Please wait a minute...
Journal of Integrative Agriculture  2019, Vol. 18 Issue (10): 2361-2368    DOI: 10.1016/S2095-3119(19)62717-6
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
Generation of recombinant rabies virus ERA strain applied to virus tracking in cell infection
ZHAO Dan-dan1*, SHUAI Lei1*, GE Jin-ying1, WANG Jin-liang1, WEN Zhi-yuan1, LIU Ren-qiang1, WANG Chong1, WANG Xi-jun1, BU Zhi-gao1, 2      
1 State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, P.R.China
2 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
The mechanism of rabies virus (RABV) infection still needs to be further characterized.  RABV particle with self-fluorescent is a powerful viral model to visualize the viral infection process in cells.  Herein, based on a reverse genetic system of the Evelyn-Rokitnicki-Abelseth (rERA) strain, we generated a recombinant RABV rERA-N/mCherry strain that stably expresses an additional ERA nucleoprotein that fuses with the red fluorescent protein mCherry (N/mCherry).  The rERA-N/mCherry strain retained growth property similar to the parent strain rERA in vitro.  The N/mCherry expression showed genetic stability during passage into mouse neuroblastoma (NA) cells and did not change the virulence of the vector.  The rERA-N/mCherry strain was then utilized as a visual viral model to study the RABV-cell binding and internalization.  We directly observed the red self-fluorescence of rERA-N/mCherry particles binding to the cell surface, and further co-localizing with clathrin in the early stage of infection in NA cells by fluorescence microscopy.  Our results showed that the rERA-N/mCherry strain uses clathrin-dependent endocytosis to enter cells, which is consistent with the well-known mechanism of RABV invasion.  The recombinant RABV rERA-N/mCherry thus appears to have the potential to be an effective viral model to further explore the fundamental molecular mechanism of rabies neuropathogenesis.
Keywords:  rabies virus        self-fluorescence       binding        internalization  
Received: 14 January 2019   Accepted:
Fund: This work was supported by the National Natural Science Fundation of China (31800138) and the National Key Research and Development Program of China (2016YFD0500403).
Corresponding Authors:  Correspondence BU Zhi-gao, E-mail:; WANG Xi-jun, E-mail:    
About author:  ZHAO Dan-dan, E-mail:; SHUAI Lei, E-mail:; * These authors contributed equally to this study.

Cite this article: 

ZHAO Dan-dan, SHUAI Lei, GE Jin-ying, WANG Jin-liang, WEN Zhi-yuan, LIU Ren-qiang, WANG Chong, WANG Xi-jun, BU Zhi-gao. 2019. Generation of recombinant rabies virus ERA strain applied to virus tracking in cell infection. Journal of Integrative Agriculture, 18(10): 2361-2368.

Albertini A A, Schoehn G, Weissenhorn W, Ruigrok R W. 2008. Structural aspects of rabies virus replication. Cellular and Molecular Life Sciences, 65, 282–294.
Betzig E, Patterson G H, Sougrat R, Lindwasser O W, Olenych S, Bonifacino J S, Davidson M W, Lippincott-Schwartz J, Hess H F. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science, 313, 1642–1645.
Boulant S, Stanifer M, Lozach P Y. 2015. Dynamics of virus-receptor interactions in virus binding, signaling, and endocytosis. Viruses, 7, 2794–2815.
Brandenburg B, Zhuang X. 2007. Virus trafficking - learning from single-virus tracking. Nature Reviews Microbiology, 5, 197–208.
Burckhardt C J, Greber U F. 2009. Virus movements on the plasma membrane support infection and transmission between cells. PLoS Pathogens, 5, e1000621.
Carette J E, Raaben M, Wong A C, Herbert A S, Obernosterer G, Mulherkar N, Kuehne A I, Kranzusch P J, Griffin A M, Ruthel G, Dal Cin P, Dye J M, Whelan S P, Chandran K, Brummelkamp T R. 2011. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature, 477, 340–343.
Davis B M, Rall G F, Schnell M J. 2015. Everything you always wanted to know about rabies virus (but were afraid to ask). Annual Review of Virology, 2, 451.
Gaudin Y. 2000. Rabies virus-induced membrane fusion pathway. The Journal of Cell Biology, 150, 601–612.
Ge J, Wang X, Tao L, Wen Z, Feng N, Yang S, Xia X, Yang C, Chen H, Bu Z. 2011. Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats. Journal of Virology, 85, 8241–8252.
Greber U F, Way M. 2006. A superhighway to virus infection. Cell, 124, 741–754.
Helmuth J A, Burckhardt C J, Koumoutsakos P, Greber U F, Sbalzarini I F. 2007. A novel supervised trajectory segmentation algorithm identifies distinct types of human adenovirus motion in host cells. Journal of Structural Biology, 159, 347–358.
Jackson A C W W. 2007. Rabies. 2nd ed. Elsevier, Academic Press, Amsterdam, Boston.
Lawson K F, Walker V C, Crawley J F. 1967. ERA strain rabies vaccine. Duration of immunity in cattle, dogs and cats. Veterinary Medicine, Small Animal Clinician, 62, 1073–1074.
Lentz T L, Burrage T G, Smith A L, Crick J, Tignor G H. 1982. Is the acetylcholine receptor a rabies virus receptor? Science, 215, 182–184.
Lewis P, Lentz T L. 1998. Rabies virus entry into cultured rat hippocampal neurons. Journal of Neurocytology, 27, 559–573.
Mahadevan A, Suja M S, Mani R S, Shankar S K. 2016. Perspectives in diagnosis and treatment of rabies viral encephalitis: Insights from pathogenesis. Neurotherapeutics: The Journal of the American Society for Experimental Neuro Therapeutics, 13, 477–492.
Minghui R, Stone M, Semedo M H, Nel L. 2018. New global strategic plan to eliminate dog-mediated rabies by 2030. The Lancet Global Health, 6, e828–e829.
Mothes W, Sherer N M, Jin J, Zhong P. 2010. Virus Cell-to-Cell Transmission. Journal of Virology, 84, 8360–8368.
Piccinotti S, Kirchhausen T, Whelan S P. 2013. Uptake of rabies virus into epithelial cells by clathrin-mediated endocytosis depends upon actin.  Journal of Virology, 87, 11637–11647.
Piccinotti S, Whelan S P. 2016. Rabies internalizes into primary peripheral neurons via clathrin coated pits and requires fusion at the cell body. PLoS Pathogens, 12, e1005753.
Roche S, Gaudin Y. 2004. Evidence that rabies virus forms different kinds of fusion machines with different pH thresholds for fusion. Journal of Virology, 78, 8746–8752.
Rupprecht C, Kuzmin I, Meslin F. 2017. Lyssaviruses and rabies: Current conundrums, concerns, contradictions and controversies. F1000research, 6, 184.
van der Schaar H M, Rust M J, Chen C, van der Ende-Metselaar H, Wilschut J, Zhuang X W, Smit J M. 2008. Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells. PLoS Pathogens, 4, e1000244.
Shuai L, Feng N, Wang X, Ge J, Wen Z, Chen W, Qin L, Xia X, Bu Z. 2015. Genetically modified rabies virus ERA strain is safe and induces long-lasting protective immune response in dogs after oral vaccination. Antiviral Research, 121, 9–15.
Shuai L, Wang X, Wen Z, Ge J, Wang J, Zhao D, Bu Z. 2017. Genetically modified rabies virus-vectored Ebola virus disease vaccines are safe and induce efficacious immune responses in mice and dogs. Antiviral Research, 146, 36–44.
Sun E, He J, Zhuang X. 2013. Live cell imaging of viral entry. Current Opinion in Virology, 3, 34–43.
Takeuchi M, Ozawa T. 2007. Methods for imaging and analyses of intracellular organelles using fluorescent and luminescent proteins. Analytical sciences: The International Journal of the Japan Society for Analytical Chemistry, 23, 25–29.
Tao L, Ge J, Wang X, Wen Z, Zhai H, Tao H, Zhao B, Kong D, Yang C, Bu Z. 2011. Generation of a recombinant rabies Flury LEP virus carrying an additional G gene creates an improved seed virus for inactivated vaccine production. Virology Journal, 8, 454–454.
Tao L, Ge J, Wang X, Zhai H, Hua T, Zhao B, Kong D, Yang C, Chen H, Bu Z. 2010. Molecular basis of neurovirulence of flury rabies virus vaccine strains: Importance of the polymerase and the glycoprotein R333Q mutation. Journal of Virology, 84, 8926–8936.
Thoulouze M I, Lafage M, Schachner M, Hartmann U, Cremer H, Lafon M. 1998. The neural cell adhesion molecule is a receptor for rabies virus. Journal of Virology, 72, 7181–7190.
Tuffereau C, Benejean J, Blondel D, Kieffer B, Flamand A. 1998. Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus. EMBO Journal, 17, 7250–7259.
Wang J L, Wang Z L, Liu R Q, Shuai L, Wang X X, Luo J, Wang C, Chen W Y, Wang X J, Ge J Y, He X J, Wen Z Y, Bu Z G. 2018. Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus. PLoS Pathogens, 14, e1007189.
Wang X, Feng N, Ge J, Shuai L, Peng L, Gao Y, Yang S, Xia X, Bu Z. 2012. Recombinant canine distemper virus serves as bivalent live vaccine against rabies and canine distemper. Vaccine, 30, 5067–5072.
Worlein J M, Baker K, Bloomsmith M, Coleman K, Koban T L. 2011. The eighth edition of the guide for the care and use of laboratory animals (2011): Implications for behavioral management. American Journal of Primatology, 73, 98–98.
Xu H J, Hao X, Wang S W, Wang Z Y, Cai M J, Jiang J G, Qin Q W, Zhang M L, Wang H D. 2015. Real-time imaging of rabies virus entry into living vero cells. Scientifc Reports, 5, 11753.
[1] HUA Jin-feng, ZHANG Lei, HAN Yong-hua, GOU Xiao-wan, CHEN Tian-yuan, HUANG Yong-mei, LI Yan-qing, MA Dai-fu, LI Zong-yun. Chromosome-level genome assembly of Cylas formicarius provides insights into its adaptation and invasion mechanisms[J]. >Journal of Integrative Agriculture, 2023, 22(3): 825-843.
[2] YE Qing-ya, LI Zhi-xing, CHEN Qing-ling, SUN Ming-xu, YIN Ming-liang, LIN Tong. Fatty acid-binding protein gene is indispensable for molting process in Heortia vitessoides (Lepidoptera: Crambidae)[J]. >Journal of Integrative Agriculture, 2023, 22(2): 495-504.
[3] WANG Ke, HE Yan-yan, ZHANG You-jun, GUO Zhao-jiang, XIE Wen, WU Qing-jun, WANG Shao-li. Characterization of the chemosensory protein EforCSP3 and its potential involvement in host location by Encarsia formosa[J]. >Journal of Integrative Agriculture, 2023, 22(2): 514-525.
[4] Muhammad Irfan WARIS, Aneela YOUNAS, Rana Muhammad Kaleem ULLAH, Fatima RASOOL, Muhammad Muzammal ADEEL, WANG Man-qun. Molecular and in vitro biochemical assessment of chemosensory protein 10 from the brown planthopper Nilaparvata lugens at acidic pH[J]. >Journal of Integrative Agriculture, 2022, 21(3): 781-796.
[5] QU Cheng, WANG Ran, CHE Wu-nan, LI Feng-qi, ZHAO Hai-peng, WEI Yi-yun, LUO Chen, XUE Ming. Identification and tissue distribution of odorant binding protein genes in Harmonia axyridis (Coleoptera: Coccinellidae)[J]. >Journal of Integrative Agriculture, 2021, 20(8): 2204-2213.
[6] CHEN Xu, CHEN Ya-qin, YIN Zhong-qiong, WANG Rui, HU Huai-yue, LIANG Xiao-xia, HE Chang-liang, YIN Li-zi, YE Gang, ZOU Yuan-feng, LI Li-xia, TANG Hua-qiao, JIA Ren-yong, SONG Xu. Kaempferol inhibits Pseudorabies virus replication in vitro through regulation of MAPKs and NF-κB signaling pathways[J]. >Journal of Integrative Agriculture, 2021, 20(8): 2227-2239.
[7] WEN Liang, GAO Gui-ping, HUANG Zhi-qiang, ZHENG Si-chun, FENG Qi-li, LIU Lin. Expression, regulation and binding affinity of fatty acid-binding protein 2 in Spodoptera litura[J]. >Journal of Integrative Agriculture, 2020, 19(6): 1492-1500.
[8] LIU Yu-xiao, ZHOU Zi-shan, LIANG Ge-mei, SONG Fu-ping, ZHANG Jie. Alanine-substituted mutant on Gly373 and Asn375 of Cry1Ai-h-loop 2 causes reduction in both toxicity and binding against Helicoverpa armigera[J]. >Journal of Integrative Agriculture, 2019, 18(5): 1064-1071.
[9] ZHANG Lei, ZHENG Xing-wei, QIAO Lin-yi, QIAO Ling, ZHAO Jia-jia, WANG Jian-ming, ZHENG Jun. Analysis of three types of resistance gene analogs in PmU region from Triticum urartu[J]. >Journal of Integrative Agriculture, 2018, 17(12): 2601-2611.
[10] WANG Yu-peng, TANG shuang-qin, WU Zhi-feng, SHI Qing-hua, WU Zi-ming. Phenotypic analysis of a dwarf and deformed flower3 (ddf3) mutant in rice (Oryza sativa L.) and characterization of candidate genes[J]. >Journal of Integrative Agriculture, 2018, 17(05): 1057-1065.
[11] ZHU Jiao, Paolo Pelosi, LIU Yang, LIN Ke-jian, YUAN Hai-bin, WANG Gui-rong. Ligand-binding properties of three odorant-binding proteins of the diamondback moth Plutella xylostella[J]. >Journal of Integrative Agriculture, 2016, 15(3): 580-590.
[12] ZHOU Xin-yu, LIU Liang-liang, JIA Wen-chao, PAN Chuan-ying. Methylation profile of bovine Oct4 gene coding region in relation to three germ layers[J]. >Journal of Integrative Agriculture, 2016, 15(3): 618-628.
[13] ZHANG Wei, LI Bei, YU Bin. Genome-wide identification, phylogeny and expression analysis of the SBP-box gene family in maize (Zea mays)[J]. >Journal of Integrative Agriculture, 2016, 15(1): 29-41.
[14] WANG Zhong-wei, ZHANG Tian-quan, XING Ya-di, ZENG Xiao-qin, WANG Ling, LIU Zhong-xian, SHI Jun-qiong, ZHU Xiao-yan, MA Ling, LI Yun-feng, LING Ying-hua, SANG Xian-chun, HE Guang-hua. YGL9, encoding the putative chloroplast signal recognition particle 43 kDa protein in rice, is involved in chloroplast development[J]. >Journal of Integrative Agriculture, 2016, 15(05): 944-953.
[15] JIN Rong, LIU Nai-yong, LIU Yan, DONG Shuang-lin. A larval specific OBP able to bind the major female sex pheromone component in Spodoptera exigua (Hübner)[J]. >Journal of Integrative Agriculture, 2015, 14(7): 1356-1366.
No Suggested Reading articles found!