这项工作是由赠款5号U54通过西方优秀的区域中心（WRCE）AI057156 - 07，R01从国家过敏和传染病研究所AI08764301 - A1，西利疫苗开发中心，在大学内部资金德克萨斯州医学科。

1。 2质粒DNA的重组的MP - 12编码NSS突变（S）的回收<ol><li>传播幼仓鼠肾（BHK）/ T7 - 15 9细胞，稳定表达T7 RNA聚合酶，到6厘米的菜最起码的基本介质中（MEM）-α（Invitrogen公司，CAT＃32561037），含10％胎牛血清（ FBS ），青霉素，链霉素（青霉素100 U /毫升，链霉素100微克/毫升）（Invitrogen公司，CAT＃15140122），和600微克/ ml的潮霉素B（Cellgro，猫＃30 - 240 - CR）。 *病毒回收效率在6厘米的菜高出35 mm培养皿中。 BHK/T7-9细胞，通过水平低的支持率较高的恢复。另外，其他的BHK细胞株稳定表达T7 RNA聚合酶可以使用4,5,16,17。 </li><li>当细胞达到70％-80％汇合，更换培养上清用新鲜的含10％胎牛血清和青霉素，链霉素（不含有潮霉素B）的MEM -α。 *细胞应该是transfected内1小时后更换介质，以避免损失的T7 RNA聚合酶表达。 </li><li>对于RVFV，编码病毒基因组RNA的全长的病毒RNA表达质粒，恢复和病毒基因的开放阅读框，编码病毒蛋白表达（ 图1和2）第二组是必需的。准备在1.5毫升管以下plasmids2（ 图2）的混合物： <ol><li> pProT7 - S与NSS基因突变（2毫克）（S）（+）：这种质粒编码的抗病毒感（正面意义）全长RVFV MP - 12的S段两侧由T7启动子和丁型肝炎病毒（HDV）核酶序列。 </li><li> pProT7 - M（+）（2毫克）：这种质粒编码的抗病毒感（积极意义）全长RVFV MP - 12米段两侧由T7启动子和HDV核酶序列。 </li><li> pProT7 - L（+）（2毫克）：这种质粒编码的抗病毒感（积极的意义上）全长RVFV MP - 12 L型段FLAnked由T7启动子和HDV核酶序列。 </li><li> PT7 - IRES - VN（2毫克）：这种质粒编码的RVFV MP - 12 N打开阅读框（ORF），T7启动子和脑心肌炎病毒（EMCV）内部核糖体进入位点（IRES）的下游。 </li><li> PT7 - IRES - VL（1毫克）：这种质粒编码的RVFV MP - 12大号的ORF下游的T7启动子和EMCV IRES的。 </li><li> pCAGGS - VG（1毫克）：这种质粒编码RVFV MP - 12的M ORF的鸡β-肌动蛋白启动子的下游。 PT7 - IRES - VN，PT7 - IRES - VL和pCAGGS - VG此外没有恢复MP - 12所必需的，但它增强了救援效率2。作者通过PT7 - IRES质粒，可能是由于缺乏核糖体AUGs漏水扫描经历GN / GC不善表达。因此，我们构建了第依赖GN / GC表达pCAGGS - VG。 </li></ol></li><li>添加30毫升过境LT1（Mirus，CAT＃MIR2300）385毫升OPTI - MEM（InvitrogeN，CAT＃31985070）在1.5 ml管，涡简要。 </li><li>在室温下5分钟孵化后，慢慢加入OPTI - MEM包含从步骤1.3脂质体的质粒混合，混合轻轻吹打，并培育在室温为15分钟。 </li><li>步骤1.2一滴一滴地（ 图3）BHK/T7-9细胞的培养基中添加脂质体和质粒混合物。 </li><li>在孵化器孵育转染细胞在37 ° C，5％CO 2 24小时，并更换培养上清新鲜MEM -α含10％胎牛血清和青霉素，链霉素（不含有潮霉素B ）。 </li><li>细胞在37 °的孵化器中的C与5％CO 2为4天（共5天孵化），并收集到15毫升管培养上清液。 *这里观察细胞病变效应（CPE），并不一定反映病毒成功复苏的结果，因为转诱导细胞死亡，出现类似病毒RNA复制或病毒蛋白的合成引起的CPE。 </li><li>在4 ° C 5分钟，在2200 XG离心的上清液。 *这一步的目的是沉淀下来的细胞碎片病毒股票。气溶胶紧离心桶建议以提高安全性。 </li><li>转移到螺丝帽的5毫升cryotubes的上清液，存储通道0（P0），于-80 ° C为进一步利用病毒的股票。 </li></ol> 2。扩增病毒的P0 <ol><li>在P0样品往往含有病毒滴度不足2下游实验。在VeroE6细胞扩增步骤，这是一个克隆的非洲绿猴肾细胞（VERO）缺乏IFN-alpha/beta基因18,19，增加病毒滴度最高水平。另外，其他细胞缺乏IFNAR1敲除小鼠21 migh如20 Hec1B细胞或MEF细胞类型，我干扰素反应T是用于这一步。蔓延到贝科的修改最低必不可少的培养基（DMEM）（Invitrogen公司，CAT＃11965092）10厘米含10％胎牛血清菜VeroE6细胞，青霉素，链霉素（青霉素：100单位/毫升，链霉素：100毫克/毫升），和孵化在37 ° C孵化器与5％的CO 2，直到他们达到80％汇合。 *重组MP - 12菌株编码突变NSS往往不能有效的I型干扰素主管细胞复制。 </li><li> 2.7毫升10％胎牛血清和青霉素链霉素的DMEM混合300毫升的P0样本。删除步骤2.1 VeroE6细胞培养基和稀释P0样品更换。在37 ° C的一个孵化1小时，5％CO2孵育。 </li><li>取出接种10毫升的DMEM添加10％胎牛血清和青霉素链霉素每道菜。 </li><li>在37 ° C孵育3〜4天，直到VeroE6细胞CPE明显。 *单层中断期间发生的MP - 12感染，whilË重组MP - 12缺乏NSS如rMP12 C13type（C13type）（ 图4），不破坏单 ​​层，但浮动细胞死亡人数出现在感染后2至3天。 </li><li>收获上清，在3至4 DPI节1.9）和1.10所述），并指定为E6P1样品。 </li></ol> 3。滴定斑块检测重组MP - 12 <ol><li> VeroE6细胞扩散到6孔板。 *每个样品的重复分析比单个分析更可靠。 </li><li>当VeroE6细胞生长至80％汇合，准备与10％FBS和10-6青霉素链霉素的DMEM 10倍系列稀释的病毒样本如下： <ul><li> 10μL的样品E6P1 + 990毫升，用10％胎牛血清和青霉素链霉素的DMEM（10-2稀释） </li><li> 10 -2样品100μL+ 900毫升的DMEM与10％胎牛血清和青霉素，链霉素（10 -3稀释） </li><li> 100μL的10 -3样品+ 900毫升的DMEM与10％胎牛血清和青霉素，链霉素（10 -4稀释） </li><li> 10-4样本100μL+ 900毫升与10％胎牛血清和青霉素链霉素的DMEM（10-5稀释） </li></ul></li><li>吸步骤3.1 6孔板中并添加每个稀释液（步骤3.2），400入井（ 图3） 。 </li><li>在37 ° C的一个孵化1小时，5％CO2孵育。 </li><li>在潜伏期，准备如下的琼脂覆盖两个15毫升管： 管（保持在42℃水浴）：7毫升1.2％的崇高琼脂水（VWR，猫＃101170-362） 管B（保持在37℃水浴）：7毫升改良Eagle培养基（MEM 2X）（Invitrogen公司，CAT＃11935046），含10％胎牛血清，青霉素，链霉素（青霉素100 U /毫升，链霉素100微克/毫升），和10％Tryptose磷酸盐肉汤（MP生物医学，CAT＃1682149）。 </li><li>孵育1 h后，删除病毒接种，并立即加入2毫升，每口井的管和管B（步骤3.5）1:1混合。 *小心添加覆盖，立即干燥井导致未受感染的细胞死亡。 </li><li>板块与5％的CO 2孵育3天的孵化器在37 ° C。 </li><li>准备管和管B再次在步骤3.5中所述。还准备500微升0.33％中性红的解决方案，每盘这也是保持在37℃水浴（西格玛Aldrich公司，CAT＃N2889 - 100ML）。 </li><li>混合管，管B和中性红溶液500毫升（终浓度为0.011％），加2毫升每口井的混合物。 *要添加的中性红溶液的量的变化很多中性红溶液，是必需的初步优化。 0.33％中性红溶液长期储存会导致沉淀。在这种情况下，可完全溶解沉淀，孵育在55 ° C为10分钟剧烈摇晃。使用沉淀中性ř弱阳性细胞ED解决方案的结果，而重新溶解中性红溶液污渍细胞以及。 </li><li>板孵育16小时（或过夜）37 ° C的一个孵化器5％的CO 2。 </li><li>统计斑块的数量，以及其中包含的10％到100％以及斑块。计算斑块的形成单位/ ml。例如，如果我们观察接种10-5稀释，28（＃斑块）×（1 ml/0.4毫升）× 10 5（稀释）= 7.0 × 10 6斑形成单位（PFU）的井28斑块/毫升（ 图5）。 </li></ol> 4。 NSS突变体的筛选缺乏I型干扰素抑制功能<ol><li>传播C57/WT MEF细胞（InvivoGen，CAT＃MEF - c57wt），编码一种分泌胚胎碱性磷酸酶（SEAP）基因诱导的NF - kB和IRF - 3 / 7（ 图6），到12孔板。 DMEM培养细胞均保持在10％胎牛血清，青霉素，链霉素（Penicillin：100单位/毫升，链霉素100微克/毫升），稻瘟素S（3毫克/毫升），和Zeocin（100微克/毫升）。 </li><li>当细胞成为融合（80％），细胞模拟感染或感染与MP - 12或重组MP - 12编码的多重感染（MOI）3或0.1（见第2和第3，金额NSS突变每接种300μL）。在感染后1小时，取出接种，并添加1毫升含10％胎牛血清，青霉素，链霉素（青霉素100 U /毫升，链霉素100微克/毫升）每口井的DMEM（稻瘟素和Zeocin不添加在这个时间）。 </li><li>在感染后14小时，收集培养上清。添加200μL定量蓝（InvivoGen，猫＃REP - QB1），每个样品50μL（一式三份），一个96孔板井。密封板在37 ° C孵育1小时 * MP - 12和重组的MP - 12缺乏NSS清楚地诱导主机包括293细胞，MRC - 5细胞和鼠标embr IFN-alpha/beta胜任细胞的翻译抑制yonic成纤维细胞（MEF）的14个小时后，在高MOI感染后的细胞。另一方面，IFN -βmRNA或ISG56 mRNA的大量积累在7至8小时后感染的类型，我干扰素胜任细胞。因此，我们选择了感染14小时后收集上清，看到的先天免疫反应诱导SEAP的积累。 </li><li>阅读使用酶标仪（ 图7）在650 nm处的OD值。 *北使用特定的鼠标ISG56基因ISG56 NSS表达的情况下（ 图 8）的mRNA的上调，这表明RNA探针的杂交获得的数据相一致的结果。 应该指出，SEAP的活动是由丰富的蛋白质可以通过主机翻译活动的影响。的mRNA水平的提高相比，一个SEAP的相对水平可能不高，因为SEAP不能SEAP基因的存在，即使在合成细胞翻译suppressed。 Northern杂交是一种更直接的检测和更准确地评估在感染的细胞比SEAP报告基因检测NSS功能的缺乏引起的mRNA量。然而，SEAP报告系统比Northern杂交更迅速，因此NSS可能缺乏主机转录抑制功能的突变体的快速筛选有用。 </li></ol> 5。代表性的成果： 反向遗传学系统不断生成可行的重组MP - 12病毒滴度大于1 × 10 6 PFU / ml的高。 C13type病毒缺乏NSS的功能形成了大量的混浊斑，而MP - 12明确斑块形成各种大小的2（图5）。模拟感染C57/WT MEF细胞或MP - 12的感染者并没有增加SEAP的水平相比，模拟病毒感染的细胞培养上清，而与C13type C57/WT MEF的感染细胞的培养上清中增加SEAP的水平，14小时后感染（HPI）（图7）。这些结果与北使用特定的鼠标ISG56 mRNA表达（图8）RNA探针的杂交获得的一致。 <img src="/files/ftp_upload/3400/3400fig1.jpg" alt="图1"/> 图1。RVFV的基因组结构 RVFV有三方负感或ambisense RNA基因组命名为S，M -和L -段。分部的S - N和NSS基因编码ambisense方式。从病毒感（负义）段的S - ñ mRNA的合成，而mRNA是合成抗病毒感（积极意义）分部的S - NSS。分部的M -编码一个M mRNA和合成5&#39;region他们合作翻译裂解22,23 M的mRNA的几个AUGs漏水扫描the78kD，NSM，GN或GC蛋白。分部L - L蛋白编码。 N和L蛋白是病毒的转录和复制所必需的，而Gn和GC病毒包膜蛋白。 NSS和NSM蛋白非结构蛋白，这是不纳入到病毒颗粒。 <img src="/files/ftp_upload/3400/3400fig2.jpg" alt="图2"/> 图2。质粒DNA重组RVFV MP - 12菌株的恢复设计 全长cDNA的反病毒检测的S，M或L段cloneddownstream T7启动子和丁型肝炎病毒（HDV）上游核酶序列，指定为pProT7 - S（+），pProT7 - M （+）或pProT7 - L（+）分别为2。 BHK/T7-9细胞中表达T7 RNA聚合酶转录的RNA编码全长S，M，或精确的基因组3&#39;末端L型部分。 N或L蛋白的开放阅读框（ORF）克隆脑心肌炎病毒（EMCV）内部核糖体进入位点（IRES），这是PT7 - IRES - VN或PT7 - IRES - VL，分别指定，允许不封顶T7 RNA转录在限额，独立的核糖体被认可凹痕的方式。的M的ORF克隆下鸡β-肌动蛋白pCAGGS质粒24，这是pCAGGS - VG指定的推动者，让78kD的合成，NSM，Gn和GC蛋白，这是从不同AUGs产生漏水扫描23。 N和L蛋白启动转录或RNA的复制，而PT7 - IRES - VN和PT7 - IRES - VL没有必要重组的MP - 12 2的恢复，可能是由于presumable的渗漏表达POL二，驱动皑皑的RNA转录编码的N - ORF和L - ORF的pProT7 - S（+）和pProT7 - L（+），分别。 <img src="/files/ftp_upload/3400/3400fig3.jpg" alt="图3"/> 图3 RVFV MP - 12质粒DNA的回收 。 pProT7 - S（+），pProT7 - M（+）pProT7 - L（+），PT7 - IRES - VN，PT7 - IRES - VL和pCAGGS - VG质粒（图BHK/T7-9细胞转染）生成传染性重组RVFV MP - 12培养上清中的应变第在5天的后转上清收集，到新鲜VERO E6细胞病毒扩增传代。通常情况下，超过1 × 10 6 PFU / ml的病毒在感染后3〜4天可以恢复。扩增的病毒（E6P1病毒）滴定使用Vero E6细胞斑块检测和表型分析和免疫原性研究。 <img src="/files/ftp_upload/3400/3400fig4.jpg" alt="图4"/> 图4段的S - MP - 12和rMP12 - C13type MP - 12的比较和rMP12 C13type（C13type）的S -段。 C13type NSS的ORF与MP - 12菌株NSS相比，被截断了69％，是相同的自然隔离克隆13 株 2,25 。 <img src="/files/ftp_upload/3400/3400fig5.jpg" alt="图5"/> 图5牌匾MP - 12（功能NSS）和C13type（非功能NSS）检测。 单层的Vero Ë6细胞在6孔板是用于检测斑块。经过3天用0.6％的琼脂覆盖，第二琼脂覆盖含有中性红溶液孵化的是。然后，感染后第4天计算斑块。 MP - 12的形式明确不同大小的斑块，而C13type形式大混浊斑块（或灶）。以及10至100斑块应用于计数。 <img src="/files/ftp_upload/3400/3400fig6.jpg" alt="图6"/> 图6的分泌型碱性磷酸酶（SEAP）记者在C57/WT MEF细胞的基因激活途径 干扰素-β启动子包括AP - 1，NF - kB和IRF - 3的结合序列。 RVFV复制激活AP - 1，NF - kB和IRF - 3 7,11。然而，抑制IFN -β启动NSS释放阻遏复杂，即使在这些转录因子的结合，从而抑制IFN -βmRNA的26的合成。此外，NSS隔绝TFIIH P44 subunits8和LSO促进TFIIH P62亚基27的降解，从而诱导普通主机的转录抑制，包括IFN -α基因和基因ISRE子下。 IFN -β的抑制功能的C13type或其他缺乏NSS突变体诱导IFN -β的合成，这反过来又激活IFN -α启动子和干扰素敏感反应元件（ISRE）发起人。 IRF - 7，然后IFN-alpha/beta刺激和支持IRF - 3 28,29的进一步上调IFN -α转录上调。在这个实验中，C57/WT MEF细胞分泌碱性磷酸酶（SEAP）编码在一个人工NF - kB的，IRF - 3和IRF - 7的结合序列下游。因此，NSS突变体，缺乏IFN -β的抑制功能上调SEAP的分泌，然后测量。 <img src="/files/ftp_upload/3400/3400fig7.jpg" alt="图7"/> 图7由C13type SEAP的感应。 C57/WT MEF细胞（InvivoGen）分别模拟感染或感染与MP - 12或rMP12 C13type（C13type）3（左侧面板）或0.01（右图）MOI。培养上清14 HPI（50μL）与200μL定量蓝（InvivoGen）在96孔板和650 nm处的OD值基板混合后测定1 h后在37 °彗星由酶标仪。 SEAP的模拟病毒感染的细胞相对增加。这些数据代表的平均+ / - 3次独立实验的标准偏差。从C13type感染的细胞培养上清显示SEAP的增加，表明主机转录抑制NSS缺乏。 <img src="/files/ftp_upload/3400/3400fig8.jpg" alt="图8"/> 与高辛标记的RNA探针Northern杂交图8。 野生型小鼠胚胎成纤维细胞（MEF）的模拟感染或感染与MP - 12或rMP12 C13type（C13type）MOI 3。总RNA用Trizol（Invitrogen公司），和北b 7 HPI大量使用地高辛标记的RNA探针的具体小鼠内源性ISG56 mRNA或MP - 12 N基因/抗病毒检测的S 段 2,30 。为了使小鼠内源性ISG56 mRNA或RVFV ñ基因/抗病毒检测的S段的探针，引物的PCR扩增片段KpnmISG56F（GGG TGG TAC总部大楼台泥法案TTC AGA海合会TTC GCA AAG CAG）和HindmISG56 （TAC AAA级GCT达GGG AGA GAA TGC TGA TGG TGA文建会GG）为ISG56 mRNA或KpnNF（AGT TGG TAC猫GGA民航局注册税务师TCA AGA GCT TGC摹）和HindNR RVFV（GGG民航局GCT TTT AGG CTG C​​TG的大老山隧道的TGT AAG） ñ基因/抗病毒检测的S段消化用 KpnⅠ 和 HindⅢ，结扎成pSPT18质粒（Roche.然后RNA与地高辛标记的探针合成，使用辛RNA标记试剂盒（SP6/T7）（罗氏猫＃1 175 025），每个样品的28S rRNA的水平也显示装载控制。野生型MEF的C13type诱导ISG56 mRNA合成受感染的细胞，而那些与MP感染-12没有诱导，暗示缺乏主机在C13type感染细胞的转录抑制。图6 SEAP检测所获得的数据是一致。

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我们什么都没有透露。

为RVFV反向遗传学系统已经开发了几个小组利用T7启动子2,4,5或鼠标3人4 POL -我子。在这个手稿中，我们描述了一个协议， 使用15个稳定表达T7 RNA聚合酶BHK/T7-9细胞产生重组RVFV MP - 12菌株。不同的病毒回收效率取决于BHK/T7-9细胞的条件，质粒量，转染细胞和数量等。我们总是放大的P0病毒在Vero E6细胞，获得高滴度实验的病毒储存。 I型干扰素主管的细胞，如人肺二倍体?...

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>最低限度的基本培养基（MEM -α） </td><td> Invitrogen公司</td><td> 32561037 </td><td></td></tr><tr><td>贝科的修改最低限度的基本培养基</td><td> Invitrogen公司</td><td> 11965092 </td><td></td></tr><tr><td>改良Eagle培养基（MEM 2X） </td><td> Invitrogen公司</td><td> 11935046 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15140122 </td><td></td></tr><tr><td>潮霉素B </td><td> Cellgro </td><td> 30 - 240 - CR </td><td></td></tr><tr><td> Tryptose磷酸盐肉汤</td><td> MP生物医学</td><td> 1682149 </td><td></td></tr><tr><td>高贵的琼脂</td><td> VWR </td><td> 101170-362 </td><td></td></tr><tr><td>中转- LT1 </td><td> Mirus </td><td> MIR2300</td><td></td></tr><tr><td> OPTI - MEM </td><td> Invitrogen公司</td><td> 31985070 </td><td></td></tr><tr><td>气溶胶紧盖子</td><td> Eppendorf公司</td><td> C - 2223 - 25 </td><td></td></tr><tr><td> 0.33％的中性红溶液</td><td>西格玛爱秩序</td><td> N2889 - 100ML </td><td></td></tr><tr><td> C57/WT MEF细胞</td><td> InvivoGen </td><td> MEF - c57wt </td><td></td></tr><tr><td>稻瘟素小号</td><td> InvivoGen </td><td> ANT - BL - 1 </td><td></td></tr><tr><td> Zeocin </td><td> InvivoGen </td><td> ANT - ZN - 1 </td><td></td></tr><tr><td>定量蓝</td><td> InvivoGen </td><td> REP - QB1 </td><td></td></tr><tr><td> BHK/T7-9细胞15 </td><td>日本岐阜大学， </td><td></td><td></td></tr><tr><td> Vero E6细胞</td><td> ATCC </td><td> CRL - 1586 </td><td></td></tr></table>

using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy

裂谷热病毒（RVFV），从而导致出血热，神经系统疾病，或在人类中的盲目性，以及高利率流产和反刍动物的胎儿畸形1，已被列为作为HHS /美国农业部重叠选择代理和风险组3病原体。它属于在家庭Bunyaviridae属Phlebovirus，是这个家庭的最致命的成员之一。自2006年以来已开发的RVFV MP - 12疫苗株2,3以及野生型RVFV 4-6株，包括ZH548和ZH501，几个反向遗传学系统。 MP - 12株（这是一个危险群2病原体和非选择代理）是由几个突变，其M -和L段高度减毒，但仍带有剧毒分部的S - 3，其编码的RNA功能的毒力因素，NSS。 rMP12 C13type（C13type）进行帧删除NSS的ORF 69％缺乏所有已知的NSS功能，而它复制为efficient作为MP - 12在VeroE6细胞缺乏I型干扰素。 NSS包括干扰素（IFN）-βmRNA的7,8诱导的转录主机关闭，并促进在翻译后水平降解双链RNA依赖的蛋白激酶（PKR）9,10 IFN -β转录干扰素调节因子3（IRF - 3），NF - kB和激活蛋白-1（AP - 1），IFN -β结合IFN-alpha/beta受体（IFNAR）上调刺激IFN -α的转录虽然IRF - 3，NF - kB和激活的基因或其他干扰素刺激基因（ISGs）11，诱导宿主的抗病毒活性，而主机包括NSS IFN -β基因的转录抑制阻止病毒复制的基因upregulations这些ISGs蛋白1（AP - 1），可以通过RVFV7激活。 。因此，NSS是一个很好的的目标，以进一步削弱MP - 12，并取消IFN -β的抑制功能，以提高主机的先天免疫反应。这里，我们描述了产生重组MP - 12的编码突变NSS的协议，并提供一种检查方法，找出缺乏的功能，抑制IFN -βmRNA的合成NSS突变体的一个例子。除了 ​​其在先天免疫系统的重要作用，I型干扰素是重要的树突状细胞诱导适应性免疫反应 12-14的成熟。因此，NSS突变体诱导I型干扰素的进一步减弱，但在同一时间在刺激宿主的免疫反应更有效率比野生型MP - 12，这使得它们接种疫苗的方法理想人选。

Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

裂谷热病毒的MP - 12疫苗株反向遗传学系统是一个有用的工具，用于创建附加的MP - 12突变体具有更高的衰减性和免疫原性。我们描述了协议的产生和表征NSS突变株。

利用反向遗传学操作的裂谷热病毒MP - 12菌株，以提高疫苗的安全性和有效性的NSS基因

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

using-reverse-genetics-to-manipulate-nss-gene-rift-valley-fever-virus

Research

JoVE Journal

Immunology and Infection

17.2K 次观看.  University of Texas Medical Branch. 裂谷热病毒的MP - 12疫苗株反向遗传学系统是一个有用的工具，用于创建附加的MP - 12突变体具有更高的衰减性和免疫原性。我们描述了协议的产生和表征NSS突变株。

视频: Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

反向遗传学感染小鼠诺沃克病毒介导的恢复

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

科研

免疫与感染

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

应用系统进化分析研究真核基因的起源

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...