Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Summary

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Abstract

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti's following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

Introduction

Mosquito-borne viruses (MBVs), a heterogeneous group of RNA viruses, can persist in mosquito vectors and subsequently spread to vertebrate hosts¹. The clinically important MBVs are majorly distributed in four virus families, namely Flaviviridae, Togaviridae, Reoviridae, and Peribunyavividae^2,3. In recent decades, these viruses have been reported all across the globe, causing public health issues. As one of the most well-known MBVs, Dengue virus (DENV) has become the most prevalent emerging or re-emerging arbovirus in over 100 countries during the last....

Protocol

1. Preparation of viruses and mosquitoes

1. Preparation of viruses
   NOTE: All processes were carried out in a biosafety level 2 (BSL-2) laboratory. The level of biosafety contaiment used should be determined by the pathogen's risk assessment and regulations specific to nations and regions. The process must be performed in a biosafety cabinet.
   1. Inoculate 1 x 10⁶ C6/36 cells into a T75 culture flask. Fill the flask with 10 mL of Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).
      NOTE: Leibovitz's L-15 m....

Discussion

The goal of this method was to provide a comprehensive risk assessment of one mosquito-borne virus by evaluating vector competence through oral feeding and intrathoracic inoculation.

In the oral-feeding experiment, engorged-mosquitoes need to be picked out and transferred to a new container, posing a severe risk to the operators. The reason for this is because any mosquito, including uninfected mosquitoes, might be a source of infection¹⁹. Consequently, mosquitoes must .......

Materials

Name	Company	Catalog Number	Comments
Aedes aegypti			Rockefeller strain
Automated nucleic acid extraction system	NanoMagBio	S-48
BHK-21 cells	National Virus Resource Center, Wuhan Institute of Virology
Buckets
C6/36 cells	National Virus Resource Center, Wuhan Institute of Virology
Carbon dioxide spray gun	wuhan Yihong	YHDFPCO2
Centrifugal machine	Himac	CF16RN
CFX96 Touch Real-Time PCR Detection System	Bio-Rad	CFX96 Touch
Ebinur Lake virus			Cu20-XJ isolation
Formaldehyde	Wuhan Baiqiandu	B0003
Glove box
Glucose	Hushi	10010518
Immersion oil	Cargille	16908-1
Insect incubator	Memmert	HPP750T7
Low Temperature Tissue Homogenizer Grinding Machine	Servicebio	KZ-III-F
Magnetic Virus Genome Extraction Kit	NanoMagBio	NMG0966-16
mesh cages (30 x 30 x 30 cm)	Huayu	HY-35
methylcellulose	Calbiochem	17851
mice feedstuff powder	BESSN	BS018
Microelectrode Puller	WPI	PUL-1000	PUL-1000 is a microprocessor controlled horizontal puller for making glass micropipettes or microelectrodes used in intracellular recording, patch clamp studies, microperfusion or microinjection.
Mosquito net meshes
Nanoject III Programmable Nanoliter Injector	Drummond	3-000-207
One Step TB Green PrimeScript PLUS RT-PCR Kit	Takara	RR096A
PBS, pH 7.4	Gibco	C10010500BT
Penicillin/streptomycin	Gibco	151140-122
Petri dishes
Plastic cupes (7 oz)	Hubei Duoanduo
Plastic cups (24 oz)	Anhui shangji	PET32-Tub-1
Plastic disposable droppers	Biosharp	BS-XG-O3L-NS
Refrigerator (-80 °C)	sanyo	MDF-U54V
Replacement Glass Capillaries	Drummond	3-000-203-G/X
RPMI medium 1640	Gibco	C11875500BT
Screw cap storage tubes (2 mL )	biofil	FCT010005
Shallow dishes
Sponge
Sterile defibrillated horse blood	Wuhan Purity Biotechnology	CDHXB413
T75 culture flask	Corning	430829
The artificial mosquito feeding system	Hemotek	Hemotek PS6
The dissecting microscope	ZEISS	stemi508
The ice plates
The mosquito absorbing machine	Ningbo Bangning
The pipette tips	Axygen	TF
Trypsin-EDTA (0.25%)	Gibco	25200056
Tweezers	Dumont	0203-5-PO