我们非常感谢博士的重要贡献。约翰安德森，安德鲁主要和Shirley蒂雷尔这项研究。这项工作是支持部分由从疾病控制和预防中心（U50/CCU116806-01-1）和美国农业部（58-6615-1-218，CONH00768，CONH00773）中心的拨款。

1。蚊子排序和识别<ol><li>在一个专门的生物安全2级（BSL - 2）的工作人员是受过训练的工作与生活的蚊子实验室，开放实验室长凳上执行下列程序。整个过程中保持一个“冷链”。 </li><li>麻醉住成蚊，灭蚊收集袋放置在-20℃冷冻5分钟。收集袋转移到一个绝缘容器干冰。关闭盖子，并揭露蚊子，以二氧化碳为1-3分钟，以确保完成击倒。 </li><li>到病床上的干冰置于预冷的泛塔麻醉蚊子。单独用细镊子和塑料部分杯地点的雌性蚊子。盖上盖板，将湿冰杯。 </li><li>确定使用描述性的分类键基础上的外部蚊虫形态，一个立体解剖显微镜（10 - 60X变焦）的援助，以物种的蚊子，安装在一个电子寒意表。 </li><li>结合确定为≤50 2毫升管理单元第的小瓶含有铜的BB的个人池蚊种。样品瓶都标有一个唯一的标识符。 </li><li>识别所有蚊子，小瓶放置在标有袋之一，在泡沫塑料含有干冰冷却器举行。 </li><li>存放在一个-80℃冷冻，直到病毒检测的蚊子池。 </li></ol> 2。生物安全考虑隔离从蚊子的病毒<ol><li>下列程序执行了一个生物安全3级（BSL - 3）实验室工作人员培训工作在这一级别遏制。实验室设施，设备，程序和做法，受到机构，州和联邦的监督和检查。寻求适当的训练，然后再尝试以下协议和批准。 </li><li>每道工序即弃保护袍，手套，面罩和呼吸器等，穿适当的个人防护装备（PPE）。 </li><li>之前和程序完成后，用70％酒精消毒工作台面。 </li><li>在II级生物安全柜（BSC）的细胞培养和潜在感染材料处理。 </li><li>蚊子池匀浆在混频器的工厂，是放置在一个BSC。 </li><li>蚊匀浆气密微量离心。如果此过程不能在BSC执行，操作人员应戴上口罩，操作和检索离心机样本时。 </li><li>浸泡在10％漂白前高压灭菌处理在BSC的材料（生物安全柜）的吸管和吸管提示。所有实验室废弃物净化处置前通过高压灭菌。 </li></ol> 3。 Vero细胞的制备<ol><li>从一个大的融合废物烧杯中含有漂白Vero细胞（克隆E6）（175厘米2）组织培养瓶中倒出文化传媒。 </li><li>洗净，加入5毫升的PBS等分和漩涡层细胞，并沿烧瓶中的双方，一定要彻底覆盖整个单层的Vero细胞。 </li><li>倒出PBS成废物烧杯。 </li><li>胰蛋白酶- EDTA加入2.5毫升，超过一定要彻底覆盖整个单层细胞层的漩涡。 </li><li>孵育5分钟，在孵化器的烧瓶中，在37 ° C，5％的CO 2 。 </li><li>轻轻摇动和漩涡烧瓶中删除从底部表面的细胞。细胞应该很容易滑出;孵育1-5分钟，如果细胞不容易滑出。 </li><li>添加5毫升（MEM），最小的重要媒体，5％胎牛血清（FBS），，采用血清学吸管和吸管起来和向下的10-20倍，打破细胞的团块。 </li><li>新增总量的22.5毫升15毫升纪念，5％FBS，拌匀。 </li><li>将40家小规模的组织培养瓶（25厘米2）直立在两个机架（20烧瓶/机架）。 </li><li>将机架含有烧瓶内的生物安全柜，并取出烧瓶帽，直接放置在前面的烧瓶的上限。 </li><li>免除到每个烧瓶中使用血清吸管4毫升5％FBS的MEM，。 </li><li>免除更换烧瓶帽到每个烧瓶（〜1:6分割比例）为0.5 mL悬浮细胞。 </li><li>添加50毫升的纪念，5％FBS回到原来的大组织培养瓶中，含有剩余的细胞悬液（约2.5 MLS），并返回烧瓶中的孵化器。同样大的组织培养瓶中，可重新使用的5代，然后一个新的瓶应设置来取代它。 </li><li>地方小组织培养瓶上的托盘堆叠。摇动托盘，以确保介质均匀覆盖flaks底部。 </li><li>在孵化器的小烧瓶置于37℃，5％的CO 2和成长至80-90％融合过夜。 </li></ol> 4。蚊子池的制备<ol><li>在测试程序，保持蚊子池冷。使用预冷冷冻调音台轧机机架和磁带，冰冷的试剂，处理蚊子池时，一个冷冻离心机。 </li><li>广场管含有蚊子成预冷冷冻机架，并添加1 - 1.5毫升PBS - G的每管蚊子。 </li><li>从冰箱中检索搅拌机磨录音带。每个卡带放入24管，然后在搅拌机磨安全。 </li><li>均质的样品，在25次/秒4分钟。混频器厂动摇以极高的速度和每一个管内金属的BB管扰乱蚊子的组织。 </li><li> 6分钟7000转离心管。 </li><li>广场放回冷冻架管，直到接种到Vero细胞。 </li></ol> 5。蚊子池匀浆接种Vero细胞<ol><li>检查从每个系列至少有一个组织培养瓶中，倒置显​​微镜下，以保证足够的汇合和细胞的健康;预计大约80-90％的覆盖​​率。 </li><li>线多达20小型组织培养瓶中，在机架上，并与每个蚊子池相应的号标签烧瓶。 </li><li>松开瓶盖和倒出的组织培养成废物烧杯烧瓶媒体。烧瓶中保留足够的媒体完全大衣细胞单层。 </li><li>分装上清100μL从每个处理成相应的组织培养瓶中的蚊子池。 </li><li>装回并拧培养瓶帽。 </li><li>躺在摇床培养瓶在35-40转5分钟（室温）。 </li><li> 20个组织培养瓶中返回直立位置在机架上，并在BSC。 </li><li>一次Uncapping 3组织培养瓶中，分配到每个烧瓶4毫升的生长介质，采用血清学移液器;取代上限。 </li><li>确保吸头不接触培养瓶的颈部或唇。根据需要更改的枪头，如果接触烧瓶。 </li><li>孵育组织培养瓶中，在37℃，5％的CO 2 。 </li></ol> 6。筛选Vero细胞病毒生长<ol><li>屏幕接种，病毒感染的迹象，细胞病变效应（CPE）组织培养瓶中，接种后3-7天。 </li><li>目视检查CPE和/或污染的细胞培养倾斜烧瓶，并审查的媒体，在烧瓶底部池。媒体会出现混浊，细胞恶化在CPE或由于酵母，真菌或细菌污染的生长。 </li><li>重新审视阴天媒体，表现出一个倒置显微镜下的细胞培养。病毒会导致细胞变成畸形，并从培养瓶中将打破松散。可见污染物，如酵母，细菌，真菌或重蚊子碎片，细胞培养，通过0.22μm的注射器过滤器通过原来的蚊子池重新测试。 </li><li>从病毒阳性的细胞培养无菌免除标记的使用血清学移液器cryotubes 1-2 MLS的媒体。 </li><li>病毒培养保存于-80 ° C直到确定使用适当的诊断方法。 </li></ol> 7。试剂的制备<ol><li> MEM，5％FBS。溶解在一个大号的dd H 2 O 4的最终体积37.6克奶粉的MEM 。免除到500毫升分装在媒体瓶和高压灭菌的解决方案。当溶液冷却后，在无菌条件下添加26毫升热灭活胎牛血清，5.2 mL的L -谷氨酰胺，5.2毫升anti-biotic/mycotic，10.4 mL 7.5％的碳酸氢钠，以每个瓶子。保存在4 ° C。 </li><li> PBS - G。溶解16克氯化钠，氯化钾0.4克，2.3克钠2 HPO 4，0.4克KH 2 PO 4，0.5％酚红和4毫升到1000毫升DD H 2 O。 2 N的氢氧化钠（如有必要）调整pH值到7.1-7.4。在一个单独的烧杯中，热量600的H 2 0毫升烧开。从热板中删除，添加明胶10克，溶解。溶解的明胶溶液，加入PBS液和调整最终体积大号用dd H 2 O 2免除成瓶和高压灭菌100毫升的溶液。当溶液冷却后，在无菌条件下加入42.8毫升热灭活兔血清和1.4毫升anti-biotic/mycotic。保存在4 ° C。 </li><li> PBS细胞洗。添加50毫升10X贝科的PBS 400毫升DD H 2 O 2 N的氢氧化钠调节pH值至7.1。调整最终体积至500 ml，用dd H 2 O 0.2uM真空过滤机的过滤解决方案。免除到5毫升分装在8毫升螺丝帽管。保存在4 ° C。 </li></ol> 8。代表性的成果九个不同的病毒从4个分类家庭康复期间连续在康涅狄格州监察从1997年至2010年（表1）收集的蚊子。五这些病毒是已知的导致人类疾病，西尼罗河病毒和东部马脑炎病毒的最重要的病原体。新发现包括：从收集到的蚊子的西尼罗病毒首次隔离北美在1999年2，波托西病毒隔离在美国东北部在2000年3，在2005年4拉克罗斯病毒在新英格兰地区的第一个隔离。 <table border="1"><tbody><tr><td> 病毒 </td><td> 家庭 </td><td> 号分离 </td><td> 号的位置 </td><td> 号年检测 </td><td> 人类疾病 </td><td> 文献。 </td></tr><tr><td>西尼罗河病毒</td><td>黄病毒</td><td> 1002 </td><td> 94 </td><td> 12 </td><td>中度至严重，发烧，脑炎</td><td> 2,5 </td></tr><tr><td>东部马脑炎病毒</td><td> Togaviridae </td><td> 292 </td><td> 41 </td><td> 10 </td><td>严重，脑炎</td><td> 6,7 </td></tr><tr><td>高地J病毒</td><td> Togaviridae </td><td> 178 </td><td> 39 </td><td> 11 </td><td>不知道</td><td> 6 </td></tr><tr><td>詹姆斯敦峡谷病毒</td><td> Bunyaviridae </td><td> 305 </td><td> 74 </td><td> 14 </td><td>轻度至中度发热，脑膜炎</td><td> 8 </td></tr><tr><td>波托西病毒</td><td> Bunyaviridae </td><td> 259 </td><td> 76 </td><td> 4 </td><td>不知道</td><td> 3 </td></tr><tr><td>缓存谷病毒</td><td> Bunyaviridae </td><td> 114 </td><td> 51 </td><td> 8 </td><td>轻度至严重，发烧，在两个有案可查的案件，神经系统疾病</td><td></td></tr><tr><td> Trivittatus病毒</td><td> Bunyaviridae </td><td> 83 </td><td> 21 </td><td> 11 </td><td>不知道</td><td></td></tr><tr><td>拉克罗斯病毒</td><td> Bunyaviridae </td><td> 1 </td><td> 1 </td><td> 1 </td><td>中度至严重，脑炎</td><td> 4 </td></tr><tr><td>佛兰德病毒</td><td> Rhabdoviridae </td><td> 4 </td><td> 4 </td><td> 2 </td><td>不知道</td><td></td></tr></tbody></table> 表1病毒检测Vero细胞培养法在现场收集蚊子。蚊子在康涅狄格州监察从1997年至2010年期间收集。

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Vero细胞培养法作为一种有效的方法现场收集的病毒的多样性屏幕蚊子。这种专门针对一个或几个病毒的分子生物学方法的对比。此外，我们发现Vero细胞培养法，同样如果不是更多的7比RT - PCR检测的敏感，并为我们提供了为今后的研究中，储存在我们的参考收集的病毒株。然而，细胞培养系统可能无法在许多情况下，适当的。这种做法会带来额外的费用和培训，要求在BSL - 3实验室中生长?...

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>产品编号</th></tr><tr><td>胎牛血清</td><td> GIBCO - Invitrogen公司</td><td> 16140 </td></tr><tr><td> Anti-biotic/mycotic </td><td> GIBCO - Invitrogen公司</td><td> 15240 </td></tr><tr><td>碳酸氢钠碳酸氢钠，7.5％Soln。 </td><td> GIBCO - Invitrogen公司</td><td> 25080 </td></tr><tr><td> L -谷氨酰胺200MM 100X </td><td> GIBCO，Invitrogen公司</td><td> 25030 </td></tr><tr><td>粉最小必需培养基（MEM） </td><td> GIBCO - Invitrogen公司</td><td> 11700 </td></tr><tr><td> 10X贝科的PBS </td><td> GIBCO - Invitrogen公司</td><td> 14080 </td></tr><tr><td>胰蛋白酶EDTA </td><td> GIBCO - Invitrogen公司</td><td> 15400 </td></tr><tr><td>兔血清</td><td> GIBCO - Invitrogen公司</td><td> 16120 </td></tr><tr><td> Vero细胞（克隆E6） </td><td> ATCC </td><td> CRL - 1586 </td></tr><tr><td>小组织培养瓶中，通风帽，25厘米2 </td><td>猎鹰 - 碧迪</td><td> 353112 </td></tr><tr><td>大型组织培养瓶，通风帽，175厘米2 </td><td>猎鹰 - 碧迪</td><td> 353108 </td></tr><tr><td>镀铜BB的，4.5毫米</td><td> Crosman </td><td> 0767 </td></tr><tr><td>调音台轧机</td><td>瑞驰</td><td> MM300 </td></tr><tr><td> Isopack冷冻齿条</td><td> Eppendorf公司</td><td> 022510240 </td></tr></tbody></table>

detection of infectious virus from field-collected mosquitoes by vero cell culture assay

蚊子都传播了不同的病毒，包括重要的人类病原体，如西尼罗河病毒，登革热病毒，和chickungunya病毒的数量。许多这些病毒已加强在其流行范围扩大到新界，需要有效的监测和控制方案，以应对这些威胁。监控病毒活动的一个战略涉及从流行网站收集大量的蚊子和测试病毒感染。在这篇文章中，我们描述了如何处理的过程中，屏幕现场收集Vero细胞培养法对传染性病毒的蚊子。由陷阱的位置和物种的蚊子进行排序，并归纳为含≤50个人的池。汇集标本匀浆缓冲使用混频器厂和水相融合Vero细胞培养（克隆E6）到接种生理​​盐水。细胞培养监测3-7后接种在细胞培养中生长的任何病毒是通过适当的诊断方法确定天的细胞病变效应。利用这种方法，我们已经分离出9种不同的病毒，在美国康涅狄格州收集蚊子，其中，5个是已知的导致人类疾病。这三种病毒（波托西病毒，西尼罗河病毒，和拉克罗斯病毒）代表北美新英格兰地区自1999年以来的新纪录。能够检测到的病毒的广泛的多样性是至关重要的监测建立和新出现的病毒在蚊子种群。

Watch this Scientific Journal Video about Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay at JoVE.com

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

我们描述了一个方法来处理和屏幕现场收集到的蚊子的病毒Vero细胞培养法的多样性。通过采用这种技术的，我们已检测到4个分类家庭在康涅狄格州收集蚊子的9种不同的病毒。

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 ...

detection-infectious-virus-from-field-collected-mosquitoes-vero-cell

Research

JoVE Journal

Immunology and Infection

18.3K 次观看.  The Connecticut Agricultural Experiment Station. 我们描述了一个方法来处理和屏幕现场收集到的蚊子的病毒Vero细胞培养法的多样性。通过采用这种技术的，我们已检测到4个分类家庭在康涅狄格州收集蚊子的9种不同的病毒。

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

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

顺行运输Alphaherpes病毒传播的活细胞成像

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

科研

免疫与感染

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

类器官的模型传染病：人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

兹卡病毒感染细胞培养系统和<em&gt;体外</em&gt;干扰素的预防作用

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

人血清抗水药物蛋白-4 免疫球蛋白 g 的细胞检测

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

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.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

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

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...