从田间收集的蚊子中分离出蚊相关病毒
Summary
由于测序技术的广泛使用，在蚊子中发现了许多新的病毒样序列。我们提供了一种使用脊椎动物和蚊子细胞系分离和扩增病毒的有效程序，这可能作为未来研究蚊子相关病毒（包括蚊媒和蚊子特异性病毒）的基础。
Abstract
随着测序技术的广泛应用，在包括蚊子在内的节肢动物中发现了许多新型病毒样序列。这些新的蚊媒病毒的两个主要类别是“蚊媒病毒（MBV）”和“蚊媒病毒（MSV）”。这些新型病毒可能对脊椎动物和蚊子都有致病性，或者它们可能只是与蚊子共生。实体病毒对于确认这些病毒的生物学特征至关重要。因此，这里描述了从现场收集的蚊子中分离和扩增病毒的详细方案。首先，制备蚊子样品作为蚊子匀浆的上清液。离心两次后，将上清液接种到蚊子细胞系C6/36或脊椎动物细胞系BHK-21中进行病毒扩增。7天后，收集上清液作为P1上清液并储存在-80°C。 接下来，P1上清液在C6 / 36或BHK-21细胞中再传代两次，同时每天检查细胞状态。当发现细胞的细胞致病作用（CPE）时，收集这些上清液并用于鉴定病毒。该协议是未来研究蚊子相关病毒（包括MBV和MSV）的基础。
Introduction
蚊子是一组重要的致病性节肢动物媒介。Culicidae1，2家族中大约有3，500种蚊子。高通量测序技术的发展导致在世界不同地区的蚊子中发现了许多新的病毒样序列3。一般来说，这些蚊子相关病毒可分为两大类：MBV和MSV。
MBV是一组不同的病毒，是许多人类或动物疾病的病原体，例如黄热病病毒（YFV）、登革热病毒（DENV）、日本脑炎病毒（JEV）、西尼罗河病毒（WNV）和裂谷热病毒（RVFV）4。它们严重威胁着公众健康，导致全世界人类和动物的严重发病率和死亡率。MBV通过从受感染的蚊子传播到幼稚的宿主，以及从病毒感染的宿主和进食的蚊子的传播，自然地维持不同宿主之间的生命周期5。因此，这些病毒可以在实验室中感染蚊子细胞系和脊椎动物细胞系1。
MSV包括宜昌病毒（YCN）、黄库蚊病毒（CxFV）和朝阳病毒（CHAOV），是昆虫特异性病毒1，6，7的一个亚组。近年来，新型MSV的发现有所增加，其中一些MSV被发现对MBV的传播有影响。例如，CxFV可能是毛库蚊的持续感染，可以在早期阶段抑制西尼罗河病毒的复制8。已发现另一种昆虫特异性黄病毒，细胞融合剂病毒（CFAV）可抑制DENV和寨卡病毒（ZIKV）在埃及伊蚊中的繁殖9。因此，该协议是分离蚊子相关病毒的有用方法，可以帮助进一步研究与蚊子相关的病原体的分布和控制蚊媒疾病的控制。
Protocol
1. 蚊虫采样和分类 通过现场的轻型捕蚊器MXA-02或二氧化碳捕蚊器诱捕成年蚊子。 通过浸入液氮10，11杀死收集的蚊子。通过冷链物流系统将它们运送到实验室12.注意：干冰主要用于冷链物流系统。 （可选）如果样品地点靠近实验室，则直接将网捕器中的活蚊子送到实验室，并通过在≤-20°C冷冻30<s…
Representative Results
用蚊子匀浆（P0）的上清液接种后，C6 / 36细胞表现出广泛的细胞间空间，并且在120小时（图1A）与未接种的细胞（对照）相比观察到脱落的细胞（图1B）同时（图1B）。将BHK-21细胞与P3上清液孵育后，与对照细胞（图1D）相比，在48小时（图1C）在BHK-21细胞中观察到可见的CPE。进行PCR以确定病毒种类。用于检测?…
Discussion
该方法的目的是提供一种使用各种细胞系分离蚊子相关病毒的实用方法。将抗生素 – 抗真菌剂（青霉素 – 链霉素 – 两性霉素）添加到蚊子匀浆的上清液中以避免被细菌或真菌污染至关重要。在田间获得的蚊子和病毒上清液必须在-80°C下冷藏，以避免反复冻融循环。
协议中的另一个关键步骤是研磨。蚊子样本应彻底粉末化并储存在冰上。地面不足的蚊子组织可诱发细胞死亡，?…
Disclosures
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
| 0.22 µm membrane filter | Millipore | SLGP033RB | Polymer films with specific pore ratings.To remove cell debris and bacteria. |
| 24-well plates | CORNING | 3524 | Containers for cell |
| 75 cm2 flasks | CORNING | 430641 | Containers for cell |
| a sterile 2 mL tube with 3 mm ceramic beads | |||
| Antibiotic-Antimycotic | Gibco | 15240-062 | Antibiotic in the medium to prevent contamination from bacteria and fungi |
| Automated nucleic acid extraction system | NanoMagBio | S-48 | |
| BHK-21 cells | National Virus Resource Center, Wuhan Institute of Virology | ||
| C6/36 cells | National Virus Resource Center, Wuhan Institute of Virology | ||
| Centrifugal machine | Himac | CF16RN | Instrument for centrifugation of mosquito samples |
| CO2 | |||
| Dulbecco’s minimal essential medium (DMEM) | Gibco | C11995500BT | medium for vertebrate cell lines |
| Ebinur Lake virus | Cu20-XJ isolation | ||
| Feta Bovine Serum (FBS) | Gibco | 10099141C | Provide nutrition for cells |
| high-speed low-temperature tissue homogenizer | servicebio | KZ-III-F | Instrument for grinding |
| incubator (28 °C) | Panasonic | MCO-18AC | Instrument for cell culture |
| incubator (37 °C) | Panasonic | MCO-18AC | Instrument for cell culture |
| PCR tube | |||
| penicillin-streptomycin | Gibco | 15410-122 | Antibiotic in the medium to prevent contamination from bacteria |
| Penicillin-Streptomycin-Amphotericin B Solution | Gibco | 15240096 | |
| Refrigerator (-80 °C) | sanyo | MDF-U54V | |
| Roswell Park Memorial Institute medium (RPMI) | Gibco | C11875500BT | medium for mosiquto cell lines |
| Screw cap storage tubes (2 mL) | biofil | FCT010005 | |
| sterile pestles | Tiangen | OSE-Y004 | Consumables for grinding |
| TGrinder OSE-Y30 electric tissue grinder | Tiangen | OSE-Y30 | Instrument for grinding |
| The dissecting microscope | ZEISS | stemi508 | |
| the light traps MXA-02 | Maxttrac | ||
| The mosquito absorbing machine | Ningbo Bangning | ||
| The pipette tips | Axygen | TF | |
| The QIAamp viral RNA mini kit | QIAGEN | 52906 | |
| Tweezers | Dumont | 0203-5-PO |
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Cite This Article
Huang, D., Ma, H., Zhao, L., Wang, X., Huang, Y., Wang, F., Yuan, Z., Xia, H. Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes. J. Vis. Exp. (186), e63852, doi:10.3791/63852 (2022).