作者要感谢克里斯汀·布拉德·费贝尔曼，米拉娜·维塞利诺维奇和克劳迪娅·鲁克特（ClaudiaRückert）的帮助，以表征克隆衍生的病毒。这项工作部分得到NIH国家过敏和传染病研究所赠款AI114675（BJG）和AI067380（GDE）的支持。

传染性克隆质粒的转化和恢复<ol><li> 使用商业转化方案（ 例如 ，NEB 5分钟转化协议）转化两种质粒（单独）并进行一些修改。两种质粒含有编码氨苄青霉素抗性的基因，因此使用氨苄青霉素或羧苄青霉素进行选择。碳青霉素是较为稳定的。 <ol><li>从-80°C冰箱中取出细胞（参见材料表），并在冰上解冻5-10分钟。在37℃下，预热溶血培养液（LB）（含有10g / L NaCl和25μg/ mL羧苄青霉素，称为LB-碳水化合物）板和具有分解代谢阻抑（SOC（无抗生素））的肉汤）。 </li><li>将1μL质粒DNA中的100pg-10ng加入含有50μL感受态细胞的管中;轻轻的轻轻捏合管子。 </li><li>在冰上孵育管5分钟。 </li><li>热冲击管在42°C 30 s在水浴中热休克后立即回冰2分钟。 </li><li>将950μL室温SOC吸入每个管中，并通过移液进行混合。将100μL细菌混合物扩散到LB-碳板上，并在37℃（大约12-14小时）下孵育过夜。 </li></ol></li><li>培养12-14小时后，从37℃培养箱中取出板。将板放置在室内黑暗的抽屉中，以使菌落继续生长（约8-9小时）。 </li><li>在细菌摇床（大约14-16小时）内，使用5mL的特殊肉汤（TB，含有25μg/ mL羧苄青霉素，称为TB-carb），每个质粒在30℃下生长5-10个小菌落。 注意:小菌落是质粒完整的指标;含有缺失，重排或突变（这里称为不稳定事件）的质粒的菌落将生长得更快，因此更大。因此，应该尝试挑选盘子上最小的殖民地，特别是没有选择最大的殖民地存在。也可以选择一些中等大小的殖民地来完成。所用的较低温度降低了细胞过度生长的可能性（OD 600大于0.6-0.8）。随着大多数研究人员进行夜间生长，它允许更多的控制和减少的过度增长的机会。 </li><li>检查液体培养物的浊度（大约OD 600为0.6-0.8）。将任何混浊的管放置在4°C，并允许其他管通过长时间生长变得浑浊。 注意:由于质粒不稳定事件可能发生，不要将细菌生长到OD 600值超过推荐值。 </li><li>用商业质粒微量稀释试剂盒（见材料表）从细菌中提取质粒DNA。在15μL预热（加热块中至70°C）洗脱缓冲液中洗脱。允许洗脱缓冲液在离心前在柱上孵育5分钟。 注意:在4℃保存至少1 mL此起始培养物进一步用于下一步。 </li><li>摘取10μL回收的质粒，以评估培养过程中是否发生质粒不稳定事件。 注意:用SalI / NheI和p2与BamHI / HindIII在37℃下将p1消化1小时。通过凝胶电泳确认消化后存在正确的条带（ 图2a ），并进入步骤2.如果通过滚环扩增（RCA）制备质粒DNA，请保留一些小批量洗脱液作为模板。 </li></ol> 2.用于连接的足够的质粒DNA的制备注意:连接和随后的电穿孔需要足够量的质粒DNA，必须通过maxiprep或RCA产生。虽然maxiprep是传统使用的方法，但RCA提供了不需要细菌的优点，从而消除了可能导致不稳定事件的细菌中质粒诱导的毒性的潜力。 <ol><li><strong&gt;执行以下maxiprep过程。 <ol><li>对于前一节中证明正确的限制酶消化模式的每个质粒，向1L TB-Carb（25μg/ mL羧苄青霉素）肉汤中加入500μL起始培养物，并在30℃（大约14-16小时） ，大约OD 600为0.6-0.8）。 注意:不要让文化成长超过OD 600值。这可能会导致质粒内的不稳定事件。 </li><li>收集细菌并按制造商的方案执行maxipreps（见材料表）。 </li></ol></li><li> 使用步骤1.6中保存的miniprep，对RCA过程执行以下操作。 <ol><li>将1μL质粒DNA转移到含有50μL样品缓冲液的管中。 </li><li>将样品在95℃下热变性3分钟，然后在4℃下孵育直到准备使用。 </li><li>准备商业放大预混料（参见材料表 ）。将50μL反应缓冲液与2μL酶混合物置于冰上的管中。 注意:放大预混物应保存在冰上，直到准备使用。通过组合足够的试剂进行所需数量的扩增反应来制备主混合物是方便的。任何未使用的预混物都必须被丢弃。 </li><li>将50μL制备的扩增预混物转移到变性样品中。 </li><li>将反应管在30℃孵育18小时。 </li><li>将管在65℃孵育10分钟以使ø29DNA聚合酶失活。 </li><li>将反应管储存在4°C或-20°C直到准备使用。 注意:此时，应通过两种方法制备的质粒DNA（使用260 nm处的吸光度），ZIKV基因组应完全进行Sanger测序，以确认在制备过程中不发生不稳定事件（缺失和重排）。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>入门名称</td><td>序列（5&#39; - 3&#39;） </td></tr><tr><td> ZIKV PRVABC59 1 For。 </td><td> AGTTGTTGATCTGTGTGAATCAGAC </td></tr><tr><td> ZIKV保护632For。 </td><td> GCCCTATGCTGGATGAGG </td></tr><tr><td> ZIKV保护692Rev。 </td><td> GGTTCCGTACACAACCCAAGTTG </td></tr><tr><td> ZIKV保存1313Rev。 </td><td> CTCCCTTTGCCAAAAAGTCCACA </td></tr><tr><td> ZIKV保护1201For。 </td><td> CCAACACAAGGTGAAGCCTAC </td></tr><tr><td> ZIKV保护1663For。 </td><td> GCAGACACCGGAACTCCACACT </td></tr><tr><td> ZIKV保护2008年。 </td><td> CAGATGGCGGTGGACATGC </td></tr><tr><td> ZIKV保存2350Rev。 </td><td> GTGAGAACCAGGACATTCCTCC </td></tr><tr><td> ZIKV保护2605For。 </td><td> TACAAGTACCATCCTGACTCCC </td></tr><tr><td> ZIKV Conserved 3499Rev。 </td><td>GCCTTATCTCCATTCCATACCA </td></tr><tr><td> ZIKV Conserved 3961Rev。 </td><td> TTGCCAACCAGGCCAAAG </td></tr><tr><td> ZIKV保护4132For。 </td><td> CATTTGTCATGGCCCTGG </td></tr><tr><td> ZIKV保护4561Rev。 </td><td> CTATTGGGTTCATGCCACAGAT </td></tr><tr><td> ZIKV Conserved 4665For。 </td><td> GACCACAGATGGAGTGTACAGAGT </td></tr><tr><td> ZIKV Conserved 5189Rev。 </td><td> AAGACAGTTAGCTGCTTCTTCTTCAG </td></tr><tr><td> ZIKV保存5219For。 </td><td> GAGAGTTCTTCCTGAAATAGTCCGTGA </td></tr><tr><td> ZIKV保守6086Rev。 </td><td> CTTGCTTCAAGCCAGTGTGC </td></tr><tr><td> ZIKV保护6119For。 </td><td> GCCTCATAGCCTCGCTCTATCG </td></tr><tr><td> ZIKV保护6721Rev。 </td><td> CCATTCCAAAGCCCATCTTCCC </td></tr><tr><td> ZIKV保护6769For。 </td><td> CCAGCCAGAATTGCATGTGTCC </td></tr><tr><td> ZIKV保存7209Rev。 </td><td> ATCATTAGCAGCGGGACTCCAA </td></tr><tr><td> ZIKV Conserved 7343For。 </td><td> GTTGTGGATGGAATAGTGGT </td></tr><tr><td> ZIKV保存8243For。 </td><td> TGCCCATACACCAGCACTATGA </td></tr><tr><td> ZIKV PRVABC59 8893Rev。 </td><td> TGCATTGCTACGAACCTTGTTG </td></tr><tr><td> ZIKV保守9133For。 </td><td> AGCCCTTGGATTCTTGAACGAGGA </td></tr><tr><td> ZIKV PRVABC59 9673Rev。 </td><td> AACGCAATCATCTCCACTGACT </td></tr><tr><td> ZIKV保护9686For。 </td><td> GATGATAGGTTTGCACATGCC </td></tr><tr><td> ZIKV保守10321Rev。 </td><td> GTCCATGTACTTTTCTTCATCACCTAT </td></tr><tr><td> ZIKV保守10455For。 </td><td> CAGGAGAAGCTGGGAAACC </td></tr><tr><td> ZIKV保护10621Rev。 </td><td> CAGATTGAAGGGTGGGGAAGGTC </td></tr></tbody></table> 表1:用于测序cDNA克隆的引物。质粒可以用所列的引物完全测序。标记为"保守"印度的引物认为它们可能能够对ZIKV的所有基因型进行序列/扩增。标记为"PRVABC59"的引物对该菌株是特异性的，但也可用于其他亚洲基因型菌株和可能的其他基因型。 3. 体外转录RNA的制备<ol><li> 建立maxiprep或RCA制备DNA的消化反应。 <ol><li>对于p1消化，混合10μL的10x反应缓冲液，3μL的ApaLI，3μL的BamHI-HF，3μL的rSAP或CIP，以及3.3μg的p1 DNA。加入ddH 2 O至100μL。 </li><li>对于p2消化，混合10μL10x反应缓冲液，3μLApaLI，3μLEcoRI-HF和13.3μgp2 DNA。加入ddH 2 O至100μL。 </li><li>在37℃孵育1-2小时。 </li><li>使用商业化的凝胶和PCR清洁试剂盒进行纯化（参见材料表）。在加热块中预热至70℃的30μL洗脱缓冲液中洗脱（孵育柱旋转5分钟）。 注意:保持1μL以后在凝胶上运行。 </li></ol></li><li> 准备连接反应。 <ol><li>结合10μL10×T4 DNA连接反应缓冲液，3μLT4 DNA连接酶（400 U /μL），29μL纯化p1和29μL纯化p2。加入ddH 2 O至100μL。 </li><li>在室温下孵育2小时或16℃过夜。 </li><li>使用商业化的凝胶和PCR清洁试剂盒进行纯化（参见材料表）。在加热块中预热至70℃的10μL洗脱缓冲液中洗脱（在纺丝前孵育柱5分钟）。 注意:保持1μL以后在凝胶上运行。 </li></ol></li><li>运行未消化的质粒，消化的质粒和在琼脂糖凝胶上连接。连接产物应该具有比单独消化的质粒更高的分子量带（约11kb），表明连接成功（ 图2b ）。</li><li> 建立T7 体外转录反应。 <ol><li>结合10μL2x ARCA / NTP Mix，2μLT7 RNA聚合酶混合液和8μL纯化的连接反应，终体积为20μL。 注意:混合中包含的ARCA是专门以正确方向结合的帽类似物。标准帽类似物可导致只有约50％的转录物具有正确方向的上限。 </li><li>在37℃下孵育4小时。 </li><li>通过紫外分光光度计测量RNA浓度。任选地，运行变性琼脂糖凝胶以证实RNA转录物的长度。 </li></ol></li></ol> 4.通过电穿孔拯救传染病毒<ol><li>在电穿孔之前几天，每个待回收的构建体准备1个T182瓶的Vero细胞（T150和T182之间是可接受的;在DMEM + 10％胎牛血清（FBS）中生长）。包括1 T182作为模拟转染控制。策lls应在70-80％的汇合处使用。 </li><li>当细胞准备就绪时，将DMEM + 15％FBS + 10mM HEPES的12mL /反应置于加热到37℃的水浴中。 </li><li>用标准方法通过胰蛋白酶消化分离细胞，并用含10％FBS的培养基回收细胞以灭活胰蛋白酶;在4℃下以150×g离心细胞5分钟。 </li><li>在1×磷酸盐缓冲盐水（PBS）中洗涤细胞，在4℃下以150×g将细胞沉淀5分钟。重复此步骤两次，共三次洗涤。 </li><li>将细胞重悬于每个样品200μL的RPMI 1640 + 10mM HEPES（无菌）中。将200μL细胞悬浮液转移到无菌管中。细胞应为〜0.5×10 7个细胞/ mL。 </li><li>在每组细胞中加入20μL水（模拟阴性对照）或20μLRNA（优选5-10μg）。轻轻的轻轻混匀，将管内容物转移到2mm间隙的电穿孔试管中。 </li><li>设一个电驱动器为170 V LV，Ω（Ω）为0和950μF（大致为625 V / cm，其他机器为20 ms时间常数）。将电阻设置为最低可用设置，0或∞（如果可用）。 </li><li>脉冲一次并立即加入600μL在步骤4.2中加热的DMEM + 15％FBS + 10mM HEPES。彻底冲洗比色皿内部以除去所有细胞。将细胞加入到含有10mL预热培养基的T75组织培养瓶中。使用比色杯附带的滴管从比色皿中除去其他介质。小心保持不育。 </li><li>旋转烧瓶将培养基和细胞混合并置于37℃培养箱中。 </li><li>检查第二天的细胞活力，并且每天都这样做，直到观察到50-75％的细胞死亡。通过与模拟电穿孔对照相比观察细胞死亡。 注意:在Vero细胞中，ZIKV细胞病变效应（CPE）是相当明显的，导致在培养基中分离和漂浮的圆形细胞。我们有观察到8-10天的范围是收获的理想选择。 </li><li>在圆锥管中收获上清液并离心以在4℃下以&gt; 3,000×g去除细胞碎片10分钟。 </li><li>将澄清的上清液移除到新管中，并补充最终浓度为20％FBS（100％原液）。另外，加入终浓度为10mM的无菌HEPES作为缓冲剂，以保存病毒感染性，同时冷冻（来自1M无菌储备）。 </li><li>通过涡旋混合病毒并将其等分成螺旋盖小瓶。储存于-80°C，以备将来使用。 </li></ol> 5.恢复病毒滴定<ol><li>在滴定前一天种上适量数量的Vero细胞的6孔或12孔板。 </li><li>从冷冻箱中取出病毒，并在管或96孔板中将DMEM + 2％FBS连续10倍稀释。 注意:来自回收克隆的预期效价为1×10 6至5×10 7 PFU / mL。 </li><li>加200 o将每次稀释液400μL分别复制到12孔或6孔板的孔中。 </li><li>将板在37℃的培养箱和岩石中每15分钟放置一次，总共约1-1.5小时的吸附周期。 </li><li>去除病毒接种物，并向每个孔中加入1mL（12孔）或2mL（6孔）DMEM-黄蓍胶覆盖物。 注意:这里可以使用琼脂糖，琼脂，甲基纤维素或其他覆盖介质。 </li><li>在37℃培养箱中孵育细胞5天。适当斑块形成的时间将根据所使用的覆盖层而变化。 </li><li>为了修复细胞，将培养箱中的板取出并轻轻倒入消毒剂中。 </li><li>向每个孔中加入足量的20％乙醇/ 0.1％结晶紫（CV），以覆盖和旋转混合。 注意:许多其他固定剂存在，可以在这里使用。另外，如果使用琼脂糖或琼脂覆盖层，则可以使用第二层覆盖层与中性红色结合使用，以便不固定地显现斑块。 20％乙醇显示为在以前的研究中有效灭活包膜病毒;不要将这个数量用于非包络病毒，因为它们不会被灭活19 。 </li><li>在室温下孵育板30-60分钟（在第二类生物安全柜中）;丢弃CV（根据当地法规），并用自来水清洗板。 </li><li>让板材干燥，并手动计数斑块。 注意:参考文献20可用作执行噬斑测定的附加参考。 </li></ol>

<ol>
	<li>Kindhauser, M. K., Allen, T., Frank, V., Santhana, R. S., Dye, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika:+the+origin+and+spread+of+a+mosquito-borne+virus.">Zika: the origin and spread of a mosquito-borne virus.</a> Bull World Health Organ. 94 (9), 675C-686C (2016).</li><li>Oehler, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+infection+complicated+by+Guillain-Barre+syndrome--case+report,+French+Polynesia,+December+2013.">Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia, December 2013.</a> Euro Surveill. 19 (9), (2014).</li><li>Li, D., Aaskov, J., Lott, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+cryptic+prokaryotic+promoter+within+the+cDNA+encoding+the+5'+end+of+dengue+virus+RNA+genome.">Identification of a cryptic prokaryotic promoter within the cDNA encoding the 5' end of dengue virus RNA genome.</a> PLoS One. 6 (3), e18197 (2011).</li><li>Pu, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+propagate+flavivirus+infectious+cDNA+clones+in+bacteria+by+introducing+tandem+repeat+sequences+upstream+of+virus+genome.">A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.</a> J Gen Virol. 95 (Pt 7), 1493-1503 (2014).</li><li>Pu, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+propagation+of+flavivirus+infectious+cDNAs+by+a+novel+method+to+reduce+the+cryptic+bacterial+promoter+activity+of+virus+genomes.">Successful propagation of flavivirus infectious cDNAs by a novel method to reduce the cryptic bacterial promoter activity of virus genomes.</a> J Virol. 85 (6), 2927-2941 (2011).</li><li>Rice, C. M., Grakoui, A., Galler, R., Chambers, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+of+infectious+yellow+fever+RNA+from+full-length+cDNA+templates+produced+by+in+vitro+ligation.">Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation.</a> New Biol. 1 (3), 285-296 (1989).</li><li>Yun, S. I., Kim, S. Y., Rice, C. M., Lee, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+reverse+genetics+system+for+Japanese+encephalitis+virus.">Development and application of a reverse genetics system for Japanese encephalitis virus.</a> J Virol. 77 (11), 6450-6465 (2003).</li><li>Gualano, R. C., Pryor, M. J., Cauchi, M. R., Wright, P. J., Davidson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+major+determinant+of+mouse+neurovirulence+of+dengue+virus+type+2+using+stably+cloned+genomic-length+cDNA.">Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA.</a> J Gen Virol. 79 (Pt 3), 437-446 (1998).</li><li>Johansen, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intron+insertion+facilitates+amplification+of+cloned+virus+cDNA+in+Escherichia+coli+while+biological+activity+is+reestablished+after+transcription+in+vivo.">Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo.</a> Proc Natl Acad Sci U S A. 93 (22), 12400-12405 (1996).</li><li>Shan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Infectious+cDNA+Clone+of+Zika+Virus+to+Study+Viral+Virulence,+Mosquito+Transmission,+and+Antiviral+Inhibitors.">An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors.</a> Cell Host Microbe. 19 (6), 891-900 (2016).</li><li>Schwarz, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+the+1947+Zika+Virus+Prototype+Strain+with+a+Cytomegalovirus+Promoter-Driven+cDNA+Clone.">Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone.</a> mSphere. 1 (5), (2016).</li><li>Tsetsarkin, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Full-Length+Infectious+cDNA+Clone+of+Zika+Virus+from+the+2015+Epidemic+in+Brazil+as+a+Genetic+Platform+for+Studies+of+Virus-Host+Interactions+and+Vaccine+Development.">A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development.</a> MBio. 7 (4), (2016).</li><li>Gadea, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+method+for+the+rapid+generation+of+recombinant+Zika+virus+expressing+the+GFP+reporter+gene.">A robust method for the rapid generation of recombinant Zika virus expressing the GFP reporter gene.</a> Virology. 497, 157-162 (2016).</li><li>Kapoor, M., Zhang, L., Mohan, P. M., Padmanabhan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+an+infectious+dengue+virus+type-2+RNA+genome+(New+Guinea+C+strain).">Synthesis and characterization of an infectious dengue virus type-2 RNA genome (New Guinea C strain).</a> Gene. 162 (2), 175-180 (1995).</li><li>Messer, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+reverse+genetic+system+for+studying+dengue+virus+serotype+3+strain+variation+and+neutralization.">Development and characterization of a reverse genetic system for studying dengue virus serotype 3 strain variation and neutralization.</a> PLoS Negl Trop Dis. 6 (2), e1486 (2012).</li><li>Kinney, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+virulence+and+thermostable+replication+of+the+North+American+strain+of+West+Nile+virus.">Avian virulence and thermostable replication of the North American strain of West Nile virus.</a> J Gen Virol. 87 (Pt 12), 3611-3622 (2006).</li><li>Chang, A. C., Cohen, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+amplifiable+multicopy+DNA+cloning+vehicles+derived+from+the+P15A+cryptic+miniplasmid.">Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.</a> J Bacteriol. 134 (3), 1141-1156 (1978).</li><li>Weger-Lucarelli, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+Recombinant+Virus+Generated+from+a+New+World+Zika+Virus+Infectious+Clone.">Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.</a> J Virol. 91 (1), (2017).</li><li>Roberts, P. L., Lloyd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+inactivation+by+protein+denaturants+used+in+affinity+chromatography.">Virus inactivation by protein denaturants used in affinity chromatography.</a> Biologicals. 35 (4), 343-347 (2007).</li><li>Baer, A., Kehn-Hall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+concentration+determination+through+plaque+assays:+using+traditional+and+novel+overlay+systems.">Viral concentration determination through plaque assays: using traditional and novel overlay systems.</a> J Vis Exp. (93), e52065 (2014).</li><li>Weger-Lucarelli, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+Recombinant+Virus+Generated+from+a+New+World+Zika+Virus+Infectious+Clone.">Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.</a> J Virol. , (2016).</li><li>Grubaugh, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Drift+during+Systemic+Arbovirus+Infection+of+Mosquito+Vectors+Leads+to+Decreased+Relative+Fitness+during+Host+Switching.">Genetic Drift during Systemic Arbovirus Infection of Mosquito Vectors Leads to Decreased Relative Fitness during Host Switching.</a> Cell Host Microbe. 19 (4), 481-492 (2016).</li></ol>

作者没有什么可以披露的。

Here we describe a method for the recovery of a bipartite infectious cDNA clone system for ZIKV. Previously described clones for ZIKV suffer from either attenuation or require the addition of introns, making plasmids larger and preventing rescue in insect cells. Infectious virus can be recovered using the two-plasmid clone system in either mammalian or insect cells (data not shown). In addition, virus recovered from this system behaves similarly to wild-type virus in several cell lines, in an immunocompromised mouse model and in mosquitoes. 
Due to the presence of cryptic bacterial promoters in flavivirus genomes3, full-length flavivirus clones are difficult to work with, commonly resulting in mutations, insertions, deletions or rearrangements in plasmids. We sought to alleviate these concerns by separating the ZIKV genome into two plasmids, disrupting the toxic sequences that are present in the viral NS1 gene. This resulted in a more stable system that is also amenable to incorporating changes. Using this clone system, we have introduced several point mutations in the ZIKV genome and inserted foreign sequences in the 3' UTR (data not shown). This system also provides an ideal starting point for studying genetic determinants of pathogenesis, construction of vaccine candidates and development of reporter viruses. Additionally, the clone-derived virus has proven to be an important tool for studying virus evolution22.
The methods described here can also be applied to new two-plasmid clone systems for other ZIKV strains or other flaviviruses, with some modifications. An important consideration for creating new virus clone systems using this approach is identifying a naturally occurring restriction enzyme site in the viral genome near the envelope-NS1 gene junction. If a suitable site cannot be found, then it is possible to generate one artificially via PCRmutagenesis. Alternatively, we have successfully rescued infectious virus by using Gibson assembly, if overlapping segments can be generated via restriction digestion (data not shown). Furthermore, it is necessary to include either a restriction enzyme cleavage site or a ribozyme sequence immediately downstream of the viral genome to ensure an authentic 3'UTR. 
By using a two-plasmid approach, we found that a stable and relatively straightforward cDNA clone system for ZIKV could be developed. The infectious virus recovered from the clone possessed wild-type characteristics in vitro and in vivo. Additionally, this approach can be adapted for any flavivirus, facilitating reverse genetics approaches for other relevant viruses.

Zika病毒（ZIKV;家庭黄病毒科: 黄病毒属）是一种蚊子传播的黄病毒，于2013-14年抵达巴西，随后发生在美洲蔓延的发热性疾病的大规模爆发1 。此外，ZIKV已经与严重的疾病结果相关联，如成人的吉兰 - 巴雷综合征和胎儿和新生儿的小头症2 。在西半球迅速传播之前，关于ZIKV的知之甚少。这包括缺乏分子工具，从而阻碍机械研究。用于病毒的分子工具，例如感染性cDNA克隆，促进疫苗和抗病毒治疗发展，并允许评估与差异病毒发病机理，免疫应答和病毒进化相关的病毒遗传因素。 已知黄病毒感染性克隆由于cr而在细菌中高度不稳定其基因组中存在的原核启动子3 。已经采用了几种方法来改善这个问题;包括在病毒序列上游插入串联重复4 ，推定的原核启动子序列的突变5 ，将基因组分裂成多个质粒6 ，低拷贝数载体（包括细菌人造染色体） 7,8和内含子在病毒基因组9中的插入。 ZIKV已经描述了一个没有修改的全长系统;然而，该克隆似乎在细胞培养物和小鼠中减弱10 。其他组已经将内含子设计到ZIKV基因组中，允许细菌中不稳定序列的破坏，其可以在体外在哺乳动物细胞中剪接以产生感染性病毒11， 12 。另外，已经成功地使用了一种名为"传染性亚基因组扩增子"的基于PCR的系统来拯救ZIKV13原型MR766菌株。这里描述的方法不需要外来序列，而是通过使用先前已经成功用于黄热病6号 ，登革热14,15和西尼罗河病毒16的多个质粒来破坏高不稳定区域的基因组。此外，在病毒基因组末端添加丙型肝炎核酶序列有助于产生真实的3&#39;端，而不需要添加线性化位点。另外，两种质粒都构建在低拷贝数载体（pACYC177，每个细胞约15个拷贝）中以减轻任何残留毒性17 。恢复的病毒显示出与之相当的增长特征包含来自哺乳动物和昆虫的各种细胞类型的8种细胞系中的亲代病毒在体外生长曲线中，并且在小鼠中表现出相同的致病性谱，并且在蚊子中具有感染，传播和传播率。 在这里，我们详细说明了如何生长感染性克隆质粒， 在体外产生全长病毒RNA（病毒基因组） 的方案，并在细胞培养物中回收感染性病毒。首先，我们描述了使用滚环扩增（RCA）的细菌在细菌或无细菌扩增中的繁殖。接下来，我们展示两种质粒如何消化，然后连接在一起以产生全长病毒。最后，我们描述转录的RNA的生产及其随后的电穿孔进入Vero细胞，然后滴定回收的病毒（ 图1 ）。描述的方法是快速的，允许fo在1-2周内恢复感染性病毒库存。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NEB Stable CompetentE. coli</td>
			<td>New England BioLabs</td>
			<td>C3040H</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin, Disodium Salt</td>
			<td>various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zyppy Plasmid Miniprep Kit</td>
			<td>Zymo Research</td>
			<td>D4036</td>
			<td></td>
		</tr>
		<tr>
			<td>ZymoPURE Plasmid Maxiprep Kit</td>
			<td>Zymo Research</td>
			<td>D4202</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI-HF</td>
			<td>New England BioLabs</td>
			<td>R3138S</td>
			<td>20,000 units/ml</td>
		</tr>
		<tr>
			<td>NheI-HF</td>
			<td>New England BioLabs</td>
			<td>R3131S</td>
			<td>20,000 units/ml</td>
		</tr>
		<tr>
			<td>ApaLI</td>
			<td>New England BioLabs</td>
			<td>R0507S</td>
			<td>10,000 units/ml</td>
		</tr>
		<tr>
			<td>EcoRI-HF</td>
			<td>New England BioLabs</td>
			<td>R3101S</td>
			<td>20,000 units/ml</td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>New England BioLabs</td>
			<td>R3136S</td>
			<td>20,000 units/ml</td>
		</tr>
		<tr>
			<td>HindIII-HF</td>
			<td>New England BioLabs</td>
			<td>R3104S</td>
			<td>20,000 units/ml</td>
		</tr>
		<tr>
			<td>illustra TempliPhi 100 Amplification Kit</td>
			<td>GE Healthcare Life Sciences</td>
			<td>25640010</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin Gel and PCR Clean-up</td>
			<td>Macherey-Nagel</td>
			<td>740609.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Shrimp Alkaline Phosphatase (rSAP)</td>
			<td>New England BioLabs</td>
			<td>M0371S</td>
			<td>1,000 units/ml</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase, Calf Intestinal (CIP)</td>
			<td>New England BioLabs</td>
			<td>M0290S</td>
			<td>10,000 units/ml</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England BioLabs</td>
			<td>M0202S</td>
			<td>400,000units/mL</td>
		</tr>
		<tr>
			<td>HiScribe T7 ARCA mRNA Kit</td>
			<td>New England BioLabs</td>
			<td>E2065S</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>ATCC</td>
			<td>CCL-81</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM 630 High Throughput Electroporation System</td>
			<td>BTX</td>
			<td>45-0423</td>
			<td>Other machines are acceptable.</td>
		</tr>
		<tr>
			<td>LB Broth with agar (Miller)</td>
			<td>Sigma</td>
			<td>L3147</td>
			<td>Can be homemade as well.</td>
		</tr>
		<tr>
			<td>Terrific Broth</td>
			<td>Sigma</td>
			<td>T0918</td>
			<td>Can be homemade as well.</td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Celltreat</td>
			<td>229693</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Tubes</td>
			<td>VWR International</td>
			<td>60818-576</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Celltreat</td>
			<td>229340</td>
			<td></td>
		</tr>
		<tr>
			<td>T182 flasks</td>
			<td>Celltreat</td>
			<td>229350</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with L-glutamine</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with L-glutamine and 4.5 g/L glucose</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlas Biologicals</td>
			<td>FP-0500-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tragacanth Powder</td>
			<td>MP Bio</td>
			<td>MP 104792</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Amresco</td>
			<td>0528-1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol Denatured</td>
			<td>VWR International</td>
			<td>BDH1156-1LP</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well plate</td>
			<td>Celltreat</td>
			<td>229111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing Oligos</td>
			<td>IDT</td>
			<td>see table 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 3.0</td>
			<td>ThermoFisher</td>
			<td>Qubit 3.0</td>
			<td>other methods are acceptable.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>ThermoFisher</td>
			<td>Q32850</td>
			<td>other methods are acceptable.</td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay Kit</td>
			<td>ThermoFisher</td>
			<td>Q32852</td>
			<td>other methods are acceptable.</td>
		</tr>
		<tr>
			<td>Class II Biosafety Cabinet</td>
			<td>Varies</td>
			<td>N/A</td>
			<td>This is necessary for live-virus work.</td>
		</tr>
	</tbody>
</table>

rescue and characterization of recombinant virus from a new world zika virus infectious clone

传染性cDNA克隆允许病毒的遗传操作，从而有助于疫苗，发病机理，复制，传播和病毒进化的工作。在这里，我们描述了Zika病毒（ZIKV）感染性克隆的构建，目前在美洲引起爆发性爆发。为了防止黄病毒衍生质粒通常观察到的对细菌的毒性，我们产生了一个双质粒系统，其将NS1基因的基因组分离，并且比不能在没有突变的情况下成功恢复的全长构建体更稳定。在消化和连接两个片段后，可以通过T7 RNA聚合酶的体外转录产生全长病毒RNA。在将转录的RNA电穿孔进入细胞后，分别在小鼠和蚊子中显示类似的体外生长动力学和体内毒力和感染表型的病毒。

这里描述的方案允许恢复感染性克隆衍生的Zika病毒。与高度不稳定的全长版本（数据未显示）相比，操作双质粒感染性克隆系统在小心执行时是简单的。在消化和连接两个不同的片段后，使用T7聚合酶的体外转录产生加帽的RNA，然后将其电穿孔至Vero细胞（ 图1 ）。在使用RCA或maxiprep进行大规模DNA生产后，可以使用限制性消化作为代谢物监测正确的质粒序列（ 图2a ）和通过Sanger测序。通过测序确认克隆后，感染性病毒的恢复仅需要消化，连接， 体外转录和最后的电穿孔。这些步骤可以在一天内完成。正确挖掘可以通过琼脂糖凝胶电泳（ 图2b ）监测茎和连接，如果需要，进行故障排除。 如图3所示 ，感染性克隆衍生的病毒在几种细胞系中在体外复制到与亲本分离物相似的水平，表明来自cDNA的回收病毒与初级分离物类似地复制。另外，传染性克隆衍生的病毒与亲本分离株之间在小鼠存活率或埃及伊蚊的感染，传播和传播速率方面没有观察到显着差异（ 图4 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55857/55857fig1.jpg" /> 图1:来自双质粒cDNA克隆的ZIKV救援工作流程。将ZIKV菌株PRVABC59的基因组克隆到两个分开的片段中成为pACYC177质粒骨架。然后将两个质粒消化并与T4DNA连接酶连接。然后通过体外转录产生包被的感染性RNA，然后通过电穿孔进入Vero细胞。 （图参考文献21 ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55857/55857fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55857/55857fig2.jpg" /> 图2:用于监测cDNA克隆的消化和连接的凝胶电泳。 （ A ）用于评估遗传稳定性的小批量衍生质粒的初始限制性消化。 （ B ）从双质粒克隆系统回收感染性病毒时的消化和连接产物的实例。 <a<html> href ="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55857/55857fig2large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55857/55857fig3.jpg" /> 图3:克隆衍生病毒的体外生长动力学。通过斑块测定法评估野生型PRVABC59和感染性克隆衍生病毒对人（SH-SY5Y，Jar和NTERA2 cI.D1），蚊子（C6 / 36，Aag2）和非人灵长类动物（Vero）细胞系的生长动力学。 n = 3误差条表示与平均值的标准偏差。 （图参考文献21 ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55857/55857fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> / 55857fig4.jpg" 图4:克隆衍生病毒在小鼠和蚊子中的体内表征。 （ A ）4周龄的雄性和雌性干扰素α，β和γ受体敲除，AG129，小鼠腹膜内接种1000PFU的PRVABC59或感染性克隆衍生的病毒，并随时间监测存活（n = 11）。给伊蚊伊蚊给予含有ZIKV的感染性血液，14天后解剖。 （ B ）使用斑块测定来评估感染性病毒的蚊体（感染），腿（传播）或唾液分泌物（传播）。速率以被测试的总蚊子的百分比表示（图中参考文献21 ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55857/55857fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone at JoVE.com

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

该方案描述了从双质粒感染性cDNA克隆中恢复感染性Zika病毒。

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 ...

rescue-characterization-recombinant-virus-from-new-world-zika-virus

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.6K 次观看.  Colorado State University. 该方案描述了从双质粒感染性cDNA克隆中恢复感染性Zika病毒。 

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

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

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 ...

科研

免疫与感染

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

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

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 ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

宣传和检测Maedi visna病毒传染性表达绿色荧光蛋白的分子克隆

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

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, ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

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 ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...