ويود المؤلفون أن يشكروا كريستين بولارد-فيبلمان وميلينا فيسيلينوفيتش وكلوديا روكيرت على مساعدتهم في وصف الفيروس المستخرج من النسخ المستنسخ. وأيد هذا العمل جزئيا من المنح المقدمة من المعهد الوطني للحساسية والأمراض المعدية، المعاهد الوطنية للصحة في إطار المنح AI114675 (بجج) و AI067380 (جدي).

<ol>
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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>

الكتاب ليس لديهم ما يكشف.

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.

فيروس زيكا (زيكف؛ عائلة فلافيفريداي : جنس فلافيفيروس ) هو فيروس منقول بالبعوض الذي وصل إلى البرازيل في 2013-14، وكان مرتبطا بعد ذلك بفاشية واسعة من مرض الحمى التي انتشرت في جميع أنحاء الأمريكتين 1 . وبالإضافة إلى ذلك، تم ربط زيكف مع نتائج المرض الشديد، مثل متلازمة غيلان باريه في البالغين وصغر الرأس في الأجنة والحديثين 2 . ولم يعرف القليل عن زيكف قبل انتشاره السريع في نصف الكرة الغربي. وشمل ذلك نقص الأدوات الجزيئية، مما أعاق البحث الآلي. الأدوات الجزيئية للفيروسات، مثل الحيوانات المستنسخة كدنا المعدية، وتسهيل اللقاح والتنمية العلاجية المضادة للفيروسات، والسماح لتقييم العوامل الوراثية الفيروسية المتعلقة المرضية الفيروسية التفاضلية، والاستجابة المناعية والتطور الفيروسي. ومن المعروف أن الحيوانات المستنسخة عدوى فيروس العوز المناعي البشري غير مستقرة للغاية في البكتيريا بسبب كريبتيك بروكاريوتيك المروجين موجودة في الجينومات الخاصة بهم 3 . وقد استخدمت عدة نهج لتحسين هذه المشكلة؛ بما في ذلك إدخال جنبا إلى جنب يكرر المنبع من تسلسل الفيروسية 4 ، طفرة من المفترضة متواليات بدائيات بدائية النواة 5 ، وتقسيم الجينوم إلى البلازميدات متعددة 6 ، ناقلات عدد النسخ المنخفضة (بما في ذلك الكروموسومات الاصطناعية البكتيرية) 7 ، 8 وإدخال الإنترونات في الجينوم الفيروسي 9 . وقد وصفت نظام كامل طول دون تعديلات ل زيكف. ومع ذلك، يبدو أن هذا الاستنساخ الموهن في ثقافة الخلايا وفي الفئران 10 . وقد قامت مجموعات أخرى بتصنيع إنترونات في جينوم زيكف، مما يسمح بتعطيل تسلسل غير مستقر في البكتيريا التي يمكن أن تقسم في خلايا الثدييات في المختبر لإنتاج الفيروس المعدية 11، 12 . بالإضافة إلى ذلك، تم استخدام نظام القائم على ير بعنوان العدوى سوبجينوميك-أمبليكونس بنجاح لإنقاذ سلالة MR766 النموذج من زيكف 13 . إن المنهج الموصوف هنا لا يتطلب أي تسلسل أجنبي، بل يعطل الجينوم في المنطقة من عدم الاستقرار العالي باستخدام البلازميدات المتعددة، والتي سبق استخدامها بنجاح مع الحمى الصفراء 6 ، حمى الضنك 14 ، 15 وفيروسات غرب النيل 16 . وعلاوة على ذلك، إضافة تسلسل ريبوزيمي التهاب الكبد D في تيرميني الجينومي الفيروسي يسهل إنشاء نهاية أصيلة 3 &#39;دون الحاجة إلى إضافة موقع الخطي. بالإضافة إلى ذلك، يتم بناء كلا البلازميدات في ناقلات عدد منخفض نسخة (pACYC177، ~ 15 نسخة لكل خلية) للتخفيف من أي سمية المتبقية 17 . فيروس تعافى يعرض لمحة النمو مماثلة لفيروس الوالدية في منحنيات نمو المختبر في 8 خطوط الخلايا التي تضم مجموعة متنوعة من أنواع الخلايا المستمدة من كل من الثدييات والحشرات، وعرضت ملامح مماثلة المسببة للأمراض في الفئران والعدوى، ونشر ونقل معدلات في البعوض 18 . هنا، ونحن بالتفصيل بروتوكول يصف كيفية زراعة البلازميدات استنساخ المعدية، وتوليد كامل طول الحمض النووي الريبي الفيروسي (الجينوم الفيروسي) في المختبر، واستعادة الفيروس المعدية في ثقافة الخلية. أولا، نحن تصف انتشار البلازميدات في البكتيريا أو خالية من البكتيريا التضخيم باستخدام المتداول دائرة التضخيم (رسيا). بعد ذلك، وتبين لنا كيف يتم هضم البلازميدات اثنين ومن ثم ليغاتد لاحقا معا لتوليد كامل طول الفيروس. وأخيرا، نحن تصف إنتاج الحمض النووي الريبي المنقولة وما يليه إليكتروبوراتيون في خلايا فيرو، تليها المعايرة من فيروس تعافى ( الشكل 1 ). النهج الموصوف هو سريع، مما يسمح فوr استعادة مخزونات الفيروسات المعدية في 1-2 أسابيع.

<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

استنساخ العدوى [كدنا] تسمح للتلاعب الجيني للفيروس، مما يسهل العمل على اللقاحات، المرضية، النسخ المتماثل، انتقال وتطور الفيروس. نحن هنا وصف بناء استنساخ المعدية لفيروس زيكا (زيكف)، الذي يسبب حاليا تفشي المتفجرات في الأمريكتين. لمنع السمية للبكتيريا التي لوحظت عادة مع البلازميدات المستمدة من فلافيروس، أنشأنا نظام البلازميد اثنين الذي يفصل الجينوم في الجين NS1 وأكثر استقرارا من البنيات كامل طول التي لا يمكن استردادها بنجاح دون الطفرات. بعد الهضم وربط للانضمام إلى شظيتين، يمكن أن تتولد كامل الحمض النووي الريبي الفيروسية طول عن طريق النسخ في المختبر مع T7 رنا البلمرة. بعد إليكتروبوراتيون من الحمض النووي الريبي كتب في الخلايا، تم استرداد الفيروس الذي عرضت مماثلة في حركية النمو المختبر وفي الجسم الحي الفوعة والأنماط الظاهرية العدوى في الفئران والبعوض، على التوالي.

بروتوكول الموصوفة هنا يسمح لاستعادة فيروس زيكا المستمدة المستنسخ استنساخ. التلاعب نظام استنساخ العدوى البلازميد اثنين هو واضح عندما يؤديها مع الرعاية، بالمقارنة مع الإصدارات كاملة الطول التي هي غير مستقرة للغاية (لا تظهر البيانات). بعد الهضم وربط قطعتين متميزة، يتم إنتاج رنا توج باستخدام النسخ في المختبر مع بوليميراز T7 الذي هو إليكتروبوراتد ثم إلى خلايا فيرو ( الشكل 1 ). يمكن رصد تسلسل البلازميد الصحيح باستخدام تقييد الهضم كما مينيبريب التالية وكيل ( الشكل 2A ) وعبر سانجر التسلسل بعد إنتاج الحمض النووي على نطاق واسع مع رسيا أو ماكسيبريب. بعد التأكد من الحيوانات المستنسخة عن طريق التسلسل، واستعادة الفيروس المعدية يتطلب الهضم فقط، ربط، في النسخ المختبر وأخيرا إليكتروبوراتيون. ويمكن تنفيذ هذه الخطوات في يوم واحد. الصحيح ديجويمكن رصد ستيون وربط بواسطة أغاروس هلام الكهربائي ( الشكل 2B )، والسماح لاستكشاف الأخطاء وإصلاحها، إذا لزم الأمر. كما هو مبين في الشكل 3 ، الفيروس المستنسخ المستنسخ المستنسخ يكرر إلى مستويات مماثلة لعزل الوالدين في المختبر في العديد من خطوط الخلايا، مما يشير إلى أن الفيروس المسترجع من [كدنا] يتكرر على غرار العزلة الأولية. بالإضافة إلى ذلك، لم يلاحظ وجود فروق ذات دلالة إحصائية بين الفيروس المستخلص المستنسخ المستنسخ وعزل الوالدين في معدلات البقاء على قيد الحياة في الفئران أو معدلات العدوى، ونشرها ونقلها في البعوض الزاعجة المصرية ( الشكل 4 ). <img alt="شكل 1" class="xfigimg" src="/files/ftp_upload/55857/55857fig1.jpg" /> الشكل 1: سير العمل لإنقاذ زيكف من استنساخ كدنا اثنين البلازميد.تم استنساخ جينوم سلالة زيكف PRVABC59 في قطعتين منفصلتين في العمود الفقري البلازميد pACYC177. ثم هضم البلازميدات اثنين و ليغاتد مع ليغاز T4 دنا. ثم تم إنتاج الحمض النووي الريبي المعدية المعبدة عن طريق النسخ في المختبر تليها إليكتروبوراتيون في خلايا فيرو. (الشكل مقتبس من المرجع 21 ). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55857/55857fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل 2" class="xfigimg" src="/files/ftp_upload/55857/55857fig2.jpg" /> الشكل 2: هلام الكهربائي لمراقبة الهضم وربط استنساخ [كدنا]. ( A ) هضم تقييد الأولي من البلازميدات المشتقة مينيبريب لتقييم الاستقرار الجيني. ( ب ) أمثلة على الهضم وربط المنتجات خلال انتعاش الفيروس المعدية من نظام استنساخ البلازميد اثنين. <a<html> هريف = &quot;https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55857/55857fig2large.jpg&quot; تارجيت = &quot;_ بلانك&quot;&gt; الرجاء النقر هنا لعرض نسخة أكبر من هذا الرقم. <img alt="الشكل 3" class="xfigimg" src="/files/ftp_upload/55857/55857fig3.jpg" /> الشكل 3: حركية النمو في المختبر من الفيروسات المستنسخة من استنساخ. تم تقييم حركية النمو للنوع البري PRVABC59 واستنساخ العدوى المستمدة الفيروس على الإنسان (ش-SY5Y، جرة و NTERA2 CI.D1)، البعوض (C6 / 36، Aag2) وخطوط الخلايا الرئيسيات غير البشرية (فيرو) من خلال فحص اللوحة. n = 3 أشرطة الخطأ تمثل الانحراف المعياري عن المتوسط. (الشكل مقتبس من المرجع 21 ). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55857/55857fig3large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> 55857fig4.jpg &quot;/&gt; الشكل 4: في توصيف الجسم الحي من الفيروسات المستنسخة من استنساخ في الفئران والبعوض. ( أ ) تم تلقيح الفئران داخل البريتوني من قبل الذكور والإناث لمدة 4 أسابيع، وبيتا وغاما خروج المغلوب المستقبلي، AG129، مع 1000 بفو من PRVABC59 أو الفيروس المستنسخ المستنسخ المستنسخ ورصدها مع مرور الوقت من أجل البقاء (ن = 11). الزاعجة المصرية البعوض أعطيت دماء المعدية التي تحتوي على زيكف وتشريح بعد 14 يوما. ( ب ) استخدمت مقايسات اللويحات لتقييم أجسام البعوض (السرطانات) أو الساقين (النشر) أو الإفرازات اللعابية (انتقال العدوى) للفيروس المعدية. وتعرض المعدلات كنسبة مئوية من مجموع البعوض التي تم اختبارها (الشكل مقتبس من المرجع 21 ). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55857/55857fig4large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</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

يصف هذا البروتوكول انتعاش فيروس زيكا المعدية من استنساخ كدنا المعدية ثنائي البلازميد.

إنقاذ وتوصيف الفيروسات المؤتلف من عالم جديد زيكا الفيروسات استنساخ العدوى

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 Views.  Colorado State University.  يصف هذا البروتوكول انتعاش فيروس زيكا المعدية من استنساخ كدنا المعدية ثنائي البلازميد. 

Video: 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.

توليد المؤتلف فيروسات الأنفلونزا من الحمض النووي البلازميد

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

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.

الكشف عن الفيروسات المعدية التي جمعت من الحقل عن طريق البعوض فيرو خلية الفحص الثقافة

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.

تصور فيروس حمى الضنك من خلال وصفها اليكسا فلور

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.

وكشف ونشر استنساخ المعدية الجزيئية لفيروس ميثة-فيزنا أن تعرب عن بروتين الفلورية الخضراء

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.

الانقاذ من الفيروسات المؤتلف مرض نيوكاسل من [كدنا]

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.

عزل وتحليل العدلة لتحديد ما دورها في الغدد اللمفاوية حساسية الخلية وكلاء العلاجية

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.

زيكا الفيروسات نظام المعدية خلية الثقافة و<em&gt; في المختبر</em&gt; وقائية تأثير الانترفيرونات

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.

اكتشاف حانمه تشخيصية محددة الفيروس Zika

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).

استخراج سريعة وآمنة وبسيطة دليل السرير الحمض النووي للكشف عن الفيروس في عينات الدم كله

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.

التحديد الكمي لتعزيز تعتمد على جسم مضاد الفيروس Zika في خلايا الإنسان الأولية

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