1. P25-8
Morphological and Molecular Changes in Murine Astrocytes Inoculated with a Neuroadapted Dengue Virus Strain
Sandra Aguilera-Rojas, Maria- Angélica Calderón-Peláez, Sigrid Camacho-Ortega, Jaime E. Castellanos, Myriam L. Velandia-Romero*.
Grupo de Virología, Vicerrectoría de Investigaciones. Universidad El Bosque, Bogotá, Colombia. *correspondance author:
BOGOTÁ-COLOMBIA
INTRODUCTION
Neurotropic flavivirus infections induce changes in phenotype and gene expression profile of astrocytes, however little is known about these items during dengue virus (DENV) infection.
OBJECTIVE
To evaluate in vitro phenotypical and molecular changes of astrocytes inoculated with two DENV variants: a clinical isolate (DENV-4) and a neuroadapted strain (D4MB-6).
Morphological changes (IFA to GFAP)
Cellular viability (Resazurin, TMRE techniques)
Cellular proliferation (MTT).
Relative expression (qPCR) and protein production (CBA) of some cytokines and adhesion molecules
Isolation, culture and purification of brain astrocytes from 7 days old Balb/C mice
Inoculated cells with the DENV-4 or D4MB-6 (MOI:1) during 24, 48 or 72 hours post-infection (hpi). Then, we evaluate:
METHODS
Cultures were characterized by immunofluorescence assay (IFA) to detect GFAP, Glutamine synthetase (GS) and glutamate transporter 1 (GLT-1) proteins
D4MB-6 inoculation induced astrocytes proliferation
RESULTS
Our cultures typically had 95% of purity and less than 1% of the cells got infected with any of the DENV variants at different hours post-infection. GS
GFAP C)
GLT-1
GFAP+DENV
Fig 1. Astrocytes culture characterization.
Cell infection was detected in a double immunostaining to detect the GFAP and the E protein of DENV (arrow) Nuclei in blue, Bar 50µm.
GLT-1
Cytokine quantitation showed that IL-6 and MCP-1 were significantly increased compared to DENV-UV inoculated cells (48 h pi)
Virus inoculation induced changes in astrocytes morphology
Mock-inoculated cells
DENV-4
D4MB-6
Non-infected cells
Fig 2. Non infected or DENV-4 inoculated cultures had star-shape cells with long and slender astrocytic end-feet (arrowhead).
Mock-inoculated cells showed hypertrophic cytoplasm (dotted box), just like the D4MB-6 inoculated cells, that showed hypertrophic cytoplasms and thick astrocytic end-feet (arrow). GFAP was detected in green, nuclei in blue, Bar 50 µm.
Virus inoculation did not affect the cellular viability
Cell viability (resazurin test)
CONCLUSION
These results showed that although astrocytes were not susceptible to DENV infection, the viral inoculum induced proliferation and morphological cell changes along with a modification in the expression of immune molecules indicating astrocytes activation as a response to DENV inoculation.
Mitochondrial membrane potential changes (TMRE technique)
ACKNOWLEDGEMENTS
The authors would like to thank to the Vicerrectoría de Investigaciones of Universidad El Bosque (project No. PCI ) and Banco de la Republica (project No ) for the funding of this work.