Visualizing Dengue Virus through Alexa Fluor Labeling

Summary

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Abstract

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development of methods to probe this interaction would be useful. Recent advancements in fluorophores development^1-3 and imaging technology⁴ can be exploited to improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, which contains a single positive strand RNA genome⁵. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer’s protocol for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block productive infection of virus and also induce cell cytotoxicity⁶. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine², making them ideal for studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue virus by virus-specific antibody and its putative receptors in host cells⁷. This method could have useful applications in virological studies.

Protocol

1. Alexa Fluor labeling of dengue virus

1. Before the labeling reaction, purify dengue virus with sucrose cushion and prepare the necessary reagents and equipment as indicated in the protocol.
2. Prepare fresh 0.2M sodium bicarbonate buffer, pH 8.5 (labeling buffer), and 1.5M hydroxylamine buffer, pH 8.3 (stop reagent), just before labeling and filter sterilize with 0.2μm syringe filters.
3. Dilute approximately 3x10⁸ plaque forming units (pfu) of purified dengue virus in 1ml of labeling buffer in a 2ml tube. This can be scaled up proportionally for batch labeling of virus.
4. Reconstitute the lyophilized Alexa Fluor 594 (AF5....

Discussion

Although AF594 dye was used in this report, a wide range of fluorophores in the Alexa Fluor succinimidyl esters series is available with similar labeling chemistry. This could extend the labeling application beyond imaging. Flow cytometry can be used as an alternative to confocal microscopy for estimating the degree of labeling for fluorophores that can be excited and detected by the FACS machine.

Alexa Fluor dyes are small molecules that react with free amino groups, primarily arginine and ly.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium bicarbonate	Sigma-Aldrich	S6297
Hydroxylamine	Sigma-Aldrich	159417
Sodium hydroxide	Merck & Co., Inc.	106498
AF594 succinimidyl esters	Molecular Probes, Life Technologies	A20004
PD-10 column	GE Healthcare	17-0851-01
Hepes	Sigma-Aldrich	H6147
NaCl	Sigma-Aldrich	S3014
EDTA	Sigma-Aldrich	E9884
M-199	Invitrogen	11150
FBS	Hyclone	SH30070.03
4-well plate	Nalge Nunc international	176740
Coverslips	Einst	0111520
Microscope slide	Sail Brand	7105
3H5 hybridoma	ATCC	HB46
10x PBS		BUF-2040-10X1L	1st Base
Saponin	Sigma-Aldrich	S4521
BSA	Sigma-Aldrich	A7906
Magnesium chloride	Sigma-Aldrich	M2670
Calcium chloride	Sigma-Aldrich	C3306
Paraformaldehye	Sigma-Aldrich	15,812-7
Mowiol 4-88	Calbiochem	475904
Dabco	Sigma-Aldrich	D27802
Tabletop centrifuge	Eppendorf	5424
Confocal microscope	Carl Zeiss, Inc.	LSM 710
To prepare M-199 growth medium, add 50ml of FBS, 5ml of sodium pyruvate and 5ml of non-essential amino acids to 500ml of M-199, sterile filter.
To prepare M-199 maintenance medium, add 15ml of FBS, 5ml of sodium pyruvate and 5ml of non-essential amino acids to 500ml of M-199, sterile filter.