Name: porcine corneal tissue explant to study the efficacy of herpes simplex virus-1 antivirals
Uploaded: 2021-09-20T15:00:00
Description: Watch this Scientific Journal Video about Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals at JoVE.com

这项研究得到了国家卫生研究院向D.S.A.提供的F30EY024710、RO1 AI139768和RO1 EY029426的资助，并得到了国家眼科研究所F30EY025981赠款的支持。研究使用从公园包装公司获得的猪角膜进行，4107阿什兰大道，新城，芝加哥，IL-60609

这项研究中使用的所有猪组织都由第三方私人组织提供，伊利诺伊大学芝加哥分校的人员没有对动物进行处理。
1. 材料
<ol><li>
试剂
	<ol>
<li>使用以下试剂进行普拉克检测：粉末甲基纤维素、杜尔贝科改良鹰的介质（DMEM）、胎儿牛血清（FBS）、青霉素和链霉素（P/S）用于普拉克检测。</li>
		<li>使用水晶紫罗兰片剂和乙醇（分子生物学等级）为斑块检测准备水晶紫...

<ol>
	<li>Liesegang, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+epidemiology+and+ocular+importance.">Herpes simplex virus epidemiology and ocular importance.</a> Cornea. 20 (1), 1-13 (2001).</li><li>Farooq, A. V., Valyi-Nagy, T., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediators+and+mechanisms+of+herpes+simplex+virus+entry+into+ocular+cells.">Mediators and mechanisms of herpes simplex virus entry into ocular cells.</a> Current Eye Research. 35 (6), 445-450 (2010).</li><li>Farooq, A. V., Shah, A., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+herpesviruses+in+ocular+infections.">The role of herpesviruses in ocular infections.</a> Virus Adaptation and Treatment. 2 (1), 115-123 (2010).</li><li>Xu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seroprevalence+and+coinfection+with+herpes+simplex+virus+type+1+and+type+2+in+the+United+States,+1988-1994.">Seroprevalence and coinfection with herpes simplex virus type 1 and type 2 in the United States, 1988-1994.</a> Journal of Infectious Diseases. 185 (8), 1019-1024 (2002).</li><li>Xu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+herpes+simplex+virus+type+1+and+type+2+seroprevalence+in+the+United+States.">Trends in herpes simplex virus type 1 and type 2 seroprevalence in the United States.</a> Journal of the American Medical Association. 296 (8), 964-973 (2006).</li><li>Koganti, R., Yadavalli, T., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+emerging+therapies+for+ocular+herpes+simplex+virus+type-1+infections.">Current and emerging therapies for ocular herpes simplex virus type-1 infections.</a> Microorganisms. 7 (10), (2019).</li><li>Lobo, A. -., Agelidis, A. M., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+herpes+simplex+keratitis:+The+host+cell+response+and+ocular+surface+sequelae+to+infection+and+inflammation.">Pathogenesis of herpes simplex keratitis: The host cell response and ocular surface sequelae to infection and inflammation.</a> Ocular Surface. 17 (1), 40-49 (2019).</li><li>Koujah, L., Suryawanshi, R. K., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+processes+activated+by+herpes+simplex+virus-1+(HSV-1)+infection+in+the+cornea.">Pathological processes activated by herpes simplex virus-1 (HSV-1) infection in the cornea.</a> Cellular and Molecular Life Sciences. 76 (3), 405-419 (2019).</li><li>Lass, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiviral+medications+and+corneal+wound+healing.">Antiviral medications and corneal wound healing.</a> Antiviral Research. 4 (3), 143-157 (1984).</li><li>Burns, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterisation+of+resistant+Herpes+simplex+virus+after+acyclovir+therapy.">Isolation and characterisation of resistant Herpes simplex virus after acyclovir therapy.</a> Lancet. 1 (8269), 421-423 (1982).</li><li>Crumpacker, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antiviral+drugs+of+herpes+simplex+virus+isolated+from+a+patient+treated+with+Acyclovir.">Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with Acyclovir.</a> New England Journal of Medicine. 306 (6), 343-346 (2010).</li><li>Yildiz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+kidney+injury+due+to+acyclovir.">Acute kidney injury due to acyclovir.</a> CEN Case Report. 2 (1), 38-40 (2013).</li><li>Fleischer, R., Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyclovir+nephrotoxicity:+a+case+report+highlighting+the+importance+of+prevention,+detection,+and+treatment+of+acyclovir-induced+nephropathy.">Acyclovir nephrotoxicity: a case report highlighting the importance of prevention, detection, and treatment of acyclovir-induced nephropathy.</a> Case Rep Med. 2010, 1-3 (2010).</li><li>Thakkar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+corneas+show+dendritic+spread+and+restrict+herpes+simplex+virus+infection+that+is+not+observed+with+cultured+corneal+cells.">Cultured corneas show dendritic spread and restrict herpes simplex virus infection that is not observed with cultured corneal cells.</a> Science Report. 7, 42559 (2017).</li><li>Pescina, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+Development+of+a+convenient+ex+vivo+model+for+the+study+of+the+transcorneal+permeation+of+drugs:+Histological+and+permeability+evaluation.">et al Development of a convenient ex vivo model for the study of the transcorneal permeation of drugs: Histological and permeability evaluation.</a> Journal of Pharmaceutical Sciences. 104, 63-71 (2015).</li><li>Jaishankar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+off-target+effect+of+BX795+blocks+herpes+simplex+virus+type+1+infection+of+the+eye.">An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye.</a> Science Translational Medicine. 10, 5861 (2018).</li><li>Duggal, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+oxide+tetrapods+inhibit+herpes+simplex+virus+infection+of+cultured+corneas.">Zinc oxide tetrapods inhibit herpes simplex virus infection of cultured corneas.</a> Molecular Vision. 23, 26-38 (2017).</li></ol>

先前的研究表明，BX795作为抗HSV-1感染的抗病毒药物具有很有前途的作用：通过抑制坦克绑定激酶1（TBK1）16。TBK1和自噬在帮助抑制HSV-1感染方面都起到了一定的作用，这表现在人类角膜上皮细胞上。BX795在浓度为10μM的抗病毒活性方面被证明是最大的有效，并且使用西方斑点分析和关键病毒蛋白的病毒斑块分析，BX795被证明可以抑制HSV-1感染，与TFT16的活动相当。?...

患有眼部感染的人经常会出现视力丧失1。由于全球血清高超常，HSV感染者患有复发性眼部感染，导致角膜疤痕、频闪角膜炎和新血管化2、3、4、5。HSV感染也显示导致较少的频率，免疫功能低下，未经治疗的患者，如脑炎和系统性发病率6，7，8的一系列严重情况。像Acyclovir（ACV）及其核苷类比这样的药物在抑制HSV-1感染甚至控制重新激活方面已经取得了一致的成功，然而这些药物的长期使用与肾衰竭、胎儿畸形和未能限制抗药性对进化的病毒株9、10、11、12、13的产生有关。与HSV-1眼部感染相关的复杂性，以前曾使用人类角膜细胞的单层和3D培养物进行体外研究，并使用穆林或兔眼感染进行体内研究。虽然这些体外模型提供了关于HSV-1感染的细胞生物成分的重要数据，但是，它们不能模仿角膜组织的复杂复杂性，也无助于照亮病毒的树突传播。相比之下，虽然体内系统在显示 HSV-1 感染期间角膜感染传播和免疫激活反应方面更有见地，但它们确实附带了警告，即它们需要训练有素的研究人员和大型动物护理设施来忽略实验。
在这里，我们使用猪角膜作为前体内模型来检查HSV-1感染诱发的伤口系统。通过组织外植培养，可以研究某些药物的潜在药理学以及感染引起的伤口系统的细胞和分子生物学。该模型也可以修改为用于其他病毒和细菌感染。在这项研究中，猪角膜被用来测试临床前小分子BX795的抗病毒功效。由于容易获得和成本效益，更倾向于使用猪角膜。此外，猪角膜模型是人眼的良好模型，角膜易于分离，适合感染和可视化，坚固耐用，可处理15。猪角膜也可与人类角膜模型在跨角膜渗透性和全身吸收15的复杂性相媲美。通过使用这个模型进行研究，我们能够阐明BX795作为HSV-1病毒感染的称职抑制剂如何值得进一步研究，并增加了将其分类为潜在的小分子抗病毒化合物16的文献。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G hypodermic needles.</td>
			<td>BD</td>
			<td>305128</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL glass bottle.</td>
			<td>Thomas Scientific</td>
			<td>844027</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycotic and Antibiotic (AA)</td>
			<td>GIBCO</td>
			<td>15240096</td>
			<td>Aliquot into 5 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Benchtop vortexer.</td>
			<td>BioDot</td>
			<td>BDVM-3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet with a Bio-Safety Level-2 (BSL-2) certification.</td>
			<td>Thermofisher Scientific</td>
			<td>Herasafe 2030i</td>
			<td></td>
		</tr>
		<tr>
			<td>Calgiswab 6&#34; Sterile Calcium Alginate Standard Swabs.</td>
			<td>Puritan</td>
			<td>22029501</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper - 25 cm</td>
			<td>Biologix BE</td>
			<td>70-1180 70-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>C6158</td>
			<td>Store the powder in a dark place</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium - DMEM</td>
			<td>GIBCO</td>
			<td>41966029</td>
			<td>Store at 4 &#176;C until use</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum -FBS</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td>Aliquot into 50 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Flat edged tweezers &#8211; 2.</td>
			<td>Harward Instruments</td>
			<td>72-8595</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezers --80 &#176;C. -</td>
			<td>Thermofisher Scientific</td>
			<td>13 100 790</td>
			<td></td>
		</tr>
		<tr>
			<td>Fresh box of blades.</td>
			<td>Thomas Scientific</td>
			<td>TE05091</td>
			<td></td>
		</tr>
		<tr>
			<td>Guaze</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td>108 square inch folder 12 ply</td>
		</tr>
		<tr>
			<td>HSV-1 17GFP</td>
			<td>grown in house</td>
			<td>-</td>
			<td>Original strain from Dr. Patricia Spears, Northwestern University. GFP expressing HSV-1 strain 17</td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium - ITS</td>
			<td>GIBCO</td>
			<td>41400045</td>
			<td>Aliquot into 5 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Magnetic stirrer.</td>
			<td>Thomas Scientific</td>
			<td>H3710-HS</td>
			<td></td>
		</tr>
		<tr>
			<td>Metallic Scissors.</td>
			<td>Harward Instruments</td>
			<td>72-8400</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 1 to 1000 &#181;L.</td>
			<td>Thomas Scientific</td>
			<td>1159M37</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium - MEM</td>
			<td>GIBCO</td>
			<td>11095080</td>
			<td>Store at 4 &#176;C until use</td>
		</tr>
		<tr>
			<td>OptiMEM&#160;</td>
			<td>GIBCO</td>
			<td>31985047</td>
			<td>Store at 4 &#176;C until use</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin.</td>
			<td>GIBCO</td>
			<td>15140148</td>
			<td>Aliquot into 5 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline -PBS</td>
			<td>GIBCO</td>
			<td>10010072</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Porcine Corneas</td>
			<td>Park Packaging Co., Chicago, IL</td>
			<td>0</td>
			<td>Special order by request</td>
		</tr>
		<tr>
			<td>Procedure bench covers - as needed.</td>
			<td>Thermofisher Scientific</td>
			<td>S42400</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Thomas Scientific</td>
			<td>P7132, P7127, P7128, P7129, P7137</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipetting equipment.</td>
			<td>Thomas Scientific</td>
			<td>Ezpette Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Carl Zeiss</td>
			<td>SteREO Discovery V20</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirring magnet.</td>
			<td>Thomas Scientific</td>
			<td>F37120</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks, T175 cm2.</td>
			<td>Thomas Scientific</td>
			<td>T1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture incubators which can maintain 5% CO2 and 37 &#176;C temperature.</td>
			<td>Thermofisher Scientific</td>
			<td>Forma 50145523</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture treated plates (6-well).</td>
			<td>Thomas Scientific</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>GIBCO</td>
			<td>25-300-062</td>
			<td>Aliquot into 10 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>American Type Culture Collection ATCC</td>
			<td>CRL-1586</td>
			<td></td>
		</tr>
	</tbody>
</table>

porcine corneal tissue explant to study the efficacy of herpes simplex virus-1 antivirals

病毒和细菌可通过角膜感染导致各种眼部表面缺陷和退化，如伤口和溃疡。由于血清超常范围为全球 60-90%，单纯疱疹病毒类型-1 （HSV-1） 通常会导致或非酸性区域的粘皮病变，这也表现为病变和感染相关失明。虽然目前的抗病毒药物是有效的，但耐药性和持久性毒副作用的出现需要开发针对这种无处不在的病原体的新型抗病毒药物。虽然体外评估提供了一些有关新兴抗病毒药物的功能数据，但它们并不显示体内眼组织具有复杂性。然而，在体内研究是昂贵的，需要训练有素的人员，尤其是当与病毒剂工作。因此，前体内模型是高效但廉价的抗病毒测试步骤。在这里，我们讨论一个协议，研究感染HSV-1使用猪角膜前体内和方法，以治疗他们使用现有的和新的抗病毒药物。我们还演示了使用 HSV-1 进行斑块检测的方法。详细的方法可用于进行类似的实验，以研究类似于HSV-1病原体的感染。

为了了解实验抗病毒药物的功效，在进行人体人体临床试验之前，需要对它们进行广泛的测试。在这方面，必须确定正控、阴性控制和测试组。三氟二甲胺（TFT）长期以来一直被用作治疗疱疹角膜炎的首选治疗方法。作为阳性控制，TFT治疗的角膜组显示感染传播较低。作为负控制，我们使用 DMSO 或溶解在 PBS 中的车辆控制。BX795，实验性临床前药物是测试组。每组共分配了4个角?...

Watch this Scientific Journal Video about Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals at JoVE.com

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

我们描述了使用猪角膜来测试实验药物的抗病毒功效。

猪角膜组织外植研究单纯疱疹病毒-1抗病毒药物的功效

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

了解药物在动物模型中的疗效是成本和劳动密集型。我们使用前体内组织外植模型的协议在进行研究所需的成本和科学人员方面都显著降低。这项技术的主要优点是使用猪眼，与其他一些小型动物模型相比，猪眼的解剖学和生理学与人眼非常相似。当我们在体外测试药物时，它们可能显示出在动物或人类模型中可能不存在的疗效。前体内组织系统是了解我们的体外系统是否可以转化为动物和人体试验的最佳方法。猪角膜系统虽然劳动密集程度较低，但需要训练有素的技能来分离、维持、感染和治疗药物，特别是将角膜与整个猪眼隔离的过程很难用语言来表达，但需要视觉演示。从合适的供应商处接收猪眼后，将组织储存在冰上直到加工，并放置适当的个人防护设备。用 70% 乙醇对工作区域进行消毒，并在工作空间上固定一个长凳盖。当工作区域准备就绪时，将眼睛放在50毫米的纱布上，用纱布一只手抓住后部。使用30度针头，小心地在眼睛上皮表面的大约中心进行一次穿刺，注意不要损坏频闪，并用锋利的绝育刀片在离角膜一毫米远的硬囊上做一个小切口。使用快速和光滑的手旋转动作，并注意，球形幽默不泄漏，切开角膜的边缘，并用扁平的钳子将角膜固定在角膜边缘，使用刀片切断剩余的系绳膜。当每个角膜被分离时，将组织面朝上放入每个井中含有两毫升角膜介质的12井板的单独井中，并在每个角膜表面的脱布里部位增加5个5倍10的微升，加入17个GFP病毒溶液的第五块斑块形成单元。收集完所有角膜后，将板材放入37摄氏度、5%的二氧化碳孵化器中72小时，并用10%的漂白剂喷洒实验中未使用的任何额外眼睛，将其安全地处理在生物危害袋中。每天在添加药物之前，将角膜板置于立体显微镜下，并选择 GFP 过滤器和 500 微秒的暴露时间。捕捉图像在最低放大倍率之前成像角膜在一系列增加的放大倍率，直到所有的病毒传播和树突形成可以清楚地可视化。当所有的角膜都被成像时，将板返回到组织培养孵化器。感染前一天，用每口10毫升PBS清洗两次汇流病毒细胞培养，并在37摄氏度下用每口井0.05%的1毫升试金石治疗细胞5至10分钟。当细胞分离后，使用整个介质的9毫升，以确保细胞完全从烧瓶表面脱落，并将产生的细胞悬浮转移到15毫升离心机管。将300微升细胞加入到新六井板的每口井中，然后向每口井添加两毫升中等，然后将板放在细胞培养孵化器中过夜。第二天早上，为了从每个角膜中收集病毒进行量化，在生物安全柜中的多个微中心管中将无菌棉签尖浸泡在500微升无血清介质中，至少5分钟。在孵化结束时，小心地将受感染的猪角膜培养物放入柜子中。使用湿棉签，顺时针进行三次旋转，从每个受感染的猪角膜中心逆时针旋转三圈，不施加过大的压力。在一系列革命的结尾，在无血清介质中顺时针和逆时针将每个拭子旋转五次，然后切割拭子，使尖端适合每个带盖子的管内。当所有的角膜都被擦拭时，以高速涡流管一分钟。要量化从每个角膜中收集的病毒量，请在微中心管中的无血清介质中准备病毒的日志101倍稀释，直到稀释10倍至负8。从每口细胞井中吸气生长介质后，将每口病毒稀释的一毫升从10倍转移到负3到镀细胞单层，并将受感染的细胞放入细胞培养孵化器中两小时。在孵化结束时，用PBS轻轻清洗每口井两次，然后向每口井加入两毫升甲基纤维素介质，进行72小时的孵化，或直到观察到斑块的形成。观察斑块后，在每个井的一角慢慢加入一毫升甲醇，在室温下孵育15分钟。在孵化结束时，慢慢地从每口井中吸出内容，而不会干扰细胞单层。接下来，用一毫升的水晶紫罗兰工作溶液给每口井贴上标签，注意所有细胞都覆盖在30分钟的潜伏期内，不受光线的照射。在孵化结束时，丢弃溶液，在吸水纸上干燥油井，然后计算最高稀释井的斑块数量，以三次量化起始溶液中的总病毒含量。正如这种立体荧光成像分析所观察到的，BX795的抗病毒功效与TFT在控制病毒传播方面相似，而用车辆治疗的角膜仅在初始病毒接种后六天内证明病毒从中央感染区传播到外围。同样，在感染后的第2天和第4天采集的眼拭子在阳性对照和BX795处理的样本中表现出对病毒的完全抑制，而在阴性控制样本中观察到感染性病毒滴答声的急剧增加。将猪角膜与整个猪眼隔离是最重要的一步。步骤 2.3 到 2.6 是此过程中最关键的步骤。我们的模型可用于了解病毒在猪角膜中的树突传播。这种方法也可以扩展到细菌和真菌感染。这种方法有助于可视化病毒在人类模型外的角膜中的树突传播。该模型有助于在进行动物和人体试验之前测试药物的功效。

Research

JoVE Journal

Immunology and Infection

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

利用反向遗传学操作的裂谷热病毒MP - 12菌株，以提高疫苗的安全性和有效性的NSS基因

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

科研

免疫与感染

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

为检验新型抗病毒药物的鉴定对蓝舌病病毒

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

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

一种测试洗手方法去除新发感染性病原体的方法

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...