وقد تم تمويل هذا البحث من قبل الزمالة ويلكوم تراست التي منحت لكبار Goodfellow إيان، وماري كوري البينية الأوروبية زمالة (FP7 مجلس البحوث الأوروبي) التي منحت لأرياس ارماندو. نود أن نشكر ايو كونغ تشيونغ، الدكتور روباي ريبيكا والدكتور الدباغ مايك لمنحنا الإذن لاستخدام هذه اجيلنت bioanalyser والمساعدة في تشغيل عينات من الحمض النووي الريبي.

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M., Chaudhry, Y., Bailey, D., Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+optimized+RNA-based+murine+norovirus+reverse+genetics+system.">Development of an optimized RNA-based murine norovirus reverse genetics system.</a> J. Virol. Methods. 169, 112-118 (2010).</li><li>Arias, A., Perales, C., Escarmis, C., Domingo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+mutants+of+VPg+reveal+new+cytopathology+determinants+in+a+picornavirus.">Deletion mutants of VPg reveal new cytopathology determinants in a picornavirus.</a> PLoS One. 5, e10735 (2010).</li><li>Werf, S. v. a. n. d. e. r., Bradley, J., Wimmer, E., Studier, F. W., Dunn, J. 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ليس لدينا ما يكشف.

هنا قد بينت أننا مختلفين النهج الوراثية العكسية التي تسمح للانتعاش من MNV المعدية في زراعة الخلايا. كلا النهجين على نحو فعال تجاوز الشرط المطلق للربط تساهمي من VPG إلى نهاية 5 &#39;من الجينوم الفيروسي RNA عن طريق توليد النصوص MNV المغطاة التي يتم التعرف عليها بعد ذلك من قبل ريبوسوم الخلوية. في النسخ المختبر يليه السد إنزيمي هو أكثر كفاءة في انتعاش MNV المعدية من النسخ من البلازميدات المعدية في الخلايا معربا عن T7 بوليميريز الحمض النووي الريبي، والتي يمكن أن توج على محاضر من الانزيمات FPV متوجا. titres فيروس تعافى مع هذه النظم الهندسة الوراثية المعكوسة هي مماثلة لتلك التي حصلت عليها ترنسفكأيشن من الحمض النووي الريبي VPG المرتبطة الفيروسية تنقيته من مزارع الخلايا المصابة 17 (الشكل 5). وترنسفكأيشن من الحمض النووي الريبي MNV توج في الخلايا Raw264.7 متساهل يجعل 1 عيار الفيروس فقط 1-سجل أقل من التجارب التي تنطوي على transfectioن من الحمض النووي الريبي من مجموع الخلايا المصابة التي تحتوي على فيروسات VPG المرتبطة الحمض النووي الريبي (الشكل 5). هذا الواقع يشجع على اجراء مزيد من التحقيقات لتحديد ما إذا كان بالإضافة إلى ذلك من جزيء VPG إلى نهاية 5 &#39;من النصوص التي تولدها هذه النظم يمكن أن يؤدي إلى زيادة غلة فيروس والتي قد تكشف عن الجوانب الفنية الكامنة وراء MNV العدوى في الخلايا المرتبطة VPG. ومع ذلك، فإننا نعتبر هذا نظام الوراثة العكسية باعتبارها واحدة ذات كفاءة عالية مقارنة للفيروسات الحمض النووي الريبي عكس غيرها من النظم المستخدمة حاليا في علم الوراثة الذي في الحمض النووي الريبي المختبر المحولة يسمح للانتعاش من titres فقط 10-100 في أقل من التهابات حقيقية مع virions 19 و 20. وعموما، فإن المنهجيات الحالية تشكل خطوة كبيرة إلى الأمام في مجال البيولوجيا الجزيئية ونوروفيروس توفر لنا الأدوات اللازمة للتحقيق في الأدوار الوظيفية للبروتينات وحفظها الزخارف في الحمض النووي الريبي الجينوم نوروفيروس. وقد تم بالفعل هذه المناهج جنبا إلى جنب مع جurrent الفأر نموذج المتاحة، وأظهرت أن MNV تعافى من [كدنا] المعدية هي قادرة على أن تسبب عدوى قاتلة من&gt; 80٪ STAT1 / - الفئران في أقل من 10 أيام 4 و 21. الاستفادة من هذا النظام انتشلنا قابلة للحياة المسوخ نوروفيروس الفئران من البروتين قفيصة والمسالك polypyrimidine المشاركة في ملزم من العوامل المضيف مختلفة (PTB وPCBP) التي تعرض الظواهر الموهن إلى حد ما في الجسم الحي 21 و 22. وبالإضافة إلى ذلك، لقد أثبتنا في الآونة الأخيرة أن الفيروسات التي تفتقر إلى القدرة على التعبير عن هذا البروتين VF1 من ORF4 بكفاءة تكرار في ثقافة الخلية ولكن مرة أخرى قد خفضت الفوعة في الفئران فيما يتعلق MNV WT 8. هذه الدراسات تشجيع لنا لتصميم إصدارات الموهن من noroviruses الإنسان على أساس دراسات MNV التي يمكن أن يتم التحقيق في مثل اللقاحات المرشحة المحتملة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> حل ليثيوم كلوريد هطول الأمطار </td><td> Ambion </td><td> AM9480 </td></tr><tr><td> الحمض النووي الريبي تخزين حل </td><td> Ambion </td><td> AM7000 </td></tr><tr><td> MEGAscript T7 النصي </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> ScriptCap m7G السد النظام </td><td> التكنولوجيات الحيوية Epicentre </td><td> SCCE0610 </td></tr><tr><td> نيون ترنسفكأيشن نظام </td><td> إينفيتروجن </td><td> MPK5000 </td></tr><tr><td> نيون عدة ترنسفكأيشن نظام </td><td> إينفيتروجن </td><td> MPK1025 </td></tr><tr><td> OPTI-MEM أنا </td><td> إينفيتروجن </td><td> 31985070 </td></tr><tr><td> الكاشف Lipofectamine ترنسفكأيشن 2000 </td><td> إينفيتروجن </td><td> 11668-027 </td></tr><tr><td> اجيلنت RNA 600 نانو عدة </td><td> اجيلنت </td><td> 5067-1511 </td></tr><tr><td> اجيلنت 2100 ثنائيةoanalyzer </td><td> اجيلنت </td><td> G2939AA </td></tr><tr><td> Illustra GFX PCR الحمض النووي وتنقية جل كيت باند </td><td> جنرال إلكتريك للرعاية الصحية </td><td> 28-9034-70 </td></tr><tr><td> RiboMAX كبير إنتاج الحمض النووي الريبي مقياس النظام-T7 </td><td> Promega </td><td> P1300 </td></tr></tbody></table>

reverse genetics mediated recovery of infectious murine norovirus

noroviruses الإنسان هي المسؤولة عن معظم حالات التهاب المعدة والأمعاء البشرية (GE) في جميع أنحاء العالم، وهي مشكلة متكررة في البيئات التي لا يمكن فيها وثيقة من شخص إلى شخص الاتصال يمكن تجنبها 1 و 2. خلال السنوات القليلة الماضية تم الإبلاغ عن حدوث زيادة في معدل انتشار المرض في المستشفيات، مما تسبب في اضطرابات كبيرة في قدرتها التشغيلية، فضلا عن خسائر اقتصادية كبيرة. وقد اقتصرت على تحديد نهج جديد للفيروسات نظرا لعدم قدرة noroviruses الإنسان لإكمال عدوى الإنتاجية في زراعة الخلايا 3. وقد فتحت ويمكن عزل مؤخرا من نوروفيروس الفئران (MNV)، ترتبط ارتباطا وثيقا نوروفيروس الإنسان (4) ولكن الذي نشر في الخلايا 5 آفاقا جديدة للتحقيق في هذه الممرضة 6 و 7. النتائج تكرار MNV في تركيب جديد الجينومية بالمعنى الإيجابي وsubgenomic جزيئات الحمض النووي الريبي، وهذه الأخيرة والتي تتطابق مع ثي مشاركةالثالث من الجينوم الفيروسي (الشكل 1). MNV يحتوي على أربعة أنواع مختلفة من إطارات القراءة المفتوحة (ORFs)، والتي تحتل معظم ORF1 من الجينوم وبترميز 7 غير الهيكلية البروتينات (NS1-7) أفرج عنه من تمهيدا polyprotein. وترد ORF2 وORF3 داخل المنطقة RNA subgenomic وترميز البروتينات قفيصة (VP1 وVP2، على التوالي) (الشكل 1). مؤخرا، حددنا أن ORF4 إضافية تداخل ORF2 لكن في إطار قراءة مختلفة وظيفية وتشفير لعامل الفوعة المترجمة الميتوكوندريا (VF1) 8. النسخ المتماثل للشعور إيجابي فيروسات الحمض النووي الريبي، بما في ذلك noroviruses، يحدث في السيتوبلازم مما أدى إلى توليف جينوم الحمض النووي الريبي جديدة لم يسبق لهم اللعب. لتشجيع ترجمة الفيروسية والفيروسات استغلال الاستراتيجيات المختلفة التي تهدف إلى توظيف تصنيع البروتين الخلوي آلية 9-11. ومن المثير للاهتمام، هو الدافع ترجمة نوروفيروس من VPG البروتين الفيروسي التمهيدي متعددة الوظائفمرتبطة تساهميا إلى نهاية 5 &#39;من كلا RNAs والجينومية وsubgenomic 12-14. هذه الآلية المتطورة للترجمة من المرجح أن يكون عاملا رئيسيا في كفاءة محدودة من الانتعاش الفيروسية بواسطة التقليدية نهج الوراثة العكسية. نحن هنا نورد استراتيجيتين مختلفتين استنادا إلى جيل من الفئران 1-نوروفيروس (المشار إليها باسم MNV طيه) محاضر توج في نهاية 5 &#39;. إحدى الطرق ينطوي على حد سواء في تركيب المختبر، ووضع حد أعلى من الحمض النووي الريبي الفيروسي، في حين أن النهج الثاني ينطوي على نسخ من MNV [كدنا] في الخلايا معربا عن T7 بوليميريز الحمض النووي الريبي. وقد وفرت من توافر هذه الأنظمة الوراثة العكسية لدراسة MNV ونموذج الحيوان الصغير قدرة غير مسبوقة لشرح دور تسلسلات فيروسية في تكرار والمرضية 15-17.

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Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Noroviruses هي أحد الأسباب الرئيسية لالتهاب المعدة والأمعاء بعد التقنيات الجزيئية لتوصيف هذه لا تزال جديدة نسبيا. نحن هنا نورد مختلفين عكس نهج الوراثة لاستعادة كفاءة نوروفيروس الفئران (MNV)، العضو الوحيد من هذا جنس والتي يمكن نشرها في ثقافة الخلية.

علم الوراثة العكسية الاسترداد بوساطة من نوروفيروس الفئران المعدية

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

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Immunology and Infection

16.5K Views.  Imperial College London. Noroviruses هي أحد الأسباب الرئيسية لالتهاب المعدة والأمعاء بعد التقنيات الجزيئية لتوصيف هذه لا تزال جديدة نسبيا. نحن هنا نورد مختلفين عكس نهج الوراثة لاستعادة كفاءة نوروفيروس الفئران (MNV)، العضو الوحيد من هذا جنس والتي يمكن نشرها في ثقافة الخلية.

Video: Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

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

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

باستخدام علم الوراثة العكسية للتلاعب الجيني NSS لفيروس حمى الوادي المتصدع MP - 12 سلالة لتحسين سلامة وفعالية اللقاحات

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

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

الفحص وحة لنوروفيروس الفئران

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

عزل والخلايا اللمفاوية المناعية والخلايا الجذعية من الفئران باير لالرقع

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Organoids كنموذج للأمراض المعدية: ثقافة الإنسان وOrganoids المعدة الفئران وهيليكوباكتر بيلوري Microinjection من

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

العزلة وتدفق Cytometric توصيف الفئران الصغيرة المعوية اللمفاويات

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

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 ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

أسلوب لتقييم وظائف المستجيب Fc بوساطة الناجم عن الإنفلونزا هيماغلوتينين أجسام مضادة محددة

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

عزل الخلايا الليمفاوية من الماوس صغيرة المعوية المناعي

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

إعداد موريني الغدة اللعابية اللهاة تستقيم مجهرية إينترافيتال

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...