus mos­quitoes, starting from the 3^rd passage. On the 4^th passage of the Aedes aegypti pool, the cytopathic effect was ob­served on the 4^th day, while the Aedes albopictus pool demonstrated it on the 3^rd day. After the additional 5^th passage on the C6/36 cells, the materials of the above pools were examined with ELISA (reagent kits from Bioservice) for presence of antigens of dengue, Chiku- ngunya, West Nile and Sindbis viruses.

In the Aedes aegypti and Aedes albopictus pools, in the materials of all the examined passages, no anti­gens of West Nile and Sindbis viruses were found (Ta­ble 2). At the same time, starting from the 4^th passage, the antigen of the Chikungunya virus was detected in the materials of the suspension obtained from the Aedes albopictus pool. Starting from the 5^th passage, the den­gue virus was detected in the materials from the Aedes aegypti pool (Figure). Thus, antigens of Chikungunya or dengue viruses were detected only in 2 out of 25 ex­amined pools of mosquitoes of different genera.

 

 

The minimum infection rate (MIR) for Aedes ae­gypti was 0.0357, for Aedes albopictus — 0.0270. Thus, the pools containing antigens of Chikungunya and den­gue viruses had a cytopathic effect on Cy6/36 on the 4^th and 5^th successive passages. The material of the 5^th passage of both pools of mosquitoes was analyzed by using RT-PCR with specific primers for dengue and Chikungunya viruses.

Discussion

In countries of Central America, the transmission of dengue and Chikungunya viruses is caused by mos­quitoes of the Aedes species. Insects are generally col­lected in areas of human residence [2-5, 9, 12]. In our study, mosquitoes of 3 species were captured outside human settlements, in the forested area. When exa­mining whether mosquitoes of these species are infect­ed with a virus, the number of mosquitoes in the studied pool is of high importance. The optimum size of the pool is a 4 mosquito pool [2, 11, 12]. PCR is used for detection of viruses in mosquito pools; if the result is positive, the obtained amplicon is sequenced. The pub­lished articles have no data about using of ELISA.

As the size of the primary suspension of mosqui­toes in our study was limited, detection of viruses after “blind” successive passages on C6/36 cells is complete­ly justified. Using ElISA and RT-PCR for detection of viruses makes it possible to perform detection by em­ploying two independent methods.

About the authors

Georgy M. Ignatyev

M.P. Chumakov Federal Scientific Center for Research and Development of Immunobiological Preparations

Author for correspondence.
Email: marburgman@mail.ru
ORCID iD: 0000-0002-9731-3681
D. Sci. (Med.), Prof., Deputy Head, Production department Россия

Konstantin V. Kaa

M.P. Chumakov Federal Scientific Center for Research and Development of Immunobiological Preparations

Email: fake@neicon.ru
ORCID iD: 0000-0002-8446-1853
postgraduate student, laboratory of molecular biology of viruses Россия

Alexey S. Oksanich

“Bioservice” Biotechnology Company

Email: fake@neicon.ru
ORCID iD: 0000-0002-8600-7347
PhD (Biol.), General Director Россия

Lilya P. Antonova

M.P. Chumakov Federal Scientific Center for Research and Development of Immunobiological Preparations

Email: fake@neicon.ru
ORCID iD: 0000-0002-1221-1134
postgraduate student, laboratory of molecular biology of viruses Россия

Tatyana G. Samartseva

Mechnikov Research Institute for Vaccines and Sera

Email: fake@neicon.ru
ORCID iD: 0000-0003-3264-6722
junior researcher Россия

Kirill M. Mefed

M.P. Chumakov Federal Scientific Center for Research and Development of Immunobiological Preparations

Email: fake@neicon.ru
ORCID iD: 0000-0002-7335-1982
PhD (Biol.), Deputy Head of Quality Management Россия

Dinora A. Yakovleva

Mechnikov Research Institute for Vaccines and Sera

Email: fake@neicon.ru
ORCID iD: 0000-0001-8771-4177
PhD (Med.), senior researcher Россия

Ekaterina N. Zhirenkina

St. Petersburg Vaccine and Sera Research Institute

Email: fake@neicon.ru
ORCID iD: 0000-0003-0810-5985
PhD (Med.), Deputy Director for science Россия

References

Supplementary files