Name: experimental viral infection in adult mosquitoes by oral feeding and microinjection
Uploaded: 2022-07-28T14:00:00
Description: Watch this Scientific Journal Video about Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection at JoVE.com

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

1. 病毒和蚊子的制备
<ol><li>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。<ol><li>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/?...

<ol>
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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...

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2，3。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6，7。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13，14。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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aedes aegypti&#160;</td>
			<td></td>
			<td></td>
			<td>Rockefeller strain</td>
		</tr>
		<tr>
			<td>Automated nucleic acid extraction system&#160;</td>
			<td>NanoMagBio</td>
			<td>S-48</td>
			<td></td>
		</tr>
		<tr>
			<td>BHK-21 cells</td>
			<td>National Virus Resource Center, Wuhan Institute of Virology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buckets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C6/36 cells&#160;</td>
			<td>National Virus Resource Center, Wuhan Institute of Virology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide spray gun&#160;</td>
			<td>wuhan Yihong</td>
			<td>YHDFPCO2</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal machine</td>
			<td>Himac&#160;</td>
			<td>CF16RN</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System&#160;</td>
			<td>Bio-Rad</td>
			<td>CFX96 Touch</td>
			<td></td>
		</tr>
		<tr>
			<td>Ebinur Lake virus</td>
			<td></td>
			<td></td>
			<td>Cu20-XJ isolation</td>
		</tr>
		<tr>
			<td>Formaldehyde&#160;</td>
			<td>Wuhan Baiqiandu</td>
			<td>B0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Glove box&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Hushi</td>
			<td>10010518</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil&#160;</td>
			<td>Cargille</td>
			<td>16908-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect incubator</td>
			<td>Memmert</td>
			<td>HPP750T7</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Temperature Tissue Homogenizer Grinding Machine&#160;</td>
			<td>Servicebio</td>
			<td>KZ-III-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Virus Genome Extraction Kit</td>
			<td>NanoMagBio</td>
			<td>NMG0966-16</td>
			<td></td>
		</tr>
		<tr>
			<td>mesh cages (30 x 30 x 30 cm)</td>
			<td>Huayu</td>
			<td>HY-35</td>
			<td></td>
		</tr>
		<tr>
			<td>methylcellulose</td>
			<td>Calbiochem</td>
			<td>17851</td>
			<td></td>
		</tr>
		<tr>
			<td>mice feedstuff powder&#160;</td>
			<td>BESSN</td>
			<td>BS018</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Puller</td>
			<td>WPI</td>
			<td>PUL-1000</td>
			<td>PUL-1000&#160;is a microprocessor controlled horizontal puller for making glass micropipettes or microelectrodes used in intracellular recording, patch clamp studies, microperfusion or microinjection.</td>
		</tr>
		<tr>
			<td>Mosquito net meshes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject III Programmable Nanoliter Injector</td>
			<td>Drummond</td>
			<td>3-000-207</td>
			<td></td>
		</tr>
		<tr>
			<td>One Step TB Green PrimeScript PLUS RT-PCR Kit&#160;</td>
			<td>Takara</td>
			<td>RR096A</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>C10010500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>151140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cupes (7 oz)&#160;</td>
			<td>Hubei Duoanduo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cups (24 oz)&#160;</td>
			<td>Anhui shangji</td>
			<td>PET32-Tub-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic disposable droppers</td>
			<td>Biosharp</td>
			<td>BS-XG-O3L-NS</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator (-80 &#176;C)</td>
			<td>sanyo</td>
			<td>MDF-U54V</td>
			<td></td>
		</tr>
		<tr>
			<td>Replacement Glass Capillaries</td>
			<td>Drummond</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640&#160;</td>
			<td>Gibco</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap storage tubes (2 mL )</td>
			<td>biofil</td>
			<td>&#160;FCT010005</td>
			<td></td>
		</tr>
		<tr>
			<td>Shallow dishes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sponge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile defibrillated horse blood</td>
			<td>Wuhan Purity Biotechnology</td>
			<td>CDHXB413</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 culture flask</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>The artificial mosquito feeding system&#160;</td>
			<td>Hemotek</td>
			<td>Hemotek PS6</td>
			<td></td>
		</tr>
		<tr>
			<td>The dissecting microscope&#160;</td>
			<td>ZEISS</td>
			<td>&#160;stemi508</td>
			<td></td>
		</tr>
		<tr>
			<td>The ice plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The mosquito absorbing machine&#160;</td>
			<td>Ningbo Bangning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The pipette tips&#160;</td>
			<td>Axygen</td>
			<td>TF</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>0203-5-PO</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental viral infection in adult mosquitoes by oral feeding and microinjection

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。

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?...

Watch this Scientific Journal Video about Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection at JoVE.com

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

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。

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

该协议可以确定肠道和甲状腺是否阻碍病毒传播，并有助于对埃及伊蚊传播的蚊媒病毒进行风险评估。我们的方案包括两种感染方法，口服喂养和胸内注射，可以有效评估虫媒病毒的媒介能力。该方案可能适用于各种蚊媒中的其他虫媒病毒感染，例如伊蚊中的登革热病毒和寨卡病毒感染，并且可能被证明是一种可行的程序。演示该程序的将是我实验室的研究助理Fei Wang。要使用人工蚊子喂养系统进行人工喂养，请将胶原蛋白膜切割成适当的大小，并用O形圈将它们固定在水库中。在储液槽中加入三毫升病毒-血液混合物，并用塑料塞密封储液器以防止泄漏。将装有蚊子的塑料杯放入手套箱。将密封的储液罐拧入 FU1 进料器，然后将一个储液器放在一个杯子上。打开动力装置，喂蚊子一小时。喂食后，用冰麻醉蚊子几秒钟，直到它们晕倒。将麻醉的蚊子倒入放在冰上的培养皿中，并迅速盖上盖子。用镊子挑选充血的雌蚊，并以每杯50只雌蚊的速度将它们转移到新的塑料杯中。用切好的蚊帐网包裹杯子，并盖上中间有孔的盖子。将海绵切成小块，然后放在杯子上。使用塑料一次性滴管将8%葡萄糖溶液添加到海绵上。将装有雌性蚊子的杯子放入27摄氏度，湿度为80%的培养箱中10天。每 72 小时更换一块新的葡萄糖饱和海绵。对于胸内接种，请按照文本手稿中的说明准备雌性蚊子。要准备传染性病毒稀释液，请从 80 摄氏度的冰箱中取出病毒原液，然后在冰上解冻。用含有 10%FBS 和 1% 青霉素-链霉素的 RPMI 1640 培养基在含有 100 至 500 个斑块形成单位病毒的 100 纳升病毒稀释液下稀释病毒原液。将病毒稀释液放在冰上。接下来，使用Puller 1000准备显微注射针，其中拔针器程序中的参数设置为热指数为450，重力（g）为110，距离（毫米为1），延迟（以秒为单位）为零。用镊子在解剖显微镜下以 16 倍放大倍率切割拉针的尖端。通过一次性无菌注射器用矿物油回填针头。按照机器上的说明将针头连接到注射器中。小心地将针头插入含有传染性病毒稀释液的管中。然后，按下填充按钮将大约四微升的病毒稀释液填充到针头中。针头填充后，请勿触摸或损坏针头。用镊子挑选大约50只雌性麻醉蚊子，并将它们放在冰板上。将板放在注射器下方，并在解剖显微镜下将针头插入蚊子的胸腔。将注射体积设置为 100 纳升，将速率设置为 50 纳升/秒。按下注射按钮，让病毒溶液流入蚊子体内。注射成功后，腹部略微隆起。将受感染的蚊子放入放在冰上的新塑料杯中。当受感染的蚊子数量足够时，用切好的蚊帐网包裹杯子，并盖上盖子。要收集受感染的雌性蚊子的唾液，请将麻醉的蚊子倒入放在冰上的培养皿中，并迅速盖上盖子。将装满浸油的 10 微升移液器吸头并排放在橡胶泥上。用镊子挑出雌性蚊子，然后把它们放在冰盘上。用镊子取出他们的腿和翅膀。将一只蚊子的口器放在每个移液管尖端上，以收集蚊子在室温下分泌到油中的唾液。45至60分钟后，将移液器吸头放入含有200微升病毒稀释剂培养基的1.5毫升管中。将试管以 5， 000 g 在 4 摄氏度下离心五分钟，以将唾液排出到试管中。将含唾液的试管储存在80摄氏度下，以便随后测定传输速率。收集唾液后，解剖受感染的雌性蚊子。使用镊子切下蚊子的头部，并将每个头放入装有200微升RPMI 1640培养基的单独管中。用镊子抓住胸部。然后，用另一个镊子抓住腹部的倒数第二段，将肠道拉出。清洁肠道中任何杂散的组织和器官。用PBS清洗肠道，并将每个肠道放入装有200微升RPMI 1640培养基的单独管中。将这些试管储存在80摄氏度，以便随后确定病毒传播和感染率。在这项具有代表性的分析中，确定了感染后10天雌性蚊子肠道，头部和唾液中的病毒RNA。胸内接种蚊子内脏、头部和唾液中埃比努尔湖病毒的病毒滴度高于口腔感染蚊子。胸内接种蚊子的感染率和传播率分别为100%和70%和38.1%。胸内接种蚊子的传播率高达90%，但口腔感染蚊子的传播率仅为4.8%，由于摄食量会影响病毒含量，因此挑选充血的雌性蚊子以保持一致性。

Research

JoVE Journal

Immunology and Infection

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

科研

免疫与感染

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

疟疾蚊子中的二维和三维染色体绘画

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...