作者想感谢的阿道夫加西亚 - 萨斯特雷和Peter Palese实验室为流感反向遗传学技术和质粒发展的过去和现在的成员。在AG - S的实验室研究的部分资金由CRIP，卓越NIAID资助流感研究和监测中心（HHSN266200700010C）NIAD补助R01AI046954，U01AI070469和P01AI058113。 LM - S的实验室的研究经费部分由NIAID的赠款RO1AI077719。

1。流感病毒救援转 A型流感病毒属于负链RNA包膜病毒的 Orthomyxoviridae家庭。 A型流感病毒的基因组由8个不同的RNA负极的基因编码，至少11病毒蛋白（图1） 4。在这份报告中，我们将重点抢救其中一个最常见的实验室菌株，使用含8流感病毒A/PR/8/34段ambisense质粒（PDZ）流感A/PR/8/34，5 （图2）。 对于抢救质粒DNA的重组流感病毒，我们建议3个独立的转染各重组病毒。如果一个以上的重组病毒救援尝试，规模以下步骤accordantly病毒的数量，被救出。下面的转染和感染协议是建立6孔板。该协议的一个示意图如图3所示。 <ol><li> OptiMEM脂质体2000（LPF2000）混合物：准备OptiMEM媒体和6-8 LPF2000每转液250μL。孵育5-10分钟，在室温（RT）。同时，准备质粒转染混合物。 </li><li>质粒转染混合物：准备在50μLOptiMEM媒体的质粒转染的鸡尾酒。我们通常使用1微克每个流感DNA质粒每救援。 PDZ质粒1μL（1微克/微升）PB2，PB1，PA，HA，NP，NA，M，和NS添加到管内含有50μLOptiMEM媒体。 </li><li> OptiMEM LPF2000 - DNA质粒混合物：步骤1.1添加到流感DNA质粒转染混合物（步骤1.2）250μL。孵育这种混合物在室温下20-30分钟。同时，准备转染293T和MDCK细胞悬浮液。 </li><li> 293T/MDCK共培养制备方法：在开始之前，带来的PBS 1X DMEM培养液10％胎牛血清的1％PS媒体，EDTA -胰蛋白酶的混合物至37 ° C。细胞的密度应在80％-90％汇合转染的一天。通常，一个融合100毫米293T和MDCK细胞融合100毫米盘菜可用于10-12抢救。我们将要使用的细胞，每孔250μL。这两种细胞株，将3 ml DMEM培养液10％胎牛血清的1％PS中的总悬浮在一个。 <ul><li>在15 mL离心管中，小心地重新悬浮在10毫升的DMEM 10％胎牛血清的1％PS的每个细胞株。你将拥有一个MDCK细胞293T细胞和单管的管。 </li><li>重悬在3毫升的DMEM 10％胎牛血清的1％PS 293T细胞重悬于时，提供3毫升MDCK细胞，这些细胞重悬。这会给你的使用您的合作文化293T和MDCK细胞的混合物。 </li><li>加入250μL每口井的293T/MDCK细胞（10-12 6井井）。 </li></ul></li><li> 20-30分钟RT潜伏期（1.3步）后，添加1毫升的DMEM 10％胎牛血清的1％PS OptiMEM - LPF2000流感DNA质粒混合物。 </li><li>入井293T/MDCK细胞250μL（步骤1.4）中加入1.3毫升（1.5步）。 </li><li>轻轻摇动6板，让转染孵育过夜（ON），在孵化器在37 ° C和5％的CO 2 。 </li><li>第二天，转染后约16-24小时，改变转染介质，并培育转染细胞48小时的PS含有1微克/毫升TPCK -胰蛋白酶的DMEM 1％，0.3％BA。 </li><li>不断变化的媒体48小时后，从转染细胞转移到离心管上清液。 </li><li>离心机在离心1-2分钟，13000转的组织培养上清。 </li><li>新鲜的MDCK细胞在6孔板（镀的前一天），或10日龄鸡胚步骤1.10离心组织培养上清液与200μL感染。在37细胞和/或蛋° C孵育2-3天。 <ol><li> 10日龄鸡胚感染的所有程序感染鸡鸡胚在无菌条件下进行。 <ol><li>蜡烛的10日龄蛋使用光烛盒看到空气囊和尿囊腔之间的接口。制作一个界面上的边界的铅笔标记。 </li><li>有了一个5毫升的注射器针头，使蛋壳中的孔。 </li><li>用1 ml注射器，每个鸡蛋200μL1.10步骤的组织培养上清感染。 </li><li>盖在融化的蜡，用棉签蛋壳的孔。 </li><li>在37οC培养2-3天，受感染的鸡蛋。 </li></ol></li><li>新鲜MDCK细胞的感染：通过前一天从组织培养上清的293T/MDCK共培养，预削减6盘菜，与MDCK细胞达到80％-90％汇合第二天。通常情况下，一个融合的100毫米的组织培养板，可分成6-8井。洗细胞两次，用PBS 1X，trypsinize，并准备在6孔板。用手轻轻摇动板才能有一个细胞的军装分布。培养的细胞，在37 ° C培养箱中培养，5％的CO 2。感染前，检查在显微镜下的细胞，以确认单层，然后，继续与感染： <ul><li>洗涤细胞两次，用1毫升的PBS 1X。 </li><li>在室温下1小时与200μL离心组织培养上清感染。不要让干细胞。岩石6板，每10分钟。 </li><li>病毒吸收1小时后，取出从MDCK细胞的感染媒体和添加2毫升的DMEM 0.3％BA 1％的PS含1微克/毫升TPCK -胰蛋白酶。 </li><li>通过后48-72小时，取决于转染效率和病毒载量，细胞病变效应（CPE）将在MDCK细胞感染的细胞中观察到。 CPE建议抢救成功。然而，房委会法（第2）应进行确认病毒在组织培养上清中存在。 </li></ul></li></ol></li><li>收获从被感染的鸡鸡胚尿囊液的所有程序，从被感染的鸡蛋收获尿囊液，无菌条件下进行。从每10日龄感染的鸡蛋，约8-12毫升的尿囊液可收获。之前收获的尿囊液，2个小时的鸡蛋（或）孵育4 ° C至杀死鸡胚胎和凝固的血。 <ul><li>用70％乙醇清洗蛋壳建立无菌条件。 </li><li>打开鸡蛋，仔细，在用勺子窃听的空气腔。与产钳的帮助下取出的碎蛋壳。 </li><li>用1毫升的针，不打破鸡蛋的蛋黄取出尿囊膜。 </li><li>稳定抹刀鸡胚你指导的尿囊液10毫升吸管。收集到尽可能多尿囊液15 mL离心管中，在不破坏或收集鸡蛋蛋黄中的任何一个冰桶冰。使用15 mL离心管中，每一个鸡蛋。 </li><li>离心5分钟，在4 ° C和尿囊液（没有考虑颗粒的红血细胞）转移到一个新的15 mL离心管。 </li><li>行管离心的尿囊液在4 ° C，直到他们获救病毒血凝（HA）检测与检查。 </li></ul></li></ol> 2。房委会的检测，以确认重组流感病毒救援血凝试验（HA）是经常用来检测获救病毒在MDCK细胞组织培养上清和/或尿囊液的收获鸡蛋的存在。另外，也可进行免疫荧光测定（IFA）的。一旦检测发现获救病毒的存在，病毒应斑块纯化病毒的遗传组成，将通过RT - PCR和测序证实。 MDCK组织培养上清和/或在从被感染的鸡蛋的尿囊液中的病毒可以宏观使用房委会的鸡（或其他来源），红细胞（RBC）的决定。病毒诱导的红细胞血凝而没有病毒的情况下允许在井底的红色颗粒（图4）的形成。在流感病毒的情况下，它是认为， 大约10 3 -10 4菌斑形成单位（PFU）需要一个积极的信号，在房委会检测，因此，可以进行独立财务顾问与房委会平行检测，以确认一个真正的负面结果。 IFA是主要的抗流感抗体比HA检测更敏感，因为少于103-104病毒可以用这种技术检测。它可能比上清或尿囊液，HA阴性阳性的IFA。在这种情况下，病毒应该由传代扩增，再次，在MDCK细胞或鸡蛋。尿囊液和/或从第二代的组织培养上清现在应该在房委会的分析显然是积极的。 医管局的检测进行了V型底96孔板。负的（例如，PBS 1X）和积极的（组织培养上清和/或流感病毒感染的尿囊液）控制​​样品应始终包含在任何HA实验来验证它。 <ul><li>免除到每个96孔板的V型底部以及50μL的PBS 1X。 </li><li>加入50μLMDCK细胞组织培养上清和/或尿囊液，从被感染的鸡蛋的第一口井，让下面的井的2倍连续稀释。丢弃从过去以及额外的50μL。 </li><li>添加0.5％-1.0％鸡红细胞（1X的PBS配制），每孔50μL。 </li><li>在冰上孵育底部的V - 96井板（30-45分钟，直到一个红点在PBS阴性对照样品的底部可见）。阅读和interpretate如在图4所示的结果。 </li></ul> 3。通道的组织培养上清液一个在房委会检测阴性结果可能是组织培养上清和/或尿囊液病毒在目前的水平低的转染效率低的结果。通过这些样本将允许在新鲜的MDCK细胞和/或鸡胚病毒的扩增（如图3所示）感染是因为以前在1.11.2节所述的执行。 4。代表性的成果积极的HA检测（图4）的存在将被证实成功的流感病毒救援。此外，在感染的细胞与组织培养上清或从尿囊液蛋中存在的CPE将建议一个积极的病毒救援。 <img src="/files/ftp_upload/2057/2057fig1.jpg" alt="图1" /> 图1。流感病毒的结构：流感病毒是含有两种病毒糖蛋白（HA，NA），脂质双层包围，离子通道蛋白，M2 。房委会是病毒附着蛋白，唾液酸结合含有受体。 NA是病毒从宿主细胞释放。下面的脂质双分子层，是一种蛋白质层，内表面信封基质蛋白1，货币供应量M1，它起着的作用，在病毒粒子的组装和出芽和核出口蛋白（NEP），病毒ribonucleocapsids的核出口所需组成。病毒的核心是由一个核糖核蛋白（RNP）复杂，组成8个单股负RNA病毒基因的病毒的核蛋白，NP encapsidated。 RNP的复杂的病毒RNA依赖的RNA聚合酶亚基的PA，PB1，和PB2。非结构蛋白NS1和PB1 - F2，NS和PB1的RNA片段，分别编码，是不是病毒粒子的结构的一部分。 <img src="/files/ftp_upload/2057/2057fig2.jpg" alt="图2" /> 图2。流感病毒拯救质粒：8个流感病毒基因的克隆到质粒PDZ ambisense表示。 PDZ质粒6，从蛋白的表达质粒pCAGGs 7派生，是一个双向的，人类的RNA聚合酶启动子和鼠标终止序列，编码的负面意义上的基因组RNA的质粒载体;在相反的方向，我团结的聚合酶，聚合酶II转录卡带（鸡β-肌动蛋白启动子和多聚腺苷酸）从相同的病毒基因编码的病毒蛋白。每个病毒段全长cDNA生成包含SAPI的限制性内切酶位点和各段的非编码区（在病毒基因年底的黑盒子）的正向和反向引物通过RT - PCR。 PCR产物克隆到与SAP，我消化的PDZ。 <img src="/files/ftp_upload/2057/2057fig3.jpg" alt="图3" /> 图3。 8质粒为基础的流感救援系统：包含8个流感病毒基因的PDZ质粒共同转染，在悬挂在293T - MDCK细胞共培养（1天）。二十四小时转染后，媒体没有胎牛血清，但含有TPCK /胰蛋白酶被替换（2天）。四十八小时后，媒体的变迁，组织培养上清收获和用于感染MDCK细胞或10日龄鸡胚（4天）。放大后48-72小时，从感染MDCK细胞或卵尿囊液的组织培养上清的收获，检测病毒的存在HA（6天）。如果没有检测到病毒，相同的上清和/或尿囊液，可以重新传代到新鲜的MDCK细胞和/或鸡胚。 <img src="/files/ftp_upload/2057/2057fig4.jpg" alt="图4" /> 图4。血凝试验（HA）：红细胞血凝病毒粒子是肉眼可见，是基础，以组织培养上清或/和尿囊液中检测病毒颗粒。虽然不区分病毒颗粒，具有传染性和退化，不再能够感染细胞的颗粒，医管局检测，检测样品中病毒的存在是一个良好的指标。一）不存在病毒在生物样品（底部）（顶），是由红细胞的存在，在盘底或他们的缺席，RESPectively。 B）病毒没有检测水平（顶部）或存在病毒（底部）代表房委会的检测结果显示。

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西奈山医学院的重组流感病毒领域的知识产权，AG - S是在这个知识产权的发明者。

质粒DNA的重组流感病毒的救援是一个简单明了的过程，一旦该协议是经常在实验室中进行，但在开始的时候，多个事情都可能出错。生成的病毒，它必须有良好的质粒准备。正确保养的细胞株（293T和MDCK）是一个成功的病毒救援的关键。传统上，一个遗传标记是插入基因编码质粒流感，沉默突变。这种沉默突变（S）的简介和创作，例如，一个新的限制性内切酶站点是用来区分野生型和经酶切重组?...

细胞系 293T（目录号CRL - 11268）和MDCK（目录号CCL - 34）细胞株维持在37℃孵化器与5％CO2的DMEM 10％FBS，1％的PS 2。细胞是形成美国典型培养物保藏（ATCC 10801大学大道，马纳萨斯，弗吉尼亚州20110-2209美国）。 鸡胚查尔斯河实验室，特定病原体费禽流供应（SPAFAS）禽流感的产品和服务，可从10日龄的鸡胚的鸡蛋。富兰克林风景，106路线32，北富兰克林，CT 06254 USA。在37 ° C前和病毒感染后的鸡蛋孵育。在病毒感染后，鸡蛋candled，以确定胚胎的可行性。死蛋前后病毒感染，这是非常重要。感染前一个死蛋，可以很容易地发现通过血管的情况下，以及胚胎的流动性的情况下。当candled，活胚胎移动。死蛋（可能与流感病毒感染）病毒感染后会由尿囊液的体积较小和血腥的蛋坏的外观很容易被发现。受感染的鸡蛋被丢弃在双高压灭菌袋和蒸压以下的标准程序。 鸡红血细胞（红细胞） 鸡红细胞可以购买从Truslow农场，谷道，201 Chestertown，MD 21620。保存在4 ° C。对于HA测定，用45毫升的PBS 1X在50 mL离心管5毫升的鸡红细胞。 1000 RPM的，RT离心5分钟。小心倒掉上清，用1:1000稀释的1X PBS中沉淀的红细胞（终浓度为0.5-1.0％红细胞）。 组织培养上清和尿囊液可以储存在4 ° C，很短的时间内，组织培养上清和尿囊液。在确认病毒救援后，病毒从细胞培养上清或尿囊液储存在-80 ° C 质粒所有质粒准备用一个质粒马克西Kit中的下列制造商的建议。质粒浓度为1微克/毫升分装在DDH 2，储存在-20 ° C短期储存，质粒可以保持在4 ° C。纯化DNA质粒的浓度是由分光光度法在260 nm，纯度估计使用260:280 nm的比例。 1.8-2.0 260:280纳米比率的准备工作被认为是病毒救援目的的拨款。此外，质粒的浓度和纯度应确认与琼脂糖凝胶色谱。 Ambisense PDZ质粒（6），其中包含8个流感A/PR/8/34病毒基因（7）如图2所示。 病毒生物安全水平（BSL）2条件下可以进行挽救流感A/PR/8/34协议。污染物质，包括组织培养上清和鸡胚，处置前应消毒。救援其他流感病毒可能需要更高的BSL条件，因此，特殊条件/安全的测量，将需要遵循。 组织文化，媒体和解决方案 DMEM培养液10％胎牛血清的1％PS：445毫升贝科改良Eagle培养基（DMEM），胎牛血清（FBS），50毫升，100X青霉素/链霉素（PS）和5毫升。保存在4 ° C。此媒体将被用来保持293T和MDCK细胞的转染以及。的DMEM 0.3％BA 1％PS：495.7毫升的DMEM，35％的牛白蛋白（BA）的4.3毫升。保存在4 ° C。使用前，TPCK处理的胰蛋白酶添加到终浓度为1微克/毫升。传染病媒体。 10X磷酸盐缓冲液（PBS）：80克氯化钠，氯化钾2克，11.5克的Na 2 HPO 4 0.7 H 2 O 2克的KH 2 PO 4。添加DDH 2到1升。调节pH值至7.3。消毒高压灭菌。在室温下储存。 1X PBS稀释10X PBS 1:10 DDH 2 O。消毒的高压灭菌器和在室温下储存。

generation of recombinant influenza virus from plasmid dna

多项流感研究小组的努力已在A型流感病毒反向遗传学的发展和改善的关键。成立于1999年<sup&gt; 1,2</sup&gt;质粒为基础的反向遗传技术，以产生重组病毒已经彻底改变了流感研究领域，因为具体问题，通过基因工程，传染病，重组流感病毒已回答。这些研究包括病毒的复制，病毒蛋白的功能，特定的突变病毒蛋白在病毒复制和/或发病机制中的贡献，此外，使用病毒载体表达外源蛋白的重组流感病毒<sup&gt; 3</sup&gt;。

Watch this Scientific Journal Video about Generation of Recombinant Influenza Virus from Plasmid DNA at JoVE.com

Generation of Recombinant Influenza Virus from Plasmid DNA

质粒DNA的A型流感病毒的救援是一个基本的和必要的实验技术，使流感的研究人员，以产生重组病毒在流感病毒的生物学特性研究的多个方面，被视为潜在的载体或疫苗使用。

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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Research

JoVE Journal

Immunology and Infection

30.2K 次观看.  University of Rochester School of Medicine and Dentistry. 质粒DNA的A型流感病毒的救援是一个基本的和必要的实验技术，使流感的研究人员，以产生重组病毒在流感病毒的生物学特性研究的多个方面，被视为潜在的载体或疫苗使用。

视频: Generation of Recombinant Influenza Virus from Plasmid DNA

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

科研

免疫与感染

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...