Summary

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Published: July 28, 2022
doi:

Summary

该方法包括经口喂养和胸内注射感染,可以有效评估中肠和/或唾液腺屏障对虫媒病毒感染的影响。

Abstract

蚊媒病毒(MBVs)是脊椎动物的传染性病原体,由许多蚊子物种传播,对公众健康构成严重威胁。一旦摄入,病毒必须克服蚊子中肠屏障才能到达血淋巴,从那里它们可能会传播到唾液腺。当蚊子叮咬时,这些病毒会传播给新的脊椎动物宿主。同样,蚊子可能会感染不同的病毒。一般来说,只有一小部分病毒可以通过肠道 进入 唾液腺。这些病毒向腺体的传播效率受到不同蚊子物种中发现的两个物理屏障的影响:中肠屏障和唾液腺屏障。该协议提出了一种在经口喂养和胸内注射感染后埃及 伊蚊唾液腺中检测病毒的方法。此外,确定肠道和/或唾液腺是否阻碍病毒传播有助于对 埃及伊蚊传播的MBV进行风险评估。

Introduction

蚊媒病毒(MBV)是一组异质的RNA病毒,可以在蚊子媒介中持续存在,随后传播到脊椎动物宿主1。临床上重要的MBV主要分布在四个病毒科,即黄病毒科、托加维里科、呼肠孤病毒科和23近几十年来,这些病毒已在全球范围内报道,导致公共卫生问题。作为最著名的MBV病毒之一,登革热病毒(DENV)在过去20年中已成为100多个国家中最流行的新兴或重新出现的虫媒病毒4。自在内陆发现寨卡病毒(ZIKV)以来,非洲大陆几乎所有热带和亚热带国家和地区都报告了人类寨卡病毒感染5。为了评估病毒传播的风险,近年来的许多研究都集中在这些病毒的蚊媒能力上67。因此,有效预防和控制病媒传播疾病至关重要。

埃及伊蚊(Ae. aegypti)是实验室中最容易饲养的蚊子之一,是登革热病毒、寨卡病毒、基孔肯雅病毒(CHIKV)和黄热病病毒(YFV)的重要载体8。长期以来, 埃及伊蚊 只在非洲大陆和东南亚发现,但近年来它几乎殖民了所有大陆9。此外, 埃及伊蚊 的全球丰度一直在不断增长,到本世纪末估计将增加20%10。从2004年到2009年,由于日常气温升高, 中国埃及伊蚊 病媒能力明显增加11. 埃及伊蚊 作为致病媒介的地位在中国显著上升。因此,为了应对这些挑战,有必要调查 埃及伊蚊传播病毒的媒介能力。

作为一种噬血节肢动物,雌性蚊子刺穿脊椎动物宿主的皮肤并以血液为食。蚊子偶尔会从感染病毒的宿主那里获取病毒,然后将病毒转移到新的宿主。因此,为了确定媒介能力,在实验室环境中通过喂食系统给蚊子喂食含有虫媒病毒的人工血粉12。单个蚊子在感染几天后被分成头部、身体和唾液分泌物。为了测量病毒感染、传播和传播速率,已通过定量逆转录 PCR (qRT-PCR) 或斑块测定法检测病毒滴度。然而,并非所有蚊子都会发生中肠感染,并且能够在血液喂养后将病毒转移到下一个宿主。它与蚊子的生理屏障有关,可以防止病原体穿透人体,并在其先天免疫力中起着至关重要的作用13。中肠屏障,特别是中肠感染屏障(MIB)和中肠逃逸屏障(MEB),会影响病毒是否可以全身感染媒介及其传播效率。它阻碍了对其他组织感染的分析,例如唾液腺也表现出唾液腺感染和逃逸屏障1314。为了更好地表征载体中内脏和唾液腺的感染,本文介绍了Ae中虫媒病毒经口喂养和胸内接种的详细方案 该方案可能适用于各种蚊媒中的其他虫媒病毒感染,例如 伊蚊 属中的DENV和ZIKV感染,并且可能被证明是一种可行的程序。

Protocol

1. 病毒和蚊子的制备 病毒的制备注意:所有过程均在生物安全2级(BSL-2)实验室进行。所使用的生物安全污染水平应由病原体的风险评估和特定于国家和地区的法规确定。该过程必须在生物安全柜中进行。将 1 x 106 C6/36 细胞接种到 T75 培养瓶中。用 10 mL 罗斯威尔公园纪念研究所 (RPMI) 1640 培养基填充烧瓶,其中含有 10% 热灭活胎牛血清 (FBS) 和 1% 青霉素/?…

Representative Results

为了通过人工喂血(病毒最终滴度为6.4 x 106 PFU/mL)和胸内注射(病毒剂量为340 PFU)检查感染蚊子体内的EBIV分布,测定感染后10天蚊子唾液、头部和肠道中的病毒RNA(dpi)。 对于埃及伊蚊,胸内接种雌性蚊子的肠道、头部和唾液中的EBIV病毒滴度远高于口腔感染的雌性蚊子(图2A-C)。胸内接种的雌性蚊子的?…

Discussion

该方法的目标是通过经口喂养和胸内接种评估媒介能力,对一种蚊媒病毒进行全面的风险评估。

在口服喂养实验中,需要挑选出充血的蚊子并将其转移到新的容器中,这对操作人员构成了严重的风险。这是因为任何蚊子,包括未感染的蚊子,都可能是感染源19。因此,必须按照协议中的规定首先麻醉蚊子,然后执行后续步骤。重要的是要注意,选择充血的蚊…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作得到了武汉市科技计划项目(2018201261638501)的支持。

Materials

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

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Wang, F., Yang, C., Wang, S., Wu, Q., Ochieng, C., Yuan, Z., Xia, H. Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection. J. Vis. Exp. (185), e63830, doi:10.3791/63830 (2022).

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