Focus-Forming Assay to Assess Zika Virus Vector Competence in Mosquitoes

Abstract

Source: Azar, S. R.,et al. Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus. J. Vis. Exp. (2020).

This video demonstrates the use of a focus forming assay to evaluate vector competence in geographically distinct Aedes aegypti populations experimentally exposed to the Zika virus. By detecting infectious virus in mosquito bodies, legs, and saliva, the assay quantifies infection, dissemination, and transmission potential. The resulting foci are used to assess how effectively each mosquito population supports and transmits the virus.

Protocollo

1. Detection of ZIKV by infectious assay

1. In a BSC, prepare 24 well tissue culture plates with Vero cells (10⁵ cells per well) 24 h prior to the onset of the infectious assay. Label each well with the identity of a single mosquito/sample.
2. If samples were frozen at -80 °C, allow to thaw.
3. Remove the media from the Vero cell plates prior to inoculation with samples one plate at a time.
4. For samples containing bodies or legs, carefully aliquot 100 μL of clarified supernatant into each well, taking care not to disturb the mosquito cell debris from the pellet.
   NOTE: Saliva samples can be diluted 1:1 (v/v) with MCM prior to inoculation onto cells to conserve samples for later titration, if necessary.
5. Move the plates into a 37 °C, 5% CO₂ incubator and incubate for 1 h.
6. Return the plates to the BSC and add ~1 mL of methylcellulose overlay to each well. Place the overlayed plates back in a 37 °C, 5% CO₂ incubator and incubate for 3−7 days (virus/strain-dependent).
7. Following incubation, remove the plates from the incubator and bring into the BSC. Discard the methylcellulose overlay into a tray pan containing 10% bleach or dual quaternary ammonium disinfectant.
8. Wash each well 2x with PBS, discarding the wash into a tray pan containing 10% bleach or dual quaternary ammonium.
9. Add ~1 mL of methanol:acetone (1:1 v:v) and allow the cells to fix onto plate for at least 30 min in the BSC at room temperature (RT). Discard methanol:acetone according to institutional policy on organic waste.
10. Visualize ZIKV using one of two methods described below.
    1. Following the removal of methanol:acetone, stain immediately with a crystal violet solution (0.25% w/v in 30% methanol) for 5 min. Rinse 2x in tap water and leave to dry, then directly visualize by eye for evidence of plaques or destruction of monolayer.
    2. Alternatively, perform focus forming assay.
       1. Allow the plates to air dry until no organic fixative remains.
          NOTE: This should take ~2−3 h outside of the BSC but can be accelerated by air-drying in a BSC or chemical fume hood.
       2. Wash each well 3x for 15 min each in nonsterile PBS (Mg²⁺ and Ca²⁺ free) on an orbital plate rocker. Remove PBS and add 1 mL of blocking solution (PBS + 3% FBS) to each well and rock for 15 min at RT.
       3. Add 100 μL per well of α-ZIKV or α-flavivirus primary antibody (e.g., flavivirus group hybridoma D1-4G2-4-15 [4G2]) at a 1:2,000 dilution in blocking solution. Incubate with rocking for a minimum of 4 h (preferably overnight, not exceeding 18 h) at RT.
       4. Remove the primary antibody and wash 3x for 15 min each with PBS (Mg²⁺ and Ca2⁺ free) on an orbital plate rocker.
       5. Add 100 µL per well of the secondary antibody (goat α mouse HRP-labeled) diluted 1:2,000 in blocking buffer. Incubate with rocking for 1 h at RT.
       6. Wash 3x for 15 min each with PBS (Mg²⁺ and Ca²⁺ free) on an orbital plate rocker.
       7. Aliquot 100 µL of substrate development reagent (Table of Materials) per well. Rock plates at RT for 15 min.
       8. Halt the reaction upon development of foci/plaques by removing the substrate and rinsing the plates 2x with tap water. Pour off tap water and allow the plates to air dry before quantifying.
       9. Count viral foci to determine the number of FFU present in the given sample.
11. Regarding scoring via a focus forming assay, quantify the positive wells by examination under a light microscope. Detection of intracytoplasmic staining of cells in a well indicates the presence of virus. Samples are scored solely as focus-positive or -negative.

Materiali

Elenco dei materiali utilizzati in questo articolo

Nome	Azienda	Numero di catalogo	Commenti
Acetone, Histological Grade, Fisher Chemicals, Poly Bottle, 4L, 4/Case	A16-P4	FisherScientific	Fixative
Adenosine 5'-triphospate disodium salt hydrat, microbial, BioReagent, suitable for cell culture	A6419-1G	MilliporeSigma	Reagent
Anti-Flavivirus Group Antigen Antibody, clone D1-4G2-4-15	MAB10216	MilliporeSigma	Primary Antibody for focus forming assay
Anti-Mouse IgG (H+L) Antibody, Human Serum Adsorbed and Peroxidase-Labeled, 1.0mL/Bottle	5450-0011	KPL/Seracare	Secondary Antibody for focus forming assay
Bleach	NC0427256	FisherScientific	Decontamination
Crystal Violet	C0775-100G	MilliporeSigma	Stain
TrueBlue Peroxidase Substrate Kit, 200mL	5510-0030	Seracare	Developing solution for focus forming assay
Vero	CCL-81	American Type Culture Collection	Mammalian cell line to amplify virus and conduct infectious assay
Vero C1008 [Vero 76, clone E6, Vero E6]	CRL-1586	American Type Culture Collection	Mammalian cell line to amplify virus and conduct infectious assay