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Molecular determination of the dengue virus serotype in clinical samples
Colombia (ASCEMCOL) - Universidad del Sinú, Elías Bechara Zainúm, Montería, Córdoba
Biomedical Research Laboratory the University of Sinú – Elías Bechara Zainúm
Requiered: English, Accepted: Spanish
Type of Research Project
- Clinical Project with Laboratory work
What is the background of the project?
Dengue is the most prevalent arbovirus, infecting nearly 50–100 million people yearly(1). Dengue fever which is the most common manifestation of the infection is caused by any of the four antigenically distinct serotypes of Dengue virus (DENV 1–4). Dispersal of the vector and an increase in the migratory flow between countries have led to large epidemics and severe clinical outcomes(2). The increasing urbanization of the population is one of the aspects that has contributed the most to the maintenance of the endemic and epidemic transmission of dengue in the Americas and Colombia, where about 80% of the population is exposed to the bite of infected A. aegypti with the different circulating serotypes of the dengue virus(3). Dengue is often considered a benign infection, however there is growing evidence that the socio-economic impact and severity of the disease have been underestimated. Due to the high incidence of dengue in the department of Córdoba, it is proposed to establish an epidemiological surveillance study from samples of patients with a clinical diagnosis of dengue, for this purpose dengue virus isolates will be obtained by culture techniques in C6/36 cells, the viral genome and the identification of serotypes will be detected through conventional reverse transcription polymerase chain reaction (RT-PCR).
What is the aim of the project?
The objective of the project is to determine the serotypes of dengue virus from clinical samples by means of viral isolates in cellular Cultures C6/36 and inspecific detection of dengue virus by means of conventional RT-PCR.
What techniques and methods are used?
Serum samples will be obtained after written informed consent from all participants. The C6/36 mosquito cell line from the Aedes albopictus adapted to grow at 28°C will culture in L15 medium (Leibovitz) supplemented with tryptose, penicillin-streptomycin and 10% fetal bovine serum. C6/36 cells were maintained in RPMI 1640 (BI, USA) supplemented with L-glutamine, penicillin, streptomycin and 10% fetal bovine serum (FBS, Gibco, USA). Vero cells were cultured in DMEM (BI, USA) suplemented in the same way as above. Vero cells were cultured at 37 °C in a 5% CO2-containing incubator, while C6/36 were cultured at 28°C in a 5% CO2-containing incubator. The DENV-2 D01090 strain Dengue virus RNA will isolate from the serum samples using the QIAamp viral RNA mini kit as per manufacturer's protocol. Extract RNA will store at − 70 °C and use for RT-PCR. The RT-PCR assay can distinguish the 4 serotypes of dengue viruses by the size of the products as described by Chien et., al. This includes a step of RT-PCR using a conserved primer pair, D1 (forward) and D2 (reverse) and a step of second-round PCR using the under ultraviolet light using a digital gel documentation system. The PCR products will electrophorese through 1.5% agarose gel, stain with ethidium bromide and examine.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The exchange student will conduct crop management of the C6/36 cell line. For the realization of this the student must prepare the means of cultivation with supervision of the tutor. The management of cells, cell count through Neubauer camera and maintenance of cell cultures with L15 Medium (Leibovitz) supplemented with tryptose, penicillin-streptomycin and fetal bovine serum at 10%. After the infection time the student will perform viral RNA extraction from commercial kits and following the manufacturer's instructions. Manipulation of strains dengue virus control conducting conventional RT-PCR and electrophoresis.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Preliminary readings about dengue: • Primary care of the patient with non-specific febrile syndrome. • Dengue pathophysiology and clinical characterics. • Viral isolation of dengue virus and molecular diagnosis.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
• Knowledge of the cellular and Molecular Biology, microbiology and virology areas. • Spanish language management (not required) and basic English knowledge of the management of Research laboratory elements and equipment • Previous readings of dengue physiopathology and clinical and molecular characteristics
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Students in biomedical fields
- Cabral-Castro MJ; Cavalcanti MG; Peralta RHS; Peralta JM. Molecular and serological techniques to detect co-circulation of DENV; ZIKV and CHIKV in suspected dengue-like syndrome patients. J Clin Virol [Internet]. 2016;82(December 2015):108–11. Available from: http://dx.doi.org/10.1016/j.jcv.2016.07.017
- Ramos-Castañeda J; Barreto dos Santos F; Martínez-Vega R; Galvão de Araujo JM; Joint G; Sarti E. Dengue in Latin America: Systematic Review of Molecular Epidemiological Trends. PLoS Negl Trop Dis. 2017;11(1):1–24.
- Mattar S; Tique V; Miranda J; Montes E; Garzon D. Journal of Infection and Public Health Undifferentiated tropical febrile illness in Cordoba ; Colombia : Not everything is dengue. J Infect Public Health [Internet]. 2017;10(5):507–12. Available from: http://dx.doi.org/10.1016/j.jiph.2016.09.014
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