Biz bu çalışmada ve USDA kullanılan kargalar sağlamak için S. Werner teşekkür etmek istiyorum, APHIS, hayvan bakımı ve izlenmesi için WS, NWRC hayvan bakım personeli. Mansiyon veya bir ürünün kullanımı USDA onaylandığı anlamına gelmez. Bu çalışma için fon USDA, APHIS, Veteriner Hizmetleri tarafından sağlanmıştır.

Bizim protokol daha önce 7 yayınlanmış bir uyarlanmıştır. Hayvanları içeren tüm işlemler Amerika Birleşik Devletleri Tarım Bakanlığı (USDA), Hayvan ve Bitki Sağlığı Denetim Servisi (Aphis), Yaban Hayatı Hizmetleri (WS), Ulusal Yaban Hayatı Araştırma Merkezi (NWRC) Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1.. Crow Gavaging <ol><li> Amerikan karga sindirim sistemi yoluyla &#39;sahte beyin malzemenin&#39; geçişi zamanı tahmin. <ol><li> Pişmiş ve (Şekil 1) gavaj iğne bir 2 kullanarak mavi boya ve sonda 1 karga ile tam pişmiş yumurta 5 ml karıştırın. </li><li> Mavi / yeşil-lekeli dışkı atılır ediliyor artık kadar Crow her 30 dakikada bir kontrol edin. </li></ol></li><li> Bulaşmamış ve ölümcül hasta RML (Chandler suşu) enfekte C57BL / 6 fare beyinleri edinin. </li><li> 1x bir kurşun blender homojenleştiricide normal bir% 20 ağırlık / hacim ve enfekte fare beyin Homojenat oluşturmakfosfat tamponlu tuzlu su (PBS) ve cam boncuklar, daha sonra daha büyük bir parçacık maddenin ayrılması için 1 dakika için 3000 x g &#39;de santrifüj. Süpernatantı ve steril PBS içinde bir% 10 ağırlık / hacim üretmek için 1x PBS eşit hacmi ile seyreltilir. Ihtiyaç kadar -80 ° C&#39;de dondurun. </li><li> Önce sonda 17 saat karga kalemler dışında, su değil gıda kaldırmak, ancak. </li><li> , Tedavi gruplarına rastgele tahsis karga ve oral gavaj iğnesi bir 2 kullanılarak, normal ya da enfekte ya da fare beyin homojenatının, 5 ml her karga sonda. </li><li> Transferi bireysel kafeslere kargalar ve 4 saat sonrası gavaj (Şekil 2) için her bir kafes içinde tüm dışkısını toplamak ve havuz. </li><li> Homojen bir doku elde edene kadar cam boncuk ile bir mermi blender homojenleştiricisinde her karga için toplandı dışkı homojenize. * Crow dışkı daha çok sıvı ve kolayca karıştırılabilir. </li><li> Her karga için, 10 ml&#39;lik toplam hacim 9.5 ml 1 x PBS içine dışkı homojenat 500 ul seyreltik. </li><li> Fekal homogena santrifüj1,400 x g&#39;de 15 dakika boyunca te ve süpernatant ekstrakte edin. </li><li> , Ikinci bir mikrobiyal enfeksiyon tehlikesini en aza indirmek homojenat 100 ul başına 100 birim 1 ul / ml penisilin ve 100 ug / ml streptomisin (Invitrogen, NY) ile tedavi edilmesi için daha sonra süpernatant azaltmak için 20 dakika boyunca oda sıcaklığında, UV ışığı altında yer viral ve bakteriyel kontaminasyon riski. UV maruz kaldıktan sonra, herhangi bir geri kalan mikropların membran bozulması için 30 saniye boyunca 70 ayar bir 3000 Mp sonikatör sonikasyon örnekleri. </li></ol> 2. Fare aşılama <ol><li> Şu tedavi gruplarında (Tablo 1) için fareler tahsis: <ol><li> Grup 1 - Tedavi ile farenin ağızdan 5 ml enfekte Fare beyinleri ile zorla ağız yolundan verilmiştir karga dışkı homojenat, 1 ml (IP) intraperitonal olarak aşılandı. </li><li> Grup 2 - negatif bir tedavi farenin ağızdan 5 ml normal fare beyinleri ile zorla ağız yolundan verilmiştir karga dışkı Homojenat 1 ml IP aşılandı. </li><li> Enfeksiyonlandı sulandırmak1:100 w / v 1x PBS için Grup 3 ve 4 için d ve normal fare beyni homojenatı. </li><li> Grup 3 - pozitif kontrol fareleri enfekte fare beyni homojenatından 1 ml IP olarak aşılanmıştır. </li><li> Grup 4 - Negatif kontrol fareleri, normal fare beyni homojenatından 1 ml IP olarak aşılanmıştır. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> Tedavi Grubu </td><td> Hayvanların sayısı </td></tr><tr><td> Grup 1 </td><td> Scrapie + Crow Dışkı </td><td> 100 </td></tr><tr><td> Grup 2 </td><td> Scrapie - Crow Dışkı </td><td> 25 </td></tr><tr><td> Grup 3 </td><td> Scrapie + Fare Beyin </td><td> 10 </td></tr><tr><td> Grup 4 </td><td> Scrapie - Fare Beyin </td><td> 5 </td></tr></tbody></table> . Tablo 1 Scrapie inokülumunun durumu (negatif pozitif +, -) ve hayvan sayısı 7 kullanılır. <ol start="2"><li> Periton fareyi aşılamak: <ol><li> Nazikçe Scruff başparmak ve işaret parmağı ile sırt boyun kürk fare ve ventral yüzü ortaya çıkarmak için döndürün. </li><li> Kafası biraz daha düşük, bu nedenle fare arka ucunu yükseltin. </li><li> 1 cm deri, orta hatta 1 cm lateraline aracılığıyla 25 G iğne yerleştirin ve 1-2 cm anterior sakroiliak eklem iğne kilitleme şırınga kullanarak. </li><li> 1 ml fare vücut boşluğuna yavaş inoküle enjekte edilir. </li></ol></li></ol> 3. Fare İzleme <ol><li> Onlar fare skrapinin klinik belirtilerini ifade kadar günlük fareler izleyin. Klinik bulgular içerebilir: kifoz, ataksi, sert kuyruk, bakım eksikliği, zayıflatma, ve uyuşukluk. </li><li> 0 görünür = yok, 1 = orta, 2 = şiddetli nerede görünür işaretler, belirgin olduğunda 6 klinik bulguların her biri için fareler skor. </li><li> Her oturum için günlük toplam puanları için 1 gün ≥ 8 ulaştığınızda fareler Euthanize, ≥ 6 sürekli olarak 3 gün boyunca, ya da 365 gün aşılama (dpi) göndermek. </li><li> Hasat beyinleri hemen aşağıdaki euthan-70 ° C&#39;de asya ve mağaza </li><li> , Bir scrapie teşhisi doğrulamak aşağıdaki gibi seyreltilmiş proteinaz-K çözeltisi (PK) içindeki bir 50 ug / ml &#39;lik 3 ul beyin örnekleri sindirimi için: 3.1. PK ul, 500 mM EDTA, 12.5 ul, pH 8, 45 ° C&#39;de 30 dakika boyunca 1x PBS 109,39 ul, daha sonra 8 ul yükleme tamponu ilave edildikten ve 5 dakika boyunca 95 ° C&#39;de inkübe edilerek örnekleri PK etkisiz hale getirirler. Bir% 12 SDS-PAGE jeli, Electrophorese ve oda sıcaklığında 1 saat boyunca PBS içerisinde% 0.2 Tween-20 içerisinde Immobilon PVDF membran ve% 5 yağsız süt ile bloğuna transfer üzerine yük örnekleri. Daha sonra, oda sıcaklığında 1 saat boyunca Superblock içerisinde seyreltilmiş Horseradish peroksidaz, konjuge edilmiş anti-PrP Bar224 monoklonal antikoru ile prob. PBS-% 0.2 Tween 20 ile, 1 saat boyunca membran durulayın. , Görselleştirmek bir G-box jel dokümantasyon sistemi üzerinde kemolüminesans ve görüntü ile Western Blot 5 dakika inkübe. </li></ol>

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Çıkar çatışması ilan etti.

Biz karga sindirim sistemi yoluyla PrP Res malzemenin geçişini belgelemek için bir prosedür ortaya koymaktadır. Biz kargalar hastalıksız coğrafi bölgelere PrP Res malzeme translokasyonunda yeteneğine sahip olup olmadığını belirlemek için geleneksel yöntemleri kullanılır. Diğerleri bunu ortadan kaldırmak için başarısız ikisi de 19-21 ve kemirgen 22,23 sindirim sıvıları, geviş PrP Res direncini değerlendirdik. Onlar da potansiyel leş PrP ...

Bulaşıcı Süngerimsi Ensefalopati (TSE) yaban hayatı etkileyen ölümcül bulaşıcı nörodejeneratif hastalıklar, evcil hayvanlar ve insanlar vardır. TSE hastalıkları, bulaşıcı madde prion proteinlerinin 1 yanlış katlanmış veya patojenik izoformu (PrP S) olduğu görülmektedir. Koyun ve keçilerde scrapie;; sığır süngersi ensefalopati (TSE Hayvan hastalıkları karaca (Odocoileus hemionus), beyaz kuyruklu geyik (Odocoileus virginianus), geyik (Cervus elaphus) ve geyik (Alces alces) kronik israf hastalığı (CWD) içerir yerli sığırlarda BSE); Yetiştirilmiş vizon transmissible vizon ensefalopati; kedilerde feline süngerimsi ensefalopati; primatlarla 8 ve süngerimsi ensefalopati; egzotik hayvanat bahçesinde egzotik toynaklı süngerimsi ensefalopati aile Bovidae ruminates. Tek bir insan TSE hastalığı, Creutzfeldt-Jakob hastalığı, nadir ve PrP Res-Kirlenmiş alıcı tarafından satın alınması için düşünülmüşated gıda 9. Kontamine sığır 10 tüketilen Benzer şekilde, eğer BSE insanları enfekte edebilir. Tüm TSE hastalıkları, scrapie ve CWD hayvandan hayvana 2,3,11 yanı sıra çevresel kaynaklardan 4-6 olduğu tahmin edilmektedir kendini idame ettiren ve salgın enfeksiyon kaynakları ile sadece iki tanesi. Araştırma çoğu TSE hastalıkları klinik bulguların tezahürü PrP Res malzemenin doğal maruz olaylardan önemli uzun kuluçka dönemleri gerektiren 2-4,6,8 ve belirgin tür engelleri en aza indirmek, ancak, türler arası iletim 12-14 potansiyelini ortadan kaldırmak olmadığını göstermektedir . Bulaşıcı prion (PrP Res) malzeme yayılması için mekanizmalar belirlenmesi TSE hastalıkları manzara karşısında nasıl hareket hakkında soruları yanıtlayan için son derece önemlidir. Deneysel araştırmalar 15,16, kümes hayvanları ve domuz 17, ve Amerikan kargaları (Corvus eşyaları böcekler düşündürmektedirhyrhynchos) 7,18 PrP Res malzemenin pasif taşıyıcılar veya dağıtıcılar bulunmaktadır. Karga sindirim sistemi yoluyla PrP Res malzemenin geçişi geçenlerde TSE hastalıkları 7 dağılması oynayabileceği rolü göstererek, belgelenmiştir. Bu sonuçlar, kargalar, bir çöpçü, karşılaşma olabilir o akla yatkın hale tüketmek, ve hastalıksız bölgelere, dışkı biriktirme yoluyla bulaşıcı malzeme taşımak. Burada göstermek prosedürler kargalar sindirim sistemi yoluyla PrP Res malzemenin geçişini belgelemek için kullanılan ve büyük ölçüde gelecekteki ilgili araştırmalarda diğer çöpçü ve etobur türe özgü modeller için bu yöntemlerin uygulanmasını kolaylaştıracaktır. Bu çalışmada, geleneksel yöntemler PrP Res malzemenin yayılması ve genel yüküne katkıda bulunabilecek PrP ​​Res malzeme kaçakçılığı bir alışılmamış araçlar araştırmak için kullanılmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

Bulaşıcı prion (PrP Res) malzeme olasılıkla ölümcül, nörodejeneratif Bulaşıcı süngerimsi ensefalopati (TSE) hastalıklar 1 nedenidir. Bu tür kronik halsizlik hastalığı (CWD) ve TSE hastalıkları, Transmission, hayvan 2,3 hayvana hem de çevresel kaynaklarından 4-6 olduğu tahmin edilmektedir. Süpürücüleri ve etobur tüketim ve CWD kirlenmiş leş boşaltım yoluyla PrP Res malzemeyi translokasyonunda potansiyele sahiptir. Son çalışma Amerikan kargalar sindirim sistemi (Corvus brachyrhynchos), ortak bir Kuzey Amerika çöpçü 7 ile PrP Res malzemenin geçişini belgelemiştir. Biz Amerikan kargalar aracılığıyla PrP Res malzemenin geçişini belgelemek için kullanılan prosedürleri açıklamaktadır. Kargalar RML-deformasyon fare adapte skrapili zorla ağız yolundan ve onların dışkı 4 saat sonrası sonda toplandı. Crow dışkı içine daha sonra bir araya getirilmiş ve periton içine enjekte edildiC57BL / 6 fareleri. Bu fare skrapinin klinik belirtileri ifade edilmiş ve daha sonra kurban edilmiştir kadar Fareler, günlük olarak izlendi. Asemptomatik fareler 365 gün Aşıdan kadar takip edildi. Western Blot analizi hastalık durumunu teyit etmek için yapılmıştır. Sonuçlar prionların testi farelerde hastalığa neden kargalar sindirim sistemi yoluyla seyahat sonra bulaşıcı kalır ve dışkı mevcut olduğunu ortaya çıkarmıştır.

Kullanılan prosedürler karga sindirim sistemi scrapie beyin homojenatı 7 arasında oral gavaj sonrası PrP Res enfekte 4 saat ortadan etmediğini göstermektedir. PrP Res malzeme ile zorla ağız yolundan verilmiştir olan tüm yirmi karga, daha sonra farelere dışkı yoluyla PrP Res malzeme aktarılır. Hastalıklı farelerin fare klinik işaretler ve scrapie hastalık onay Western blot analizi ile tamamlandı tezahürü ile belirlenmiştir. K...

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Süpürücüleri hastalıksız bölgelere kendi dışkılarıyla bulaşıcı bulaşıcı süngerimsi ensefalopati prionları translokasyonunda potansiyele sahiptir. Biz fare adapte skrapi prıonlar Amerikan kargalar sindirim sisteminde (Corvus brachyrhynchos), ölü hayvanların ortak bir tüketici olsa geçtikten sonra bulaşıcı kalırsa belirlemek için kullanılan ayrıntı yöntemleri.

Bir Kuş Türlerinin Sindirim Sistemi geçtikten sonra Bulaşıcı prions belirlenmesi için prosedürler

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Research

JoVE Journal

Immunology and Infection

11.0K Görüntüleme.  USDA.  Süpürücüleri hastalıksız bölgelere kendi dışkılarıyla bulaşıcı bulaşıcı süngerimsi ensefalopati prionları translokasyonunda potansiyele sahiptir. Biz fare adapte skrapi prıonlar Amerikan kargalar sindirim sisteminde (Corvus brachyrhynchos), ölü hayvanların ortak bir tüketici olsa geçtikten sonra bulaşıcı kalırsa belirlemek için kullanılan ayrıntı yöntemleri. 

Video: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

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.

Çoğaltım ve Yeşil floresan proteini ifade Maedi-visna Virüs bir Enfeksiyöz Moleküler Clone algılama

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

İmmünoloji ve Enfeksiyon

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prionlar protein misfolding Döngüsel Amplifikasyon

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

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.

Kullanımı Geleneksel Hikaye ve Roman Yerleşimi Sistemleri: Plak Tahliller sayesinde viral Konsantrasyon Tayini

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

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

bir (A) Güvenlik Önlemleri ve Çalışma Usul BSL-4 Laboratuvar: 1. Biyogüvenlik Seviye 4 Suit Laboratuvar Suite Giriş ve Çıkış İşlemleri

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

bir (A) Güvenlik Önlemleri ve Çalışma Usul BSL-4 Laboratuvar: 2. Genel Uygulamalar

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

bir (A) Güvenlik Önlemleri ve Çalışma Usul BSL-4 Laboratuvar: 4. Tıbbi Görüntüleme İşlemleri

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

bir (A) Güvenlik Önlemleri ve Çalışma Usul BSL-4 Laboratuvar: 3. Aerobiyoloji

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

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.

Zika Virüs Bulaşıcı hücre kültürü sistemine ve<em&gt; İn Vitro</emİnterferonların&gt; profilaktik etkisi

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

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.

Lenfositler fare küçük bağırsak bağışıklık sistemi yalıtma

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

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.