Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Summary

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Abstract

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Introduction

Vector competence is defined as the ability at the level of species, population, and even an individual, of a given arthropod such as a mosquito, tick, or phlebotomine sand fly, to acquire and transmit an agent biologically with replication or development in the arthropod^1,2. With respect to mosquitoes and arthropod-borne viruses (i.e., arboviruses), the agent is imbibed from a viremic host by a female mosquito. Following ingestion, the virus must productively infect one of a small population of midgut epithelial cells³, overcoming various physiological obstacles such as proteolytic deg....

Protocol

All procedures performed in these protocols were performed in full compliance with protocols approved by the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee of the University of Texas Medical Branch at Galveston.

1. Amplify ZIKV in Vero cells

1. Grow Vero cells (CCL-81 or VeroE6) in Dulbecco’s modification of Eagle’s minimal essential medium (DMEM) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS), and 1% (v/v) p.......

Discussion

The methods described here provide a generalized workflow to conduct vector competence analyses. As a general framework, many of these methodologies are conserved throughout the literature. However, there is substantial room for modifications (reviewed in Azar and Weaver¹). Virus (e.g., viral lineage, storage of challenge virus, viral passage history), entomology (e.g., laboratory colonization of mosquito populations, innate immunity, the mosquito microbiome/virome), and experimental variables (e........

Materials

Name	Company	Catalog Number	Comments
3mL Standard Reservoir	R37P30	Hemotek Ltd	Insectary Equipment
7/32" Stainless Steel 440 Grade C Balls	4RJH9	Grainger	Grinding Media
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
Corning, Cell Culture Treated Flasks, 150cm2, Vented Cap, Case of 50	10-126-34	FisherScientific	Cell culture consumable
Costar Cell Culture Plates, 24-well, 5/bag, 100/case, Corning	07-200-740	FisherScientific	Cell culture consumable
Costar Cell Culture Plates, 96-well, 5/bag, 100/case, Corning	07-200-91	FisherScientific	Cell culture consumable
Crystal Violet	C0775-100G	MilliporeSigma	Stain
Eppendorf Snap Cap Microcentrifuge Safe-Lock 2mL Tubes, 500/Case	05-402-7	FisherScientific	Plastic consumable
Falcon 15mL Conical Centrigue Tubes	14-959-70C	FisherScientific	Plastic consumable
Falcon 50mL Conical Centrigue Tubes	14-959-49A	FisherScientific	Plastic consumable
Falcon Disposable Polystyrene Serological 10mL Pipets, 200/Case	13-675-20	FisherScientific	Plastic consumable
Falcon Disposable Polystyrene Serological 1mL Pipets, 1000/Case	13-675-15B	FisherScientific	Plastic consumable
Falcon Disposable Polystyrene Serological 25mL Pipets, 200/Case	13-675-30	FisherScientific	Plastic consumable
Falcon Disposable Polystyrene Serological 5mL Pipets, 200/Case	13-675-22	FisherScientific	Plastic consumable
Falcon Standard Tissue Culture Dishes	08-772B	FisherScientific	Plastic consumable
Fetal Bovine Serum-Premium, 500mL	S11150	Atlanta Biologicals	Cell culture reagent
Fisherbrand Economy Plain Glass Microscope Slides	12-550-A3	FisherScientific	Immobilization of Mosquitos
FU1 Feeder	FU1-0	Hemotek Ltd	Insectary Equipment; feeding units
Gibco DPBS with Calcium and Magnesium, 10 x 500mL Bottles	140-040-182	FisherScientific	Cell culture reagent
Gibco Fungizone, Amphotericin B, 250μg/mL, 50mL/Bottle	15-290-026	Fisher Scientific	Cell culture reagent
Gibco Penicillin-Streptomycin (10,000 U/mL), 100mL/Bottle, 20 Bottles/Case	15-140-163	FisherScientific	Cell culture reagent
Gibco, Tryptsin-EDTA (.25%), Phenol red, 20 x 100mL Bottles	25-200-114	FisherScientific	Cell culture reagent
Gibcom DMEM, High Glucose, 10 x 500mL Bottles	11-965-118	FisherScientific	Cell culture reagent
Human Blood, Unspecified Gender, Na-Citrate, 1 Unit	7203706	Lampire	Bloodmeal preparation
InsectaVac Aspirator	2809B	Bioquip	Insectary Equipment
Methanol, Certified ACS, Fisher Chemicals, Amber Glass Bottle, 4L, 4/Case	A412-4	FisherScientific	Fixative
Methyl cellulose, viscosity: 3,500-5,600 cP, 2 % in water(20 °C), 250g/Bottle	M0512-250G	MilliporeSigma	Cell culture reagent
Micro-chem Plus Disinfectant Detergent	C849T34	Thomas Scientific	Decontamination; working dilution of dual quaternary ammonium
Mineral Oil, BioReagent, for molecular biology	M5904-5X5ML	MilliporeSigma	Immobilization of Mosquitos
O-rings	OR37-25	Hemotek Ltd	Insectary Equipment
Plastic Plugs	PP5-250	Hemotek Ltd	Insectary Equipment
PS6 Power Unit (110-120V)	PS6120	Hemotek Ltd	Insectary Equipment; power source
Rubis Forceps, Offset blades, superfine points	4525	Bioquip	Insectary Equipment
Sarstedt Inc, 2mL Screw Cap Microtube, Conical Bottom, O-ring Cap, Sterile, 1000/Case	50-809-242	FisherScientific	Plastic consumable
Sucrose, BioUltra, for molecular biology	84097-250G	MilliporeSigma	Reagent
ThermoScientific, ART Barrier Low Retention 1000μL Pipette Tips, 100 tips/Rack, 8 Racks/Pack, 4 Packs/Case	21-402-487	FisherScientific	Plastic consumable
ThermoScientific, ART Barrier Low Retention 200μL Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case	21-402-486	FisherScientific	Plastic consumable
ThermoScientific, ART Barrier Low Retention 20μL Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case	21-402-484	FisherScientific	Plastic consumable
ThermoScientific, ART Barrier Low Retention, Extended Reach 10μL Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case	21-402-482	FisherScientific	Plastic consumable
TissueLyser II	85300	QIAGEN	Homogenization
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