Name: live imaging and quantification of viral infection in k18 hace2 transgenic mice using reporter-expressing recombinant sars-cov-2
Uploaded: 2021-11-05T13:00:00
Description: Watch this Scientific Journal Video about Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2 at JoVE.com

我们要感谢我们研究所（德克萨斯生物医学研究所）的成员在COVID-19大流行期间为保持我们的设施全面运营和安全所做的努力。我们还要感谢我们的机构生物安全委员会（IBC）和spell（IACUC）以省时的方式审查我们的协议。我们感谢西奈山伊坎医学院的Thomas Moran博士提供SARS-CoV交叉反应性1C7C7核衣壳（N）蛋白单克隆抗体。马丁内斯-索布里多实验室的SARS-CoV-2研究目前得到NIAID/ NIH拨款RO1AI161363-01，RO1AI161175-01A1和R43AI165089-01的支持;国防部（DoD）拨款W81XWH2110095和W81XWH2110103;圣安东尼奥精准治疗伙伴关系;德克萨斯生物医学研究所论坛;德克萨斯大学圣安东尼奥健康科学中心;圣安东尼奥医学基金会;并由流感发病机制和传播研究中心（CRIPT）提供，该中心是NIAID资助的流感研究和反应卓越中心（CEIRR，合同号75N93021C00014）。

涉及K18 hACE2转基因小鼠的方案由德克萨斯生物医学研究所（TBRI）机构生物安全委员会（IBC）和机构动物护理和使用委员会（IACUC）批准。所有实验都遵循国家研究委员会40号实验动物护理和使用指南中的建议。与小鼠一起工作时，需要适当的个人防护设备（PPE）。
1. K18 hACE2转基因小鼠的使用
<ol><li>在特定无病原体条件下，在生物安全水平（BSL?...

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	<li>V'Kovski, P., Kratzel, A., Steiner, S., Stalder, H., Thiel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronavirus+biology+and+replication:+implications+for+SARS-CoV-2.">Coronavirus biology and replication: implications for SARS-CoV-2.</a> Nature Reviews Microbiology. 19 (3), 155-170 (2021).</li><li>Pal, M., Berhanu, G., Desalegn, C., Kandi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+acute+respiratory+syndrome+coronavirus-2+(SARS-CoV-2):+An+update.">Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2): An update.</a> Cureus. 12 (3), 7423 (2020).</li><li>Su, 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=Epidemiology,+genetic+recombination,+and+pathogenesis+of+coronaviruses.">Epidemiology, genetic recombination, and pathogenesis of coronaviruses.</a> Trends in Microbiology. 24 (6), 490-502 (2016).</li><li>Cui, J., Li, F., Shi, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+evolution+of+pathogenic+coronaviruses.">Origin and evolution of pathogenic coronaviruses.</a> Nature Reviews Microbiology. 17 (3), 181-192 (2019).</li><li>Lu, R., 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=Genomic+characterisation+and+epidemiology+of+2019+novel+coronavirus:+implications+for+virus+origins+and+receptor+binding.">Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.</a> Lancet. 395 (10224), 565-574 (2020).</li><li>Evans, J. P., Liu, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+host+factors+in+SARS-CoV-2+entry.">Role of host factors in SARS-CoV-2 entry.</a> Journal of Biological Chemistry. 297 (1), 100847 (2021).</li><li>Petersen, E., 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=Comparing+SARS-CoV-2+with+SARS-CoV+and+influenza+pandemics.">Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics.</a> Lancet Infectious Diseases. 20 (9), 238-244 (2020).</li><li>Alfaraj, S. 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=Clinical+predictors+of+mortality+of+Middle+East+Respiratory+Syndrome+Coronavirus+(MERS-CoV)+infection:+A+cohort+study.">Clinical predictors of mortality of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection: A cohort study.</a> Travel Medicine and Infectious Disease. 29, 48-50 (2019).</li><li>Harvey, W. T., 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=SARS-CoV-2+variants,+spike+mutations+and+immune+escape.">SARS-CoV-2 variants, spike mutations and immune escape.</a> Nature Reviews Microbiology. 19 (7), 409-424 (2021).</li><li>Dong, E., Du, H., Gardner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+interactive+web-based+dashboard+to+track+COVID-19+in+real+time.">An interactive web-based dashboard to track COVID-19 in real time.</a> Lancet Infectious Diseases. 20 (5), 533-534 (2020).</li><li>Bar-On, Y. M., Flamholz, A., Phillips, R., Milo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARS-CoV-2+(COVID-19)+by+the+numbers.">ARS-CoV-2 (COVID-19) by the numbers.</a> Elife. 9, 57309 (2020).</li><li>Roussel, Y., 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=SARS-CoV-2:+fear+versus+data.">SARS-CoV-2: fear versus data.</a> International Journal of Antimicrobial Agents. 55 (5), 105947 (2020).</li><li>Scialo, 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=SARS-CoV-2:+One+year+in+the+pandemic.+What+have+we+learned,+the+new+vaccine+era+and+the+threat+of+SARS-CoV-2+variants.">SARS-CoV-2: One year in the pandemic. What have we learned, the new vaccine era and the threat of SARS-CoV-2 variants.</a> Biomedicines. 9 (6), 611 (2021).</li><li>. Coronavirus (COVID-19) update: FDA authorizes additional monoclonal antibody for treatment of COVID-19 Available from: <a target="_blank" href="https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-monoclonal-antibody-treatment-covid-19">https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-monoclonal-antibody-treatment-covid-19</a> (2021)</li><li>Dougan, 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=Bamlanivimab+plus+Etesevimab+in+Mild+or+Moderate+Covid-19.">Bamlanivimab plus Etesevimab in Mild or Moderate Covid-19.</a> New England Journal of Medicine. 385 (15), 1382-1392 (2021).</li><li>Ledford, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COVID+antibody+treatments+show+promise+for+preventing+severe+disease.">COVID antibody treatments show promise for preventing severe disease.</a> Nature. 591 (7851), 513-514 (2021).</li><li>Tuccori, 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=An+overview+of+the+preclinical+discovery+and+development+of+bamlanivimab+for+the+treatment+of+novel+coronavirus+infection+(COVID-19):+reasons+for+limited+clinical+use+and+lessons+for+the+future.">An overview of the preclinical discovery and development of bamlanivimab for the treatment of novel coronavirus infection (COVID-19): reasons for limited clinical use and lessons for the future.</a> Expert Opinion on Drug Discovery. , 1-12 (2021).</li><li>Phan, A. T., Gukasyan, J., Arabian, S., Wang, S., Neeki, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergent+inpatient+administration+of+casirivimab+and+imdevimab+antibody+cocktail+for+the+treatment+of+COVID-19+pneumonia.">Emergent inpatient administration of casirivimab and imdevimab antibody cocktail for the treatment of COVID-19 pneumonia.</a> Cureus. 13 (5), 15280 (2021).</li><li>O'Brien, M. P., 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=Subcutaneous+REGEN-COV+antibody+combination+in+early+SARS-CoV-2+infection.">Subcutaneous REGEN-COV antibody combination in early SARS-CoV-2 infection.</a> medRxiv. , (2021).</li><li>Beigel, 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=Remdesivir+for+the+treatment+of+Covid-19+-+Final+report.">Remdesivir for the treatment of Covid-19 - Final report.</a> New England Journal of Medicine. 383 (19), 1813-1826 (2020).</li><li>Li, L., 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=Effect+of+convalescent+plasma+therapy+on+time+to+clinical+improvement+in+patients+with+severe+and+life-threatening+COVID-19:+A+randomized+clinical+trial.">Effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening COVID-19: A randomized clinical trial.</a> Journal of the American Medical Association. 324 (5), 460-470 (2020).</li><li>Polack, F. P., 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=Safety+and+efficacy+of+the+BNT162b2+mRNA+Covid-19+vaccine.">Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine.</a> New England Journal of Medicine. 383 (27), 2603-2615 (2020).</li><li>Oliver, S. E., 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+advisory+committee+on+immunization+practices'+interim+recommendation+for+use+of+Pfizer-BioNTech+COVID-19+vaccine+-+United+States,+December+2020.">The advisory committee on immunization practices' interim recommendation for use of Pfizer-BioNTech COVID-19 vaccine - United States, December 2020.</a> Morbidity and Mortality Weekly Report. 69 (50), 1922-1924 (2020).</li><li>Oliver, S. E., 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+advisory+committee+on+immunization+practices'+interim+recommendation+for+use+of+Janssen+COVID-19+vaccine+-+United+States,+February+2021.">The advisory committee on immunization practices' interim recommendation for use of Janssen COVID-19 vaccine - United States, February 2021.</a> Morbidity and Mortality Weekly Report. 70 (9), 329-332 (2021).</li><li>Zhao, 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=Emission+spectra+of+bioluminescent+reporters+and+interaction+with+mammalian+tissue+determine+the+sensitivity+of+detection+in+vivo.">Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo.</a> Journal of Biomedical Optics. 10 (4), 41210 (2005).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nature Methods. 2 (12), 905-909 (2005).</li><li>Nogales, A., 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+novel+fluorescent+and+bioluminescent+bireporter+Influenza+A+Virus+to+evaluate+viral+infections.">A novel fluorescent and bioluminescent bireporter Influenza A Virus to evaluate viral infections.</a> Journal of Virology. 93 (10), 00032 (2019).</li><li>Nogales, A., 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=Replication-competent+fluorescent-expressing+influenza+B+virus.">Replication-competent fluorescent-expressing influenza B virus.</a> Virus Research. 213, 69-81 (2016).</li><li>Welsh, D. K., Noguchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bioluminescence+imaging.">Cellular bioluminescence imaging.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Tran, V., Moser, L. A., Poole, D. S., Mehle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+real-time+in+vivo+imaging+of+an+influenza+reporter+virus+reveals+dynamics+of+replication+and+spread.">Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread.</a> Journal of Virology. 87 (24), 13321-13329 (2013).</li><li>Schoggins, J. W., 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=Dengue+reporter+viruses+reveal+viral+dynamics+in+interferon+receptor-deficient+mice+and+sensitivity+to+interferon+effectors+in+vitro.">Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (36), 14610-14615 (2012).</li><li>Luker, G. 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=Noninvasive+bioluminescence+imaging+of+herpes+simplex+virus+type+1+infection+and+therapy+in+living+mice.">Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice.</a> Journal of Virology. 76 (23), 12149-12161 (2002).</li><li>Li, X., 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=Development+of+a+rapid+antiviral+screening+assay+based+on+eGFP+reporter+virus+of+Mayaro+virus.">Development of a rapid antiviral screening assay based on eGFP reporter virus of Mayaro virus.</a> Antiviral Research. 168, 82-90 (2019).</li><li>Kirui, J., Freed, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+validation+of+a+highly+sensitive+bioluminescent+HIV-1+reporter+vector+that+simplifies+measurement+of+virus+release.">Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release.</a> Retrovirology. 17 (1), 12 (2020).</li><li>Shang, 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=Development+and+characterization+of+a+stable+eGFP+enterovirus+71+for+antiviral+screening.">Development and characterization of a stable eGFP enterovirus 71 for antiviral screening.</a> Antiviral Research. 97 (2), 198-205 (2013).</li><li>Zou, G., Xu, H. Y., Qing, M., Wang, Q. Y., Shi, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+stable+luciferase+dengue+virus+for+high-throughput+screening.">Development and characterization of a stable luciferase dengue virus for high-throughput screening.</a> Antiviral Research. 91 (1), 11-19 (2011).</li><li>Ye, 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=Rescue+of+SARS-CoV-2+from+a+single+bacterial+artificial+chromosome.">Rescue of SARS-CoV-2 from a single bacterial artificial chromosome.</a> mBio. 11 (5), 02168 (2020).</li><li>Avila-Perez, G., Park, J. G., Nogales, A., Almazan, F., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+recombinant+Zika+virus+from+a+bacterial+artificial+chromosome+cDNA+clone.">Rescue of recombinant Zika virus from a bacterial artificial chromosome cDNA clone.</a> Journal of Visualized Experiments: JoVE. (148), e59537 (2019).</li><li>Chiem, 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+bifluorescent-based+assay+for+the+identification+of+neutralizing+antibodies+against+SARS-CoV-2+variants+of+concern+in+vitro+and+in+vivo.">A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo.</a> Journal of Virology. , (2021).</li><li>Committee for the update of the guide for the care and use of laboratory animals., Institute for laboratory animal research (U.S) &amp; National Academies Press (U.S.). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+for+the+care+and+use+of+laboratory+animals.+8th+edn.">Guide for the care and use of laboratory animals. 8th edn.</a> National Research Council (US). , (2011).</li><li>Ye, 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=Analysis+of+SARS-CoV-2+infection+dynamic+in+vivo+using+reporter-expressing+viruses.">Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (41), (2021).</li></ol>

该协议证明了使用这些表达报告基因的rSARS-CoV-2来监测体内病毒感染 的可行性。两种表达报告基因的重组病毒都为研究 体内SARS-CoV-2感染提供了极好的工具。所描述的 离体 （rSARS-CoV-2 / Venus）和 体内 （rSARS-CoV-2 / Nluc）成像系统代表了了解SARS-CoV-2感染动力学，病毒发病机制以及在病毒感染后不同时间识别受感染细胞/器官的绝佳选择。为了进行 离体 （rSARS-CoV-2 / Ven...

严重急性呼吸系统综合征冠状病毒2（SARS-CoV-2）是一种包膜，阳性，单链RNA病毒，属于冠状病毒科1中的β冠状病毒谱系。这种病毒家族分为α-，β-，Gamma-和Delta-coronavirus1。α-和β-冠状病毒主要感染哺乳动物，而γ-和δ-冠状病毒几乎只感染鸟类2。迄今为止，已有七种冠状病毒（CoV）跨越了物种屏障，成为人类冠状病毒（HCoV）：两种α冠状病毒（HCoV-229E和HCoV-NL63）和五种β-CoV（HCoV-OC43，HCoV-HKU1，SARS-CoV，中东呼吸综合征冠状病毒[MERS-CoV]和SARS-CoV-2）3，4，5，6。SARS-CoV、MERS-CoV和SARS-CoV-2具有高致病性，可引起严重的下呼吸道感染7。在SARS-CoV-2出现之前，有两次由CoV引起的流行病爆发：2002-2003年在中国广东普罗维登斯的SARS-CoV，病死率（CFR）约为9.7%;2012年至今中东中东呼吸综合征冠状病毒，病死率约为34%7，8。SARS-CoV-2的总体CFR在3.4%-49%之间，基础疾病导致更高的CFR8，9。自2019年12月在中国武汉发现SARS-CoV-2以来，已造成全球超过2.42亿人感染和超过490万人死亡7，10，11，12。值得注意的是，自2020年底以来，新的SARS-CoV-2关注变体（VoC）和感兴趣的变体（VoI）影响了病毒特征，包括传播和抗原性9，13，以及COVID-19大流行的总体方向。对于SARS-CoV-2感染的治疗，目前只有一个美国（美国）美国食品和药物管理局（FDA）治疗性抗病毒药物（瑞德西韦）和一种紧急使用授权（EUA）药物（巴瑞替尼，与瑞德西韦联合给药）14。还有6种批准的EUA单克隆抗体：REGEN-COV（卡西维单抗和依德维单抗，一起给药），索曲维单抗，托珠单抗，巴姆拉尼昔单抗和雌虫昔单抗一起给药15，16，17，18，19。目前只有一种FDA批准的预防性疫苗辉瑞-BioNTech，另外两种预防性疫苗（Moderna和Janssen）已获得EUA批准的20，21，22，23，24。然而，随着感染率不受控制以及VoC和VoI的出现，SARS-CoV-2仍然对人类健康构成威胁。因此，迫切需要新的方法来确定有效的预防和治疗方法，以控制SARS-CoV-2感染和仍在进行的COVID-19大流行。
研究SARS-CoV-2需要费力的技术和次要方法来识别感染细胞和/或经过验证的感染动物模型中病毒的存在。反向遗传学的使用允许重组病毒的产生来回答病毒感染生物学中的重要问题。例如，反向遗传学技术提供了揭示和理解病毒感染，发病机制和疾病的方法。同样，反向遗传学方法为设计缺乏病毒蛋白的重组病毒铺平了道路，以了解它们在病毒发病机制中的贡献。此外，反向遗传学技术已被用于生成表达报告基因的重组病毒，用于 体外 和 体内 应用，包括确定用于治疗病毒感染的预防和/或治疗方法。荧光和生物发光蛋白是最常用的报告基因，因为它们具有灵敏度，稳定性和基于新技术改进的易于检测25，26。 在体外，荧光蛋白已被证明是病毒在受感染细胞中定位的更好选择，而荧光素酶更便于定量研究27，28，29。 在体内，荧光素酶优于荧光蛋白用于整个动物成像，而荧光蛋白优选用于鉴定感染细胞或 离体 成像30，31，32。使用报告基因表达重组病毒已成为研究许多家族病毒的有力工具，其中包括黄病毒，肠道病毒，甲型病毒，慢病毒，沙粒病毒和流感病毒28，33，34，35，36。
为了克服对研究SARS-CoV-2的次要方法的需求并表征 体内实时SARS-CoV-2感染，我们产生了复制能力强的重组（r）SARS-CoV-2，该重组体表达生物发光（纳米酰化酶，Nluc）或荧光（金星）蛋白，使用我们之前描述的基于细菌人造染色体（BAC）的反向遗传学，这些基因在 大肠杆菌 中作为单个拷贝保存 为了尽量减少病毒序列在细菌中繁殖过程中的毒性37，38。值得注意的是，rSARS-CoV-2 / Nluc和rSARS-CoV-2 / Venus 在体内显示出rSARS-CoV-2 / WT样致病性。来自rSARS-CoV-2 / Venus的高水平金星表达允许使用 体内 成像系统（IVIS）检测受感染的K18 hACE2转基因小鼠肺部的病毒感染39。Venus表达水平与肺部检测到的病毒滴度密切相关，证明了使用Venus表达作为SARS-CoV-2感染的有效替代物的可行性。使用rSARS-CoV-2 / Nluc，我们能够实时跟踪病毒感染的动态，并使用相同的IVIS方法在K18 hACE2转基因小鼠中纵向评估 体内 SARS-CoV-2感染。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% Triton X-100</td>
			<td>J.T.Baker</td>
			<td>X198-07</td>
			<td>Store at room temperature (RT)</td>
		</tr>
		<tr>
			<td>1% DEAE-Dextran</td>
			<td>MP Biomedicals</td>
			<td>195133</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Formalin solution, neutral buffered</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Oxoid</td>
			<td>LP0028</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S-5761</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>African green monkey kidney epithelial cells (Vero E6)</td>
			<td>ATCC</td>
			<td>CRL-1586</td>
			<td></td>
		</tr>
		<tr>
			<td>Ami HT</td>
			<td>Spectral Instruments Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aura Imaging Software 3.2.0</td>
			<td>Spectral Instruments Imaging</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), 35%</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Corning</td>
			<td>25-055-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Corning Cellgro</td>
			<td>15-013-CV</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anesthesia gas machine</td>
			<td>Veterinary Anesthesia Systems, Inc.</td>
			<td>VAS 2001R</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Seradigm</td>
			<td>1500-050</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Four- to six-week-old female K18-hACE2 transgenic mice</td>
			<td>The Jackson Laboratory</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism Version 9.1.0</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>1001936040</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>MARS Data Analysis Software</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MB10 tablets</td>
			<td>QUIP Laboratories</td>
			<td>MBTAB1.5</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Nano-Glo Luciferase Assay Reagent</td>
			<td>Promega</td>
			<td>N1110</td>
			<td>This reagent is used to measure Nluc activity. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well Microplates</td>
			<td>ThermoFisher Scientific</td>
			<td>269620</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well Microplates</td>
			<td>ThermoFisher Scientific</td>
			<td>269620</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/L-Glutamine (PSG) 100x</td>
			<td>Corning</td>
			<td>30-009-CI</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>PHERAstar FSX</td>
			<td>BMG LABTECH</td>
			<td>PHERAstar FSX</td>
			<td></td>
		</tr>
		<tr>
			<td>Precelleys Evolution homogenizer</td>
			<td>Bertin Instruments</td>
			<td>P000062-PEVO0-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft tissue homogenizing CK14 &#8211; 7 mL</td>
			<td>Bertin Instruments</td>
			<td>P000940-LYSK0-A</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 EasYFlask</td>
			<td>ThermoFisher Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASTAIN ABC-HRP Kit, Peroxidase</td>
			<td>Vector Laboratories</td>
			<td>PK-4002</td>
			<td>ABC kit and DAB Peroxidase Substrate kit</td>
		</tr>
	</tbody>
</table>

live imaging and quantification of viral infection in k18 hace2 transgenic mice using reporter-expressing recombinant sars-cov-2

2019年新型冠状病毒肺炎（COVID-19）大流行是由严重急性呼吸系统综合征冠状病毒2（SARS-CoV-2）引起的。迄今为止，SARS-CoV-2已造成全球超过2.42亿例感染和490多万人死亡。与其他病毒类似，研究SARS-CoV-2需要使用实验方法来检测受感染细胞和/或动物模型中病毒的存在。为了克服这一限制，我们产生了复制能力强的重组（r）SARS-CoV-2，其表达生物发光（纳米脱硫酶，Nluc）或荧光（Venus）蛋白。这些报告基因表达rSARS-CoV-2允许基于Nluc和Venus报告基因的表达在 体外 和 体内 跟踪病毒感染。在这里，该研究描述了使用rSARS-CoV-2 / Nluc和rSARS-CoV-2 / Venus来检测和跟踪先前描述的K18人血管紧张素转换酶2（hACE2）转基因小鼠感染模型中的SARS-CoV-2感染，使用 体内 成像系统（IVIS）。这种rSARS-CoV-2 / Nluc和rSARS-CoV-2 / Venus在 体内显示出rSARS-CoV-2 / WT样致病性和病毒复制。重要的是，Nluc和Venus的表达使我们能够直接跟踪受感染小鼠 体内和离体的病毒感染。这些rSARS-CoV-2 / Nluc和rSARS-CoV-2 / Venus代表了研究SARS-CoV-2 体内生物学，了解病毒感染和相关COVID-19疾病以及确定有效的预防和/或治疗治疗以对抗SARS-CoV-2感染的绝佳选择。

k18 hACE2转基因小鼠的rSARS-CoV-2 / Nluc感染（图1 和2） 图1A显示了用于评估体内感染的rSARS-CoV-2 / WT（顶部）和rSARS-CoV-2 / Nluc（底部）的示意图。图1B显示了用于评估K18 hACE2转基因小鼠中rSARS-CoV-2 / Nluc感染动态的示意图流程图。4至6周龄的雌性K18 hACE2转基因小鼠（N = 4）在鼻内模拟感染1x PBS或感染105...

Watch this Scientific Journal Video about Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2 at JoVE.com

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

该协议描述了在K18 hACE2转基因小鼠中使用荧光素酶和荧光表达重组（r）SARS-CoV-2和 体内 成像系统（IVIS）的病毒感染动力学，以克服研究 体内SARS-CoV-2感染所需的次要方法的需求。

使用报告基因表达重组SARS-CoV-2对K18 hACE2转基因小鼠的病毒感染进行实时成像和定量

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

我们的实验方案描述了在SARS-CoV-2感染的K18人ACE2转基因小鼠模型中使用荧光或发光表达重组SARS-CoV-2进行体内和离体活体成像病毒检测和病毒追踪。该技术的主要优点是病毒检测和跟踪在K18人ACE2转基因小鼠模型中SARS-CoV-2的体内和离体研究中的应用和可行性。在K18人ACE2转基因小鼠模型中使用SARS-CoV-2的体内成像代表了评估治疗体内SARS-CoV-2感染的治疗方法的绝佳选择。实施报告者SARS-CoV-2代表了评估体内致病性，病毒复制和营养作用的绝佳选择。对于表达SARS-CoV-2的荧光素酶的体内研究，建议在底物注入后剃须小鼠并立即成像以改善生物发光信号。通过启动体内成像系统或IVIS开始生物发光监测，并设置参数，单击图像模式到生物发光，然后打开滤镜并将曝光时间设置为自动。接下来，从隔离室中取出已经剃光和麻醉的小鼠，并使用25号针头向后轨道施用100微升在PBS中稀释1至10的荧光素酶底物给小鼠。然后将鼠标放回隔离室，将隔离室转移到IVIS机器上，该机器配备了加热组件以防止体温过低，并通过在软件程序中选择“获取”来关闭IVIS成像器门以初始化成像。要分析采集的生物发光图像，请使用软件中的ROI或感兴趣区域工具来指定精确信号并测量通量。启动成像软件并设置参数。单击图像模式到荧光，将激发设置为500纳米，将发射滤光片设置为530纳米，并将曝光时间设置为自动。将切除的肺放入含有两毫升1X PBS的6孔板中，冲洗以除去多余的血液。使用70%乙醇对样品之间的手术工具进行消毒和清洁。初始化IVIS机器后，将肺部放在黑色薄片上，并将组织彼此分开。然后将托盘放在生物安全柜内的隔离室内，然后将隔离室转移到IVIS。关闭IVIS成像仪门，然后单击“采集”以启动成像系统。要分析荧光，请使用ROI工具并在每个单独的肺周围绘制ROI。手动测量每个ROI，然后使用给定的平均辐射效率值，并从模拟感染的小鼠中减去。成像完成后，将组织放入冷冻管中进行干冰冷冻，以零下80摄氏度储存，以便以后处理。利用从组织匀浆中获得的上层，进行十倍连续稀释，并用一毫升每个上清液稀释液一式三份感染Vero E6细胞的融合单层。让病毒在37摄氏度下在加湿的5%二氧化碳培养箱中吸附一小时。完成后，用一毫升1X PBS洗涤细胞，然后将细胞在两毫升含有1%琼脂的感染后培养基中在37摄氏度的加湿的5%二氧化碳培养箱中孵育72小时。孵育后，在10%中性缓冲福尔马林中在4摄氏度下灭活板24小时，确保整个板浸没。取出生物安全3级的板，在室温下用一毫升0.5%Triton X-100洗涤细胞三次10分钟。接下来，用一毫升2.5牛血清白蛋白或BSA和PBS在37摄氏度下阻断透性细胞一小时，然后在一毫升每毫升一微克的SARS-CoV核衣壳蛋白交叉反应性单克隆抗体1C7C7中孵育，在2.5 BSA中在37摄氏度下稀释一小时。用一毫升1X PBS洗涤细胞三次，并根据制造商的说明使用ABC和二氨基苯或DAB，过氧化物酶底物试剂盒显现斑块，然后将病毒滴度计算为每毫升斑块形成单位。在研究中，观察到生物发光纳米酸化酶或Nluc很容易被检测到，并且感染了rSARS-CoV-2 Nluc的小鼠，但那些感染了rSARS-CoV-2野生型或模拟感染的小鼠却没有。定量分析显示感染后不同日子的Nluc强度。感染rSARS-CoV-2 Nluc的小鼠肺表面的肉眼病变与rSARS-CoV-2野生型感染组相当。此外，在感染后不同日期，检测到的病毒滴度和rSARS-CoV-2 Nluc感染小鼠在所有器官中与感染rSARS-CoV-2野生型的小鼠相当。同时，仅在rSARS-CoV-2 Nluc感染小鼠的器官中检测到Nluc活性。另一组模拟感染和病毒感染的小鼠被监测12天，以观察体重和存活率的变化。感染rSARS-CoV-2 Nluc和rSARS-CoV-2野生型的小鼠体重减轻了高达25%的体重，并且在感染后七到八天内全部死于病毒感染。在rSARS-CoV-2 Venus感染后，在所有肺部很容易检测到感染rSARS-CoV-2 Venus的小鼠的金星表达，但那些感染rSARS-CoV-2野生型或模拟感染的小鼠则不然。定量分析表明，Venus强度在感染后两天达到峰值，并在感染过程中和受感染小鼠的肺部下降。肺表面图像显示感染rSARS-CoV-2 Venus的小鼠的严重病变与rSARS-CoV-2野生型感染小鼠的严重病变相当。此外，感染rSARS-CoV-2 Venus导致病毒滴度与在所有器官中感染rSARS-CoV-2野生型的小鼠中观察到的病毒滴度相当。感染rSARS-CoV-2 Venus和rSARS-CoV-2野生型的小鼠体重减轻了25%，并在感染后第九天死于病毒感染，没有存活。报告者表达SARS-CoV-2的结果已被证明是病毒感染的有效替代品。因此，这可能导致快速确定和评估治疗SARS-CoV-2感染的对策。

Research

JoVE Journal

Immunology and Infection

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Production and Titering of Recombinant Adeno-associated Viral Vectors

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

使用假病毒感染的高通量荧光成像检测SARS-CoV-2中和抗体

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

使用基于HiBiT的生物报告器检测SARS-CoV-2受体结合域抗体

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

利用开源移液机器人的高效SARS-CoV-2定量逆转录酶PCR唾液诊断策略

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Studying Host-Virus Interactions with Pseudotyped Viruses (Video) | JoVE

假型病毒作为通过中和测定监测针对 SARS-CoV-2 的体液免疫反应的分子工具

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...