This study is supported by funding from Taipei Medical University Hospital (102TMU-TMUH-19) and the Ministry of Science and Technology of Taiwan (MOST103-2320-B-038-031-MY3).

ملاحظة: تأكد من أن تتم جميع الإجراءات التي تنطوي على زراعة الخلايا وعدوى فيروس في اغطية شهادة السلامة الأحيائية التي هي مناسبة لمستوى السلامة الحيوية من العينات التي يجري التعامل معها. لغرض وصف البروتوكولات، يتم استخدام Gaussia luciferase المراسل HCV-الموسومة مراسل ك فيروس طراز 32. في سياق نتائج ممثل، يتم استخدام حمض المركبات chebulagic (CHLA) وpunicalagin (PUG) كما مضادات الفيروسات المرشحة التي تستهدف التفاعلات بروتين سكري الفيروسية مع الجليكوسامينوجليكان سطح الخلية خلال دخول الفيروس في وقت مبكر خطوات 31. الهيبارين، والذي يعرف لتتداخل مع دخول العديد من الفيروسات 30،31،33،34، ويستخدم كعلاج مراقبة إيجابية في هذا السياق. لالأساسية عن تقنيات علم الفيروسات، والإكثار من الفيروسات، وتحديد عيار الفيروس، والتعبير عن الجرعة المعدية في وحدات اللوحة تشكيل (PFU)، والتركيز حدات تشكيل (FFU)، أو تعدد العدوى (وزارة الداخلية)، والقارئ هو إعادةferred مرجع 35. للحصول على أمثلة السابقة والظروف الأمثل لاستخدام الفيروسات هو مبين في نتائج ممثل، وعلى القارئ الرجوع إلى المراجع 30-32،36-39 فضلا عن تفاصيل المدرجة في الجدول 1، الشكل 1A، والشكل 2A. الثقافة 1. الخليوي، إعداد المجمع، والسمسة مجمع <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو خط الخلية المعنية لنظام عدوى فيروس ليتم تحليلها (الجدول 1). لHCV، وتنمو الخلايا هوه 7.5 في تعديل متوسطة النسر Dulbecco و(DMEM) تستكمل مع المصل 10٪ بقري جنيني (FBS)، و 200 U / البنسلين مل G، 200 ميكروغرام / مل الستربتومايسين، و 0.5 ميكروغرام / مل الأمفوتريسين B. </li><li style=";text-align:right;direction:rtl"> إعداد اختبار مركبات وضوابط استخدام المذيبات كل منهما: على سبيل المثال، حل CHLA وPUG في سلفوكسيد ثنائي ميثيل (DMSO)؛ إعداد الهيبارين في المياه المزدوج المقطر المعقم. لجميع التخفيفات لاحقة، استخدام وسائل الإعلام الثقافة. ملاحظة: تناسق النهائيالتموينية من DMSO في العلاجات اختبار المركب هو أقل من 1٪ في التجارب. يتم تضمين 1٪ DMSO كعلاج سيطرة سلبية في المقايسات للمقارنة. </li><li style=";text-align:right;direction:rtl"> تحديد السمية الخلوية من المركبات اختبار (على سبيل المثال، CHLA وPUG) على الخلايا لعدوى فيروسية باستخدام بقاء الخلية تحديد كاشف مثل XTT (2،3 مكرر [2-ميثوكسي-4-نيترو-5-sulfophenyl] -5-phenylamino) -carbonyl] هيدروكسيد -2H-نتروبلو): <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لHCV، البذور هوه 7.5 الخلايا في لوحة 96-جيدا (1 × 10 4 خلايا لكل بئر)، واحتضان عند 37 درجة مئوية في 5٪ CO 2 حاضنة O / N للحصول على أحادي الطبقة. </li><li style=";text-align:right;direction:rtl"> تطبيق السيطرة DMSO (1٪) أو زيادة تركيزات من CHLA مركبات اختبار وPUG (مثلا 0، 10، 50، 100، و 500 ميكرومتر) إلى الآبار الثقافة في ثلاث نسخ. </li><li style=";text-align:right;direction:rtl"> احتضان عند 37 درجة مئوية لمدة 72 ساعة، ثم تجاهل المتوسطة في لوحة وغسل الخلايا مع 200 ميكرولتر من الفوسفات مخزنة المالحة (PBS) مرتين. </li><li style=";text-align:right;direction:rtl"> إضافة 100 ميكرولتر من assayinز حل من عدة في المختبر فحص السموم على أساس XTT إلى كل بئر واحتضان لوحات عند 37 درجة مئوية لمدة ساعة أخرى 3 للسماح XTT إنتاج formazan. </li><li style=";text-align:right;direction:rtl"> تحديد الامتصاصية مع قارئ صفيحة ميكروسكوبية في الطول الموجي الاختبار من 492 نانومتر، والطول الموجي إشارة من 690 نانومتر. </li><li style=";text-align:right;direction:rtl"> حساب النسبة المئوية للبقاء الخلايا باستخدام الصيغة التالية: بقاء الخلية (٪) = في / و× 100٪ &quot;، وفي&quot; حيث و&#39;باسم&#39; الرجوع إلى امتصاص المركبات اختبار ومراقبة المذيبات (مثلا DMSO 1٪. ) العلاجات، على التوالي. تحديد تركيز للخلايا الخلوية 50٪ (CC 50) من المركبات اختبار من البرامج التحليلية مثل GraphPad بريزم وفقا لبروتوكول الشركة الصانعة. </li></ol></li></ol> 2. قراءات من العدوى الفيروسية ملاحظة: قراءات من عدوى فيروسية تعتمد على نظام فيروس يستخدم ويمكن أن تشمل أساليب مثل المقايسات البلاك أو الشرق الأوسط وأفريقياسورينغ إشارات مراسل من الفيروسات الموسومة مراسل. وصفت طريقة للكشف عن الإصابة مراسل HCV على أساس النشاط luciferase المراسل أدناه. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع supernatants من الآبار المصابة وتوضيح في 17000 x ج في microcentrifuge لمدة 5 دقائق في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> مزيج 20 ميكرولتر من اختبار طاف 50 ميكرولتر من luciferase المراسل الركيزة من Gaussia عدة luciferase الفحص وقياس مباشرة مع luminometer وفقا لتعليمات الشركة الصانعة. </li><li style=";text-align:right;direction:rtl"> التعبير عن HCV العدوى كما سجل 10 وحدات الخفيفة النسبية (RLU) لتحديد تثبيط الفيروسي (٪) وحساب تركيز 50٪ فعالة (EC 50) من المركبات اختبار ضد فيروس التهاب الكبد الوبائي باستخدام خوارزميات من برنامج GraphPad بريزم وفقا لبروتوكول الشركة الصانعة. </li></ol> 3. الفيروسية التعطيل الفحص ملاحظة: أمثلة من فترة الحضانة وجرعة الفيروسية لمختلف الفيروسات لإعادة المدرجة في الشكل 1A. ويمكن أيضا اختبار تركيزات أعلى من الفيروس عن طريق زيادة وزارة الداخلية / PFU. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> البذور هوه 7.5 الخلايا في لوحة 96-جيدا (1 × 10 4 خلايا لكل بئر)، واحتضان عند 37 درجة مئوية في 5٪ CO 2 حاضنة O / N للحصول على أحادي الطبقة. </li><li style=";text-align:right;direction:rtl"> احتضان مركبات اختبار أو ضوابط (تركيزات النهائي هي: CHLA = 50 ميكرومتر؛ PUG = 50 ميكرومتر؛ الهيبارين = 1000 ميكروغرام / مل؛ DMSO = 1٪) مع جزيئات HCV عند 37 درجة مئوية (الشكل 1A، &quot;طويل الأجل&quot; ) في نسبة 1: 1. على سبيل المثال، إلى اللقاح فيروس 100 ميكرولتر تحتوي على 1 × 10 4 FFU، إضافة 100 ميكرولتر من تمييع العمل 100 ميكرومتر CHLA. ينتج هذا العلاج CHLA بتركيز نهائي 50 ميكرومتر. </li><li style=";text-align:right;direction:rtl"> تمييع الخليط الفيروس للأدوية إلى (غير فعالة) التركيز &quot;شبه العلاجي&quot; للمركبات الاختبار. على سبيل المثال، وتركيز غير الفعال للCHLA وPUG ضد HCV هو في 1 ميكرومتر 31؛ لذاوهذا يتطلب التخفيف 50 أضعاف من خليط الفيروس للأدوية التي يمكن أن يتحقق مع 9.8 مل من القاعدية المتوسطة (متوسطة زراعة الخلايا مع 2٪ FBS). ملاحظة: تخفيف لتركيز شبه العلاجية يمنع تفاعل كبير بين مركبات اختبار وسطح الخلية المضيفة ويسمح فحص تأثير العلاج على virions خالية من الخلايا. لاحظ أن هذا التخفيف تعتمد على الاستجابة للجرعة مضادة للفيروسات من المركبات اختبار ضد عدوى فيروسية معينة، ويتم تحديد مسبق لأداء معين هذا الاختبار 31. </li><li style=";text-align:right;direction:rtl"> وعلى سبيل المقارنة، مزيج الفيروس مع مركبات الاختبار وعلى الفور تمييع (أي فترة الحضانة) لتركيز الفرعية العلاجي قبل الإصابة (الشكل 1A، &quot;قصير الأجل&quot;). </li><li style=";text-align:right;direction:rtl"> إضافة 100 ميكرولتر من المخفف خليط HCV-المخدرات على هوه 7.5 الخلية أحادي الطبقة (كمية الفيروس الآن في 1 × 10 2 FFU؛ النهائية زارة الداخلية = 0.01)، واحتضان لمدة 3 ساعة على 37 درجة مئوية للسماح الفيروسيةالامتزاز. </li><li style=";text-align:right;direction:rtl"> بعد الإصابة، وإزالة قائح المخفف وغسل بلطف الآبار مع 200 ميكرولتر من برنامج تلفزيوني مرتين. ملاحظة: إجراء يغسل بلطف لتجنب رفع الخلايا. </li><li style=";text-align:right;direction:rtl"> تطبيق 100 ميكرولتر من المتوسط ​​القاعدية إلى كل بئر واحتضان عند 37 درجة مئوية لمدة 72 ساعة. </li><li style=";text-align:right;direction:rtl"> تحليل العدوى الناجم عن يعاير وطاف لluciferase النشاط كما هو موضح في &quot;2. قراءات من العدوى الفيروسية. </li></ol> 4. الفيروسية الفحص مرفق ملاحظة: يتم سرد أمثلة من فترة الحضانة وجرعة الفيروسية لمختلف الفيروسات في الشكل 2A، &quot;مرفق&quot;. ويمكن أيضا اختبار تركيزات أعلى من الفيروس عن طريق زيادة وزارة الداخلية / PFU. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> البذور هوه 7.5 الخلايا في لوحة 96-جيدا (1 × 10 4 خلايا لكل بئر)، واحتضان عند 37 درجة مئوية في 5٪ CO 2 حاضنة O / N للحصول على أحادي الطبقة. </li><li style=";text-align:right;direction:rtl"> قبل البرد الطبقات الوحيدة الخلية في لوحات في 4 درجات مئوية FOص 1 ساعة. </li><li style=";text-align:right;direction:rtl"> شارك في علاج الخلايا مع HCV اللقاح (وزارة الداخلية = 0.01) واختبار مركبات أو ضوابط (تركيزات النهائي هي: CHLA = 50 ميكرومتر؛ PUG = 50 ميكرومتر؛ الهيبارين = 1000 ميكروغرام / مل؛ DMSO = 1٪) في 4 درجات مئوية ل 3 ساعة. على سبيل المثال، إلى اللقاح فيروس 90 ميكرولتر تحتوي على 1 × 10 2 FFU، إضافة 10 ميكرولتر من تمييع العمل 500 ميكرومتر CHLA. ينتج هذا العلاج CHLA بتركيز نهائي 50 ميكرومتر والعدوى عن طريق HCV في وزارة الداخلية = 0.01 على أحادي الطبقة الخلية. ملاحظة: من المهم لتنفيذ التجربة في 4 ° C لأنه يسمح للفيروس ملزم ولكن يحول دون دخول الذي يحدث بشكل أكثر فعالية في 37 ° C. نفذ إضافة الفيروسات واختبار المركبات على الجليد والحضانة التي تلت ذلك في C الثلاجة 4 درجات للتأكد من أن درجة الحرارة هو الحفاظ على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> إزالة طاف وغسل بلطف أحادي الطبقة الخلية مع 200 ميكرولتر من برنامج تلفزيوني الجليد الباردة مرتين. ملاحظة: إجراء يغسل بلطف لتجنب رفع الخلايا &lt;./ لى&gt; <li style=";text-align:right;direction:rtl"> تطبيق 100 ميكرولتر من المتوسط ​​القاعدية إلى كل بئر واحتضان عند 37 درجة مئوية لمدة 72 ساعة. </li><li style=";text-align:right;direction:rtl"> تحليل العدوى الناجم عن يعاير وطاف لluciferase النشاط كما هو موضح في &quot;2. قراءات من العدوى الفيروسية. </li></ol> 5. الفيروسية الدخول / فيوجن الفحص ملاحظة: يتم سرد مثال على فترات الحضانة وجرعة الفيروسية لمختلف الفيروسات في &#39;دخول / فيوجن &quot;الشكل 2A. ويمكن أيضا اختبار تركيزات أعلى من الفيروس عن طريق زيادة وزارة الداخلية / PFU. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> البذور هوه 7.5 الخلايا في لوحة 96-جيدا (1 × 10 4 خلايا لكل بئر)، واحتضان عند 37 درجة مئوية في 5٪ CO 2 حاضنة O / N للحصول على أحادي الطبقة. </li><li style=";text-align:right;direction:rtl"> قبل البرد الطبقات الوحيدة الخلية في لوحات في 4 درجة مئوية لمدة 1 ساعة. </li><li style=";text-align:right;direction:rtl"> تصيب الخلايا مع فيروس (سي) (MOI = 0.01) في 4 درجة مئوية لمدة 3 ساعات. على سبيل المثال، استخدام اللقاح الفيروس 100 ميكرولتر تحتوي على 1 × 10 2 FFU. ملاحظة: نفذ عدينشوئها من اللقاح الفيروسي على الجليد والحضانة التي تلت ذلك في C الثلاجة 4 درجات للحفاظ على درجة حرارة 4 درجات مئوية، والذي يسمح دخول ملزم ولكن لا الفيروسي. </li><li style=";text-align:right;direction:rtl"> إزالة طاف وغسل بلطف الطبقات الوحيدة الخلية مع 200 ميكرولتر من برنامج تلفزيوني الجليد الباردة مرتين. ملاحظة: إجراء يغسل بلطف لتجنب رفع الخلايا. </li><li style=";text-align:right;direction:rtl"> علاج الآبار مع مركبات الاختبار أو ضوابط (تركيزات النهائي هي: CHLA = 50 ميكرومتر؛ PUG = 50 ميكرومتر؛ الهيبارين = 1000 ميكروغرام / مل؛ DMSO = 1٪)، واحتضان عند 37 درجة مئوية لمدة 3 ساعات. على سبيل المثال، إضافة 10 ميكرولتر من تمييع العمل 500 ميكرومتر CHLA إلى 90 ميكرولتر من وسائل الإعلام، وخلط، ومعالجة الآبار. ينتج هذا العلاج CHLA بتركيز نهائي 50 ميكرومتر. ملاحظة: إن التحول من 4 ° C إلى 37 درجة مئوية يسهل الآن الفيروسي حالة دخول / الانصهار، وبالتالي تتيح تقييم تأثير مركبات اختبار &#39;على هذه الخطوة معينة. </li><li style=";text-align:right;direction:rtl"> نضح طاف التي تحتوي على المخدرات وإزالة غير المنضوية-الفيروسات خارج الخلية إما عن طريق غسل 200 ميكرولتر من العازلة سيترات (50 ملي سيترات الصوديوم، 4 ملي كلوريد البوتاسيوم، ودرجة الحموضة 3.0) أو برنامج تلفزيوني. تطبيق 100 ميكرولتر من المتوسط ​​القاعدية قبل يحتضنها عند 37 درجة مئوية لمدة 72 ساعة. </li><li style=";text-align:right;direction:rtl"> تحليل العدوى الناجم عن يعاير وطاف لluciferase النشاط كما هو موضح في &quot;2. قراءات من العدوى الفيروسية. </li></ol>

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The authors declare that no competing interests exist.

In this report the methods to identify and evaluate antiviral compounds based on a mechanistic approach of dissecting the early viral entry events were described. Specifically, the assays allowed us to examine the effect of test compounds on free virus particles, viral attachment, and viral entry/fusion. Critical steps were implemented to distinctly evaluate the drug effect on the specific stage of early viral entry. For instance, in the &#8216;viral inactivation assay&#8217;, the dilution of the virus-drug mixture to sub-therapeutic concentration prevents significant interaction between the test compound and the host cell surface by &#8216;titrating out&#8217; the drug. This ensures that the inhibitory effect observed on the subsequent infection of the host cell is due to a direct interaction between the test compound and the cell-free virions, rather than an effect from the test compound on host cell membrane or membrane-associated molecules, including viral receptors30. Similarly, the shift in temperature between 4 &#176;C (which allows for virus binding but not entry) and 37 &#176;C (which facilitates virus entry/fusion) in the &#8216;viral attachment assay&#8217; and &#8216;viral entry/fusion assay&#8217; are crucial to determine the test compound&#8217;s effect on each of these specific events. This is feasible due to the temperature sensitivity of enveloped viruses during these steps in the infection24-29. It is therefore important that the assays are performed at the indicated temperature to ensure the accuracy of the results; for example, by carrying out the experiment on ice to maintain at 4 &#176;C and by placing the sample directly in a 37 &#176;C incubator for the temperature shift. In addition, the use of negative (ex. DMSO solvent for drug preparation) and positive (ex. heparin treatment) controls also help further establish the assays&#8217; accuracy. The utility and applicability of such methods have been demonstrated in many antiviral studies26,30,31,40,41. Note that while heparin is included as a control for all three assays in the context of the representative results, it typically blocks the initial virus binding rather than the ensuing fusion/entry step (as reflected by the data in Figure 2). Additional controls could also be used, such as neutralizing antibodies directed against the virus (for viral inactivation assay), antibodies that mask the cell surface receptors for the virus (for viral attachment assay), and membrane fusion inhibitors (for viral fusion/entry assay). 
The assays described in this report, which are specific to the early stages of the viral infection, are useful in terms of application as secondary tests to characterize the mechanism of action of candidate drugs from primary screens which typically target the viral infection more broadly. Alternatively, they could also be incorporated in primary screens if one is specifically looking for inhibitors of early viral entry, including virus inactivating agents, viral attachment antagonists, and inhibitors to viral entry/fusion. In this case, their use allows a more focused and precise screen analysis for the identification of mechanism-specific antiviral candidates, which, in turn, would expedite downstream drug development. 
The use of cell-based assays in identifying antiviral agents provides several important advantages compared to biochemical assays, including revealing potential off-target effects (such as cytotoxicity) and adding physiological relevance to the bioactivity of the test agents42. These issues are important considerations for deciding whether a candidate agent is of value for continuation in subsequent phases of drug development. Similarly, the early viral entry-specific assays described in this report allow examination of the drug effect on the distinct viral entry stage at the cellular level, and more specifically in the context of an authentic viral infection in vitro. The results obtained from such assays would therefore help better predict the antiviral efficacy of the test compounds and also identify potentially unwanted off-target effects against the host cell. One potential limitation though, is that an in vitro cell-based assay may not completely reflect the actual in vivo entry step in the context of a natural viral infection. Nonetheless, the assays presented in this protocol do serve as an analytical platform for mechanism-based identification and evaluation of novel antiviral agents.
The development of reporter viruses or reporter cell systems to quantitate the amount of viral infection has greatly facilitated cell-based screening and evaluation of antiviral compounds. Examples include the use of recombinant viruses carrying a reporter gene or by means of recombinant human cell lines containing a reporter gene driven by the specific virus promoter31,43. In this report, the infection from luciferase-tagged HCV can be easily monitored by quantitating the reporter signal, thus facilitating data analysis. By incorporating these useful reporter-based tools, the early viral entry assays described here can essentially be adapted into high-throughput format for mechanism-based screening of small molecule libraries.
In conclusion, a protocol was described for assays dissecting the early viral entry as a means of identifying and evaluating mechanism-specific antiviral compounds. Such assays would be useful for discovering novel antagonists/inhibitors to viral entry and help expand the scope of antiviral agents for development as prophylactic and/or therapeutic treatments.

Viral infections are a constant threat to the public health and a significant cause of epidemic diseases, morbidity, and deaths worldwide. Specific modes of control against viral infections include vaccine development and antiviral therapies. While vaccine efforts have proven successful in immunizing against several viruses, many viral pathogens remain without a protective vaccine including dengue virus (DENV), human cytomegalovirus (HCMV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV)1-5. Antivirals, on the other hand, play an important role for the management of these viral infections when prophylactic vaccines are unavailable. However, to date, only few licensed and cost-effective antiviral drugs are available compared to the number of viral pathogens that threatens the public health. More importantly, due to an increase in global travel and rapid urbanization, the situation is aggravated by risks of epidemic outbreaks from emerging/re-emerging viral infections that are being introduced into non-indigenous areas6. Recent outbreaks caused by severe acute respiratory syndrome (SARS) virus, influenza viruses (H1N1, H5N1, H3N2, and H7N9), DENV, West Nile virus (WNV), measles virus (MV), Middle East Respiratory Syndrome (MERS) virus, and Ebola virus6-12 are among the examples reflecting the need for antivirals development when immunization and/or therapeutics are unavailable. In addition, there is always a potential risk of generating drug-resistant infections with currently used antivirals. Thus, the continuous development and expansion of the scope of antivirals to these emerging/re-emerging viral infections are necessary to provide better management strategies and safeguard the public health.
Most antiviral therapies consist of direct acting antivirals (DAAs) which target a specific viral protein or cofactor that mediates important steps in the viral life cycle. For example, the nucleoside analogue Acyclovir inhibits herpesvirus DNA polymerase, protease inhibitors Boceprevir and Telaprevir antagonize the HCV NS3, and Oseltamivir and Zanamivir are neuraminidase inhibitors that block the release of influenza virus particles from infected cells13-15. There are however very few licensed viral entry inhibitors including Enfuvirtide, which targets HIV gp41 to prevent fusion, and Maraviroc, which blocks the HIV co-receptor CCR5, thereby preventing viral entry16. Exploring novel antagonists/inhibitors to viral entry could help provide additional therapeutics for prophylactic or therapeutic use, such as in combination with other antivirals with a different mechanism of action to better manage viral infections17-19. 
Identification of antivirals can involve structure-guided drug design and candidate drug screening-based strategy. Methods for assessing antiviral activity of test agents include biochemical assays of enzymatic activity and evaluation by cell-based systems20-23. In cell-based systems, the viral life cycle can be dissected into distinct stages of infection, such as entry events (attachment, fusion, uncoating), the replication phase (viral genome replication and protein translation), and virion egress (assembly, maturation, and release). Since the assays can be adapted to investigate each specific stage using various tools and methods, this approach allows identification/examination of potential candidate antivirals with a specific mechanism of action targeting the distinct stage analyzed. For instance, to analyze drug effect specifically on the free virus particles prior to binding to the host cell, a &#8216;viral inactivation assay&#8217; can be performed. In this assay, the virus is allowed to incubate with the test drug and then diluted to titrate out the drug before infecting a cell monolayer. Additional steps such as viral attachment and entry/fusion stages can also be analyzed individually, by shifting the temperature during the infection. For many enveloped viruses, viral entry/fusion at the host cell membrane is greatly facilitated at 37 &#176;C, but is typically precluded at 4 &#176;C which does not affect virus binding24-29. Finally, the use of reporter viruses or cell systems could facilitate these studies and permit a high-throughput analysis.
We previously employed the cell-based approach and dissected the early entry of various enveloped viruses for the examination of antiviral compounds that potentially antagonize viral entry30,31. Herein, the various methods used, including viral inactivation, viral attachment, and viral entry/fusion assays, are described.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>GIBCO</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>In vitro toxicology assay kit, XTT-based</td>
			<td>Sigma</td>
			<td>TOX2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4&#160;</td>
			<td>GIBCO</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Bio-Tek Instrument, Inc.</td>
			<td>ELx800&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002420</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLux Gaussia luciferase assay kit</td>
			<td>New England Biolabs</td>
			<td>E3300L&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax-20/20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate, dihydrate</td>
			<td>Sigma</td>
			<td>71402</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
	</tbody>
</table>

early viral entry assays for the identification and evaluation of antiviral compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

في الشكل 1، تم إجراء &quot;فحص التثبيط الفيروسي &#39;لدراسة ما إذا كانت مدة محددة مركبات CHLA الطبيعي وPUG يمكن إبطال نشاط الفيروسات يلفها مختلفة في الدولة خالية من الخلايا ومنع العدوى لاحق. وقد تم تحديد السمية الخلوية وفيروسات الاستجابة للجرعة من هذه المركبات قبل إجراء دراسة الآلية 31. الفيروسات وعولج قبل مع مركبات اختبار ومن ثم تم تخفيفه الخلطات الفيروس للأدوية لتركيزات الفرعية العلاجية قبل التلقيح على أحادي الطبقة خلية منها لكل نظام الفيروس. كما هو مبين في الشكل 1، سواء CHLA وPUG يبدو للتفاعل مع virions خالية من الخلايا، مما يؤدي إلى آثار لا رجعة فيها محمية أحادي الطبقة الخلية من عدوى لاحقة. حققت مركبات اختبار اثنين في القريب 100٪ تثبيط ضد HCMV، HCV، وDENV-2، في حين أن 60 - وقد لوحظ كتلة 80٪ مقابل MV وRSV. هذه النتائج سوجمؤسسة أن CHLA وPUG يكون لها تأثير مباشر على هذه الجسيمات فيروس مجانا عن طريق تعطيل لهم وتحييد العدوى بها. في الشكل 2، وأجريت المرفق ودخول / المقايسات الانصهار لاستكشاف تأثير CHLA وPUG ضد هذه الأحداث ذات الصلة دخول الفيروسية في وقت مبكر من HCMV، HCV، DENV-2، MV، وRSV. كلا CHLA وPUG منعت بشكل فعال ملزم للفيروسات التحقيق على الخلية المضيفة كما هو مبين من خلال تثبيط على العدوى الفيروسية الناتجة (الشكل 2، &quot;مرفق&quot;: أشرطة رمادي فاتح). وكان تأثير كابح بشكل خاص على مرفق الفيروس عن طريق كل من مركبات مماثلة ضد HCMV (الشكل 2B)، HCV (الشكل 2C)، DENV-2 (الشكل 2D)، وRSV (الشكل 2F)، تتراوح 90-100٪. من ناحية أخرى، يبدو PUG أن يكون أكثر فعالية من CHLA ضد MV ملزم (الشكل 2E)، مع معدل تثبيط من TWس المركبات تتراوح بين 50-80٪. الهيبارين معاملة السيطرة، والذي يعرف لمنع دخول العديد من الفيروسات، وأيضا مرفق تحول دون لHCMV، DENV-2، RSV، إعلان MV، ولكن أقل كفاءة ضد HCV. فحص تلت ذلك &quot;الفيروسي دخول / الانصهار فحص&quot; ما إذا كان CHLA وPUG الاحتفاظ نشاطهم خلال مرحلة دخول فيروس / الانصهار (الشكل 2، &quot;دخول / فيوجن&quot;: أشرطة رمادية داكنة). مرة أخرى، لوحظت على حد سواء CHLA وPUG مخلا بشكل فعال الفيروسية خطوة دخول / الانصهار من الفيروسات فحص (الشكل 2B - F)، مما أسفر عن 50 - 90٪ تأثير وقائي على أحادي الطبقة خلية منها. الهيبارين أيضا تحول دون دخول أكثر قدرة / الانصهار في DENV-2 والتهابات RSV، ولكن كان أقل فعال ضد HCMV، HCV، وMV (&lt;40٪ تثبيط في المتوسط). <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> فيروس </td><td> نوع من الخلايا </td></tr><tr><td> HCMV </td> &lt;td&gt; HEL </td></tr><tr><td> HCV </td><td> هاه 7.5 </td></tr><tr><td> DENV-2 </td><td> فيرو </td></tr><tr><td> MV </td><td> CHO-SLAM </td></tr><tr><td> RSV </td><td> التهاب الكبد-2 </td></tr></tbody></table> الجدول 1: المضيف نوع من الخلايا لعدوى فيروسية يشار إلى نوع من الخلايا المستخدمة لكل نظام عدوى فيروسية موضح في نتائج ممثل. تفاصيل إضافية بشأن الخلايا يمكن العثور في اشارة 31. <img alt="الشكل 1" src="/files/ftp_upload/53124/53124fig1.jpg" /> الشكل 1. تثبيط الالتهابات الفيروسية التي CHLA مركبات اختبار وPUG تم علاج الفيروسات المختلفة مع مركبات الاختبار لفترة طويلة. (المحتضنة لمدة 1،5-3 ساعة قبل المعايرة؛ القضبان الرمادية الخفيفة) أو فترة قصيرة (مخفف على الفور، الرمادي الداكن القضبان) عند 37 درجة مئوية قبل التخفيف إلى concentra الفرعية العلاجيةنشوئها وتحليل لاحقة للإصابة في الخلايا المضيفة المعنية. (A) الخطط التجربة (كما هو موضح على اليسار) مع تركيز الفيروس النهائي (PFU / جيد أو وزارة الداخلية)، على المدى الطويل فترة حضانة الفيروس للأدوية (ط)، وفيما بعد فترة حضانة (ب) أشار لكل فيروس في الجدول على اليمين. تحليلات ل(B) HCMV، (C) HCV، (D) DENV-2، وأشار (E) MV، و (F) RSV في كل لوحة إضافية. يتم رسم النتائج ضد DMSO العلاج سيطرة سلبية لعدوى فيروس والبيانات المعروضة هي الوسائل ± الأخطاء المعيارية للمتوسط ​​(SEM) من ثلاث تجارب مستقلة. تم تعديل هذا الرقم من إشارة 31. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig1highres.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الرقم 2" src="/files/ftp_upload/53124/53124fig2.jpg" /> الشكل 2. تقييم الأنشطة المضادة للفيروسات من CHLA مركبات اختبار وPUG ضد مرفق فيروس ودخول / الانصهار. (A) وإجراء التجارب، وتركيز الفيروس (PFU / جيد أو وزارة الداخلية)، ووقت الجمع والمعالجة مع مركبات الاختبار (الأول والثاني والثالث) يتم عرض لكل الفيروس في الخطط والجداول المرتبطة بها. في التحليل المرفق فيروس (أشرطة رمادي فاتح)، كانت الطبقات الوحيدة من أنواع مختلفة من الخلايا برود قبل في 4 درجات مئوية لمدة 1 ساعة، ثم تعامل المشترك مع الفيروسات منها واختبار مركبات في 4 ° C (1،5-3 ساعة؛ ط) قبل غسل قبالة inoculates والمركبات اختبار للحضانة لاحقة (37 ° C؛ الثاني) وفحص عدوى الفيروس. في دخول الفيروس / تحليل الانصهار (أشرطة رمادية داكنة)، كانت الطبقات الوحيدة الخلية المصنفة مسبقا المبردة في 4 درجة مئوية لمدة 1 ساعة ثم تحدت مع الفيروسات منها في 4 درجة مئوية لمدة 1،5-3 ساعة (ط). كانت خلايا ثمغسلها وتعامل مع مركبات الاختبار لفترة حضانة إضافية (II) والتي تم خلالها تحول درجة الحرارة إلى 37 درجة مئوية لتسهيل الفيروسي حالة دخول / الانصهار. في نهاية الحضانة، وإزالة الفيروسات خارج الخلية إما عازلة سيترات (درجة الحموضة 3.0) أو يغسل PBS وحضنت الخلايا كذلك (ج) لتحليل عدوى الفيروس. نتائج (B) HCMV، (C) HCV، (D) DENV-2، وأشار (E) MV، و (F) RSV في كل لوحة إضافية. يتم رسم البيانات ضد DMSO العلاج سيطرة سلبية من عدوى فيروس ويتم عرضها كما يعني ± SEM من ثلاث تجارب مستقلة. تم تعديل هذا الرقم من إشارة 31. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig2highres.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a>

Watch this Scientific Journal Video about Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds at JoVE.com

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

الفيروسية في وقت مبكر فحوصات دخول لتحديد وتقييم المركبات المضادة للفيروسات

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

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JoVE Journal

Immunology and Infection

30.0K Views.  Taipei Medical University Hospital. Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Video: Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

نوع التحاليل الكمية للجميع الأنفلونزا من Hemagglutinins الفيروسية وNeuraminidases باستخدام الاجسام المضادة العالمي في فتحة بسيطة فحوصات لطخة

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

تضخيم وقياس HIV - 1 الحمض النووي الريبي في الأفراد المصابين بالايدز مع الأحمال الفيروسية أقل من الحد كشفها بواسطة فحوصات السريرية القياسية

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

فحوصات لتحديد رواية الأدوية المضادة للفيروسات ضد فيروس اللسان الأزرق

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

تحديد تركيز الفيروسي من خلال البلاك فحوصات: نظم تراكب عن طريق التقليدية والرواية

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

عالية الإنتاجية الكمي في الوقت الحقيقي RT-PCR الفحص لتحديد ملامح التعبير من أنواع الأول والثالث الانترفيرون الأنواع الفرعية

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

فحوصات النوعية والكمية للكشف وتوصيف مضادات الميكروبات البروتين

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

تطوير بوساطة حلقة-المجففة بالتبريد متساوي التضخيم الكواشف للكشف عن<em&gt; الكوكسيلا البورنيتية</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

مسحة طريقة أخذ العينات للكشف عن نوروفيروس الإنسان على السطوح

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

تنقية الحمض النووي الفيروسي للتعرف على البروتينات الفيروسية والخلوية المرتبطة بها

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

تقييم الاستجابات الخاصة بفيروس زيكا تي خلية في أجهزة إيمونوبريفيليجيد من الفئران المصابة Ifnar1-/-

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...