Name: isolation and quantification of zika virus from multiple organs in a mouse
Uploaded: 2019-08-15T13:30:00
Description: Watch this Scientific Journal Video about Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse at JoVE.com

Pinto博士的资金来自圣路易斯大学医学院的种子赠款和圣路易斯大学医学院的启动基金。Brien博士的资助来自NIH NIAID的K22AI104794早期调查员奖以及圣路易斯大学学院的种子补助金。对于所有受资助的个人,资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有作用。

本研究的所有程序均符合圣路易斯大学动物护理和使用委员会制定的指导方针。SLU 获得国际实验室动物护理评估与认证协会 (AAALAC) 的完全认可。
1. 器官隔离
注:病毒在室温 (RT) 下不稳定,因此必须仔细规划一次收获的动物数量,以保存病毒性定位剂。
<ol><li>根据所需的表型,使用所选剂量和路线感染小鼠。对于此方案,感染8-10周大的雄性?...

<ol>
	<li>Lazear, H. M., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus:+New+Clinical+Syndromes+and+Its+Emergence+in+the+Western+Hemisphere.">Zika Virus: New Clinical Syndromes and Its Emergence in the Western Hemisphere.</a> Journal of Virology. 90 (10), 4864-4875 (2016).</li><li>Simpson, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infection+in+Man.">Zika Virus Infection in Man.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 58, 335-338 (1964).</li><li>Fagbami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiological+investigations+on+arbovirus+infections+at+Igbo-Ora,+Nigeria.">Epidemiological investigations on arbovirus infections at Igbo-Ora, Nigeria.</a> Tropical and Geographical Medicine. 29 (2), 187-191 (1977).</li><li>McCrae, A. W., Kirya, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yellow+fever+and+Zika+virus+epizootics+and+enzootics+in+Uganda.">Yellow fever and Zika virus epizootics and enzootics in Uganda.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 76 (4), 552-562 (1982).</li><li>Rodhain, 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=Arbovirus+infections+and+viral+haemorrhagic+fevers+in+Uganda:+a+serological+survey+in+Karamoja+district,+1984.">Arbovirus infections and viral haemorrhagic fevers in Uganda: a serological survey in Karamoja district, 1984.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 83 (6), 851-854 (1984).</li><li>Wikan, N., Smith, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus:+history+of+a+newly+emerging+arbovirus.">Zika virus: history of a newly emerging arbovirus.</a> Lancet Infect Dis. 16 (7), e119-e126 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+outbreaks+in+the+Americas.">Zika virus outbreaks in the Americas.</a> The Weekly Epidemiological Record. 90 (45), 609-610 (2015).</li><li>Brien, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunological basis of age-related vulnerability to viral infection. , (2007).</li><li>Brien, J. D., Uhrlaub, J. L., Nikolich-Zugich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus-specific+CD4+T+cells+exhibit+direct+antiviral+cytokine+secretion+and+cytotoxicity+and+are+sufficient+for+antiviral+protection.">West Nile virus-specific CD4 T cells exhibit direct antiviral cytokine secretion and cytotoxicity and are sufficient for antiviral protection.</a> Journal of Immunology. 181 (12), 8568-8575 (2008).</li><li>Brien, J. D., Uhrlaub, J. L., Hirsch, A., Wiley, C. A., Nikolich-Zugich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+role+of+T+cell+defects+in+age-related+vulnerability+to+West+Nile+virus.">Key role of T cell defects in age-related vulnerability to West Nile virus.</a> Journal of Experimental Medicine. 206 (12), 2735-2745 (2009).</li><li>Brien, J. 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=Genotype-specific+neutralization+and+protection+by+antibodies+against+dengue+virus+type+3.">Genotype-specific neutralization and protection by antibodies against dengue virus type 3.</a> Journal of Virology. 84 (20), 10630-10643 (2010).</li><li>Shrestha, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+therapeutic+antibodies+that+neutralize+homologous+and+heterologous+genotypes+of+dengue+virus+type+1.">The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1.</a> PLoS Pathogens. 6 (4), e1000823 (2010).</li><li>Brien, J. 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=Interferon+regulatory+factor-1+(IRF-1)+shapes+both+innate+and+CD8(+)+T+cell+immune+responses+against+West+Nile+virus+infection.">Interferon regulatory factor-1 (IRF-1) shapes both innate and CD8(+) T cell immune responses against West Nile virus infection.</a> PLoS Pathogens. 7 (9), e1002230 (2011).</li><li>Pinto, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+temporal+role+of+type+I+interferon+signaling+in+CD8++T+cell+maturation+during+acute+West+Nile+virus+infection.">A temporal role of type I interferon signaling in CD8+ T cell maturation during acute West Nile virus infection.</a> PLoS Pathogens. 7 (12), e1002407 (2011).</li><li>Brien, J. D., Lazear, H. M., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+quantification,+detection,+and+storage+of+West+Nile+virus.">Propagation, quantification, detection, and storage of West Nile virus.</a> Current Protocols in Microbiology. 31, 11-15 (2013).</li><li>Brien, J. 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=Protection+by+immunoglobulin+dual-affinity+retargeting+antibodies+against+dengue+virus.">Protection by immunoglobulin dual-affinity retargeting antibodies against dengue virus.</a> Journal of Virology. 87 (13), 7747-7753 (2013).</li><li>Messaoudi, I., 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=Chikungunya+virus+infection+results+in+higher+and+persistent+viral+replication+in+aged+rhesus+macaques+due+to+defects+in+anti-viral+immunity.">Chikungunya virus infection results in higher and persistent viral replication in aged rhesus macaques due to defects in anti-viral immunity.</a> PLoS Neglected Tropical Diseases. 7 (7), e2343 (2013).</li><li>Pinto, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hydrogen+peroxide-inactivated+virus+vaccine+elicits+humoral+and+cellular+immunity+and+protects+against+lethal+West+Nile+virus+infection+in+aged+mice.">A hydrogen peroxide-inactivated virus vaccine elicits humoral and cellular immunity and protects against lethal West Nile virus infection in aged mice.</a> Journal of Virology. 87 (4), 1926-1936 (2013).</li><li>Sukupolvi-Petty, 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=Functional+analysis+of+antibodies+against+dengue+virus+type+4+reveals+strain-dependent+epitope+exposure+that+impacts+neutralization+and+protection.">Functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that impacts neutralization and protection.</a> Journal of Virology. 87 (16), 8826-8842 (2013).</li><li>Pinto, A. K., 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=Deficient+IFN+signaling+by+myeloid+cells+leads+to+MAVS-dependent+virus-induced+sepsis.">Deficient IFN signaling by myeloid cells leads to MAVS-dependent virus-induced sepsis.</a> PLoS Pathogens. 10 (4), e1004086 (2014).</li><li>Pinto, A. K., 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=Defining+New+Therapeutics+Using+a+More+Immunocompetent+Mouse+Model+of+Antibody-Enhanced+Dengue+Virus+Infection.">Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection.</a> MBio. 6 (5), (2015).</li><li>Pinto, A. K., 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=Human+and+Murine+IFIT1+Proteins+Do+Not+Restrict+Infection+of+Negative-Sense+RNA+Viruses+of+the+Orthomyxoviridae,+Bunyaviridae,+and+Filoviridae+Families.">Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families.</a> Journal of Virology. 89 (18), 9465-9476 (2015).</li><li>Hassert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+T+cells+mediate+protection+against+Zika+associated+severe+disease+in+a+mouse+model+of+infection.">CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection.</a> PLoS Pathog. 14 (9), e1007237 (2018).</li><li>Pinto, A. K., 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=Deficient+IFN+signaling+by+myeloid+cells+leads+to+MAVS-dependent+virus-induced+sepsis.">Deficient IFN signaling by myeloid cells leads to MAVS-dependent virus-induced sepsis.</a> PLoS Pathog. 10 (4), e1004086 (2014).</li><li>Brien, J. D., Lazear, H. M., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+quantification,+detection,+and+storage+of+West+Nile+virus.">Propagation, quantification, detection, and storage of West Nile virus.</a> Curr Protoc Microbiol. 31, (2013).</li><li>Hassert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+T+cells+mediate+protection+against+Zika+associated+severe+disease+in+a+mouse+model+of+infection.">CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection.</a> PLoS Pathogens. 14 (9), e1007237 (2018).</li><li>Fuchs, A., Pinto, A. K., Schwaeble, W. J., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lectin+pathway+of+complement+activation+contributes+to+protection+from+West+Nile+virus+infection.">The lectin pathway of complement activation contributes to protection from West Nile virus infection.</a> Virology. 412 (1), 101-109 (2011).</li><li>Lazear, H. M., Pinto, A. K., Vogt, M. R., Gale, M., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+interferon+controls+West+Nile+virus+infection+and+pathogenesis+in+mice.">Beta interferon controls West Nile virus infection and pathogenesis in mice.</a> Journal of Virology. 85 (14), 7186-7194 (2011).</li></ol>

ZIKV感染可引起神经系统疾病,因此目前动物模型研究的发病机制、免疫反应和疫苗和抗病毒药物的保护功效需要专注于中枢神经系统内的病毒控制。关注中枢神经系统疾病的挑战之一是,它往往以研究周围感染为代价。这里提出的器官分离方法侧重于需要快速评估在外围和CNS的ZIKV感染,以便评估中枢介ZIKV相关疾病,并建立抗病毒药物临床前测试模型,治疗和疫苗。该技术的另一个好处是,它还允许高度?...

寨卡病毒(ZIKV)是一种属于浮病毒家族的病毒,包括重要的神经侵入性人类病原体,如波瓦桑病毒(POWV)、日本脑炎病毒(JEV)和西尼罗病毒(WNV)1。在隔离和鉴定后,非洲和亚洲定期报告人类ZIKV感染2、3、4、5和中美洲和南美洲的流行病(在参考6。然而,直到最近,ZIKV才被认为会导致严重的疾病7。现在有成千上万的神经系统疾病和出生缺陷与ZIKV感染有关。ZIKV的迅速出现引发了许多问题:为什么疾病严重程度增加,对ZIKV感染的免疫反应是什么,以及是否有病毒和/或免疫介导的病理与神经学的增加有关表现和先天缺陷。现在人们急于了解与ZIKV相关的中枢神经系统(CNS)相关疾病,以及快速测试抗病毒药物和疫苗对ZIKV的疗效的必要性。正是在这种背景下,我们开发了使用ZIKV特定焦点形成测定(FFA)对外围和CNS中ZIKV定子进行快速分析的方法。
小型动物模型对于了解疾病进展和早期评估疫苗、治疗和抗病毒药物非常重要。我们已经建立了研究阿尔博病毒病的小动物模型,利用各种小鼠菌株来模拟人类感染,并保护病毒病原体8,9,10,11, 12,13,14,15,16,17,18,19,20 21,22.利用这个以往的经验,我们开始修改用于评估WNV和登革热病毒的技术,这是一种相关的黄病毒,用于评估两个外围器官的ZIKV丁打剂以及CNS21、2324.这些方法比其他测定的优点是:1) 它们结合收集外周和CNS器官进行分析的能力;2) 该方法适用于流式细胞测定,用于测量先天和适应性免疫反应,以及同一器官中同一动物的病毒定子;3)收获技术适用于组织学分析;4)ZIKV FFA是一种快速的高通量方法,用于病毒性皮底分析;5)这些方法可用于评估感染大多数病原体的动物器官中的病毒性定子25。

<table>
	<tbody>
		<tr>
			<td>1-bromo-3-chloropropane (BCP)</td>
			<td>MRC gene</td>
			<td>BP151</td>
			<td></td>
		</tr>
		<tr>
			<td>10cc syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>BD 309642</td>
			<td></td>
		</tr>
		<tr>
			<td>18G needle</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-557-145</td>
			<td></td>
		</tr>
		<tr>
			<td>1cc TB syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-823-16H</td>
			<td></td>
		</tr>
		<tr>
			<td>20cc syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-561-66</td>
			<td></td>
		</tr>
		<tr>
			<td>24 tube beadmill</td>
			<td>Thermo Fisher Scientific</td>
			<td>15 340 163</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm stainless steel beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9084634</td>
			<td></td>
		</tr>
		<tr>
			<td>37C Tissue Culture incubator</td>
			<td>Nuair</td>
			<td>5800</td>
			<td></td>
		</tr>
		<tr>
			<td>4G2 antibody</td>
			<td>in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well flat bottom plates</td>
			<td>Midsci</td>
			<td>TP92696</td>
			<td></td>
		</tr>
		<tr>
			<td>96well round bottom plates</td>
			<td>Midsci</td>
			<td>TP92697</td>
			<td></td>
		</tr>
		<tr>
			<td>Basix 1.5ml eppendorf tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-682-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated Germicidal Bleach</td>
			<td>Staples</td>
			<td>30966CT</td>
			<td></td>
		</tr>
		<tr>
			<td>CTL S6 Analyzer</td>
			<td>CTL</td>
			<td>CTL S6 Universal Analyzer</td>
			<td></td>
		</tr>
		<tr>
			<td>curved cutting scissors</td>
			<td>Fine Science Tools</td>
			<td>14061-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium - high glucose With 4500 mg/L glucose</td>
			<td>MilliporeSigma</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (molecular biology-grade)</td>
			<td>MilliporeSigma</td>
			<td>e7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>MilliporeSigma</td>
			<td>F0926-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11036-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>MilliporeSigma</td>
			<td>537020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse HRP-labeled antibody</td>
			<td>MilliporeSigma</td>
			<td>8924</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>MilliporeSigma</td>
			<td>H3537-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (molecular biology-grade)</td>
			<td>MilliporeSigma</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine/Xylazine cocktail</td>
			<td>Comparative Medicine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>MilliporeSigma</td>
			<td>g7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Magmax RNA purification kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1830</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>MilliporeSigma</td>
			<td>M0512</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Ependorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniCollect 0.5ml EDTA tubes</td>
			<td>Bio-one</td>
			<td>450480</td>
			<td></td>
		</tr>
		<tr>
			<td>o-ring tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>21-403-195</td>
			<td></td>
		</tr>
		<tr>
			<td>one step q RT-PCR mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4392938</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>EMS- 15713-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>MilliporeSigma</td>
			<td>d8537-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline multichannel pipettes</td>
			<td>Sartorius</td>
			<td>72230/72240</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline single channel pipettes</td>
			<td>Sartorius</td>
			<td>728230</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse free water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10-977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAzol BD</td>
			<td>MRC gene</td>
			<td>RB192</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Platform</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-676-333</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Fisher</td>
			<td>MT10040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>MilliporeSigma</td>
			<td>s7900</td>
			<td></td>
		</tr>
		<tr>
			<td>spoon/spatula</td>
			<td>Fine Science Tools</td>
			<td>10090-17</td>
			<td></td>
		</tr>
		<tr>
			<td>straight cutting scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>MilliporeSigma</td>
			<td>t8787</td>
			<td></td>
		</tr>
		<tr>
			<td>True Blue Substrate</td>
			<td>VWR</td>
			<td>95059-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>MilliporeSigma</td>
			<td>T3924-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and quantification of zika virus from multiple organs in a mouse

提出的方法展示了从寨卡病毒感染动物中分离器官和定量病毒载量的实验室程序。该程序的目的是量化小鼠在感染后不同时间点或不同实验条件下的周围和中枢神经系统区域的病毒性色度,以确定调节寨卡病毒感染的病毒学和免疫学因素。所演示的器官分离程序允许对病毒性定位剂进行焦点形成测定定量和定量PCR评估。快速器官分离技术是为保存病毒性皮炎而设计的。通过聚焦形成测定对病毒进行量度定量,可快速评估寨卡病毒的通量。重点形成测定的好处是评估传染性病毒,这种测定的局限性是降低检测极限的器官毒性的潜力。病毒性排名评估与定量PCR相结合,使用重组RNA拷贝控制病毒基因组拷贝数在器官内评估检测限值低。总体而言,这些技术为分析寨卡病毒感染动物的外围寨卡病毒分子和中枢神经系统提供了准确的快速高通量方法,并可用于评估大多数感染动物器官中的病毒性定子病原体,包括登革热病毒。

使用上述协议评估ZIKV针,通过皮下(SC)注射到脚垫上,小鼠感染了ZIKV(PRVABC59)。 在这里,给ZIKV的1 x 105 FFU给8-12周大的Ifnar1-/-小鼠SC不是致命的,但病毒可以在外围和CNS中复制。此剂量和途径用于研究宿主病原体免疫反应和致病性。给ZIKV的1 x 105 FFU注射8-12周大的Ifnar1-/-小鼠静脉注射(IV)是80至100%的致命性,动物在病毒注射后8至14天之间?...

Watch this Scientific Journal Video about Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse at JoVE.com

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

从小鼠多个器官分离和定量寨卡病毒

该协议的目的是通过分离和量化寨卡病毒,从感染后小鼠的多个器官中分离和量化,来展示用于调查病毒性疾病的技术。

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

在这段视频中，我们描述了一套程序，旨在询问病毒感染的影响，以及由此产生的使用寨卡感染的Murine模型的免疫反应。该协议是重要的，因为它使我们能够发展对感染和免疫的机械理解，通过确定病毒在全身传播的程度，跨体屏障和访问组织。这些技术允许对病毒发病机制进行详细了解，并提供了剖析疫苗或治疗提供的保护机制的能力。在这里，我们演示了使用小鼠寨卡病毒感染的这种方法，但这种方法可以应用于一系列病毒感染，包括其他病毒，如登革热病毒和α病毒，如基孔肯雅病毒。采集中枢神经系统的器官可能具有挑战性，因此一个人计划一个小实验，并准备事先练习技术是重要的。该协议允许在小动物模型中快速解剖病毒传播，能够可视化、捕获和存储实验数据。重要的是要直观地演示这种方法，让实验者了解如何收获中枢神经系统器官。同样重要的是，要看到细胞汇合水平和病毒性细菌的强度。首先，使用钳子去除受感染小鼠的皮毛。用剪刀斩首老鼠的头，然后切除胳膊和腿。要收获大脑，使用锯齿状的拉格兰奇剪刀切割头骨通过前额巨无霸。用钳子剥下头骨，用铲子挖出大脑。使用强钝剪刀，去除脊柱周围的骨头，然后切开骨盆骨，在腰椎水平上露出椎前科。然后，去除脊柱周围尽可能多的肌肉。小心地将针头的斜头放在椎前体内，避免过度压力，防止针头侵入椎体。握住，对椎体和针头施加压力，然后按注射器柱塞将绳索排出到培养皿中。立即将脊髓转移到标记管中，并放在干冰浴中，以进一步均匀化。要进行均质化，请添加一毫升 DMEM，并在珠节拍器中均匀化样品。在确保器官均质化后，将离心机和等同样品储存在零下80摄氏度，直到需要。在测定当天，从零下80摄氏度的冰柜中取出均质样品，并允许其解冻。将寨卡病毒的样品稀释十倍，进入稀释板的第一井，并使用多通道移液器在板上进行十倍连续稀释，每次更换尖端。通过将 20 微升样品添加到 180 微升生长介质中，更改每次稀释之间的移液器尖端。通过从准备好的平底 96 井板上拆下覆盖维罗细胞的介质来准备焦点成形板。为了防止单层干燥，立即在振动板的每孔中加入100微升的病毒稀释剂。使用同一组提示从最低浓度添加到最高浓度。岩石板一侧两到四次，在37摄氏度下孵育5%的二氧化碳一至两个小时。将2%甲基纤维素预热至室温。然后，以大约2比1的比例稀释生长介质中的2%甲基纤维素溶液。孵育后，在96孔板的每个孔中加入125微升的甲基纤维素生长介质。在32至40小时中，在37摄氏度的二氧化碳中再次孵育。为了修复玻璃细胞，在生物安全柜中，在96孔板的每个孔的甲基纤维素层顶部添加50微升5%的甲醛。在室温下孵育60分钟。或者，用 Parafilm 盖住盘子，并在夜间将板放在 4 摄氏度。之后，使用移液器将覆盖物和介质从细胞中移入生物安全柜内的一次性容器中。每井用 150 微升 PBS 轻轻清洗。从板上拆下 PBS，然后从生物安全柜中拆下板。重复PBS洗涤两次，然后每井添加150微升FFA洗涤缓冲液，在室温下离开5至10分钟，使固定细胞渗透。接下来，使用原发性寨卡抗体检测感染。在FFA染色缓冲液中，以每毫升1微克的浓度准备原抗体4G2。从板中轻拂FFA洗涤缓冲液，并在FFA染色缓冲液中加入50微升原抗体。然后用帕拉膜或塑料薄膜密封板，并在摄氏四度下孵育过夜。在FFA染色缓冲液中，以1至5000浓度准备二次去抗小鼠HRP标记抗体。去除抗体溶液后，用每井150微升FFA洗涤缓冲液洗三次细胞。每次轻拂到水槽中，拆下洗涤缓冲液。FFA染色缓冲液中的二级抗体将细胞污渍，每井50微升，在室温下孵育一至两个小时。之后，使用 FFA 洗涤缓冲液洗三次细胞，每次轻拂到水槽中去除洗涤缓冲液。在每个井中加入 50 微升的真蓝底材。等待 2 到 15 分钟，直到用最少的背景完全定义斑点。斑点可见后，用手轻轻用水清洗，以保护单层免受流水力的污染。在纸巾上轻拍干。不久之后，使用剖析范围手动计算每个井中的点数，或使用自动点计数器。在 ImmunoCapture 软件中，为每个样品选择易于区分的 20 到 200 个孔的稀释液，并使用重复井的平均值计算每毫米聚焦单位中的滴度。在这项实验中，在伊格纳小鼠皮下感染寨卡病毒后，在周围和中枢神经系统组织中显示了病毒负担。根据器官，检测的限值是每克100至500FFU。执行对焦形成分析时，技术错误可能导致结果欠佳。器官毒性，由高浓度的肝脏驱动，改变了测定的敏感性。当器官中的病毒滴度低于毒性时，FFA无法准确记录病毒滴度。肾细胞说明毒性与高器官浓度，但克服病毒滴度在低器官浓度。大力移液或清洗可以去除单层，导致数据丢失不准确的滴度结果报告。实验室工作台吸水纸中的纤维或毛，如果使用自动计数程序，可能会污染单个孔并造成重大错误。细胞密度是另一个问题，它可以显著影响焦点形成测定的成功。在测定开始时，大约60%汇合的细胞，与90%汇合时镀的细胞相比，显示出对焦形成测定的显著差异。规划实验的规模和范围，使器官收获高效完成，是手术中最重要的部分，然后开始用高质量的细胞进行聚焦形成测定。这个程序已经用于量化多种病毒家庭的病毒。该协议也可以适应对治疗化合物的筛选和量化中和抗体滴答剂。这些技术用于量化活病毒，应根据 BMBL 中规定的准则采取适当的安全步骤。

Research

JoVE Journal

Immunology and Infection

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

科研

免疫与感染

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

兹卡病毒感染细胞培养系统和<em&gt;体外</em&gt;干扰素的预防作用

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

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Zika Virus Specific Diagnostic Epitope Discovery

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

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

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

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

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

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

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