Email updates

Keep up to date with the latest news and content from Parasites & Vectors and BioMed Central.

Open Access Highly Accessed Research

Detection of dengue group viruses by fluorescence in situ hybridization

Vincent Raquin1, Martin Wannagat12, Karima Zouache3, Catherine Legras-Lachuer1, Claire Valiente Moro1 and Patrick Mavingui1*

Author Affiliations

1 UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, 43 boulevard du 11 Novembre 1918, Villeurbanne cedex, 69622, France

2 PRABI, Pôle Rhône-Alpes de Bioinformatique, Villeurbanne, France

3 Institut Pasteur, Department of Virology, Arboviruses and Insect Vectors, 25–28 rue du Dr Roux, Paris cedex 15, 75724, France

For all author emails, please log on.

Parasites & Vectors 2012, 5:243  doi:10.1186/1756-3305-5-243

Published: 30 October 2012

Abstract

Background

Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays.

Methods

Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy.

Results

The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes.

Conclusion

Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays.

Keywords:
Dengue; Virus; Fluorescence In Situ Hybridization; Aedes albopictus