Cytotoxic effect of dual fluorescent-labeled oncolytic herpes simplex virus type 1 on mouse tumorigenic cell lines

Shahriyar Abdoli , Farzin Roohvand, Ladan Teimoori-Toolabi, Sara Shayan, Mohammad Ali Shokrgozar


The increasing incidences of cancer at the global scale have recently resulted in the invention of various biotechnology approaches among which the oncolytic virotherapy is a new strategy for the treatment of multiple tumors. Herpes simplex virus (HSV) based vectors are one of the most studied oncolytic agents, worldwide. Moreover, syngeneic animal models are the principal parts of the oncolytic virotherapies investigation. The effects of a dual fluorescent γ34.5 deleted vector-HSV-GR- on three mouse tumor                  cell lines were studied in this work. We previously generated a dual fluorescent labeled oncolytic                         HSV-HSV-GR- (both copies of γ34.5 were inactivated by insertion of two distinct fluorescent dyes, GFP and mCherry) in our laboratory; subsequently, they were used as oncolytic viruses. The three 4T1, TC-1, and CT26 cell lines were infected with HSV-GR. The infection efficacy and the elimination potency of HSV-GR were analyzed by photomicrography and flow cytometry methods. HSV-GR showed a significant efficiency to infect the cell lines examined. Flow cytometry analyses demonstrated that HSV-GR infected 89.3%, 86.1%, and 92.4% of 4T1, TC-1, and CT26 cells, respectively. Moreover, propidium iodide (PI) staining of infected cells indicated that HSV-GR could kill 27.9%, 21.2%, and 21.3% of 4T1, TC-1, and CT26 cells, respectively. Interestingly, HSV-GR infected cells were capable of expressing both GFP and mCherry at the same time. The promising effects of the oncolytic virus HSV-GR in the mouse syngeneic tumor cell system have shed more light on the therapeutic potential of this anti-cancer approach.


Flow cytometry; Oncolytic HSV; Oncolytic virotherapy; Syngeneic tumor cell lines.

Full Text:



Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA: a cancer journal for clinicians. 2016;66(1):7-30.

DeSantis CE, Lin CC, Mariotto AB, Siegel RL, Stein KD, Kramer JL, et al. Cancer treatment and survivorship statistics, 2014. CA: a cancer journal for clinicians. 2014;64(4):252-71.

Friedman GK, Pressey JG, Reddy AT, Markert JM, Gillespie GY. Herpes Simplex Virus Oncolytic Therapy for Pediatric Malignancies. Mol Ther. 2009;17(7):1125-35.

Singh PK, Doley J, Kumar GR, Sahoo A, Tiwari AK. Oncolytic viruses & their specific targeting to tumour cells. The Indian journal of medical research. 2012;136(4):571.

Seymour LW, Fisher KD. Oncolytic viruses: finally delivering. Br J Cancer. 2016;114(4):357-61.

Huang F, Wang B-R, Wu Y-Q, Wang F-C, Zhang J, Wang Y-G. Oncolytic viruses against cancer stem cells: A promising approach for gastrointestinal cancer. World Journal of Gastroenterology. 2016;22(35):7999-8009.

Meerani S, Yao Y. Oncolytic viruses in cancer therapy. Eur J Sci Res. 2010;40(156171.3).

Argnani R, Lufino M, Manservigi M, Manservigi R. Replication-competent herpes simplex vectors: design and applications. Gene Ther. 2005;12(S1):S170-S7.

Speranza MC, Kasai K, Lawler SE. Preclinical Mouse Models for Analysis of the Therapeutic Potential of Engineered Oncolytic Herpes Viruses. ILAR journal. 2016;57(1):63-72.

Kanai R, Zaupa C, Sgubin D, Antoszczyk SJ, Martuza RL, Wakimoto H, et al. Effect of γ34. 5 deletions on oncolytic herpes simplex virus activity in brain tumors. Journal of virology. 2012;86(8):4420-31.

Li Y, Zhang C, Chen X, Yu J, Wang Y, Yang Y, et al. ICP34. 5 protein of herpes simplex virus facilitates the initiation of protein translation by bridging eukaryotic initiation factor 2α (eIF2α) and protein phosphatase 1. Journal of biological chemistry. 2011;286(28):24785-92.

Campadelli‐Fiume G, De Giovanni C, Gatta V, Nanni P, Lollini PL, Menotti L. Rethinking herpes simplex virus: the way to oncolytic agents. Reviews in medical virology. 2011;21(4):213-26.

Falls T, Roy DG, Bell JC, Bourgeois-Daigneault M-C. Murine Tumor Models for Oncolytic Rhabdo-Virotherapy. ILAR journal. 2016;57(1):73-85.

Zhao Q, Zhang W, Ning Z, Zhuang X, Lu H, Liang J, et al. A Novel Oncolytic Herpes Simplex Virus Type 2 Has Potent Anti-Tumor Activity. PLOS ONE. 2014;9(3):e93103.

Arulanandam R, Batenchuk C, Varette O, Zakaria C, Garcia V, Forbes NE, et al. Microtubule disruption synergizes with oncolytic virotherapy by inhibiting interferon translation and potentiating bystander killing. 2015;6:6410.

Thomas DL, Fraser NW. HSV-1 therapy of primary tumors reduces the number of metastases in an immune-competent model of metastatic breast cancer. Molecular Therapy. 2003;8(4):543-51.

Bennett JJ, Malhotra S, Wong RJ, Delman K, Zager J, St-Louis M, et al. Interleukin 12 Secretion Enhances Antitumor Efficacy of Oncolytic Herpes Simplex Viral Therapy for Colorectal Cancer. Annals of Surgery. 2001;233(6):819-26.

Fabiani M, Limongi D, Palamara AT, De Chiara G, Marcocci ME. A Novel Method to Titrate Herpes Simplex Virus-1 (HSV-1) Using Laser-Based Scanning of Near-Infrared Fluorophores Conjugated Antibodies. Frontiers in microbiology. 2017;8:1085.

Blaho JA, Morton ER, Yedowitz JC. Herpes simplex virus: propagation, quantification, and storage. Current protocols in microbiology. 2005;Chapter 14:Unit 14E.1.

Abdoli S, Roohvand F, Teimoori-Toolabi L, Shokrgozar MA, Bahrololoumi M, Azadmanesh K. Construction of Various gamma34.5 Deleted Fluorescent-Expressing Oncolytic herpes Simplex type 1 (oHSV) for Generation and Isolation of HSV-Based Vectors. Iranian biomedical journal. 2017;21(4):206-17.

Sanderson MJ, Smith I, Parker I, Bootman MD. Fluorescence Microscopy. Cold Spring Harbor Protocols. 2014;2014(10):pdb.top071795.

Zhu H. Cell Cycle Analysis Using Propidium Iodide Staining with GFP Detection. Bio-protocol. 2012;2(7):e199.

Riccardi C, Nicoletti I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nature Protocols. 2006;1:1458.

Yura Y. Presage of oncolytic virotherapy for oral cancer with herpes simplex virus. The Japanese dental science review. 2017;53(2):53-60.

DiGiulio S. FDA Approves First Oncolytic Virus Therapy—Imlygic for Melanoma. Oncology Times. 2015.

Peters C, Rabkin SD. Designing herpes viruses as oncolytics. Molecular Therapy—Oncolytics. 2015;2.

Popov S, Mirshahidi S, Essono S, Song R, Wang X, Ruprecht RM. Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection. DNA and Cell Biology. 2009;28(3):103-8.

Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science (New York, NY). 1991;252(5007):854-6.

Learmonth K, Braidwood L, Woll P, Conner J. Immune responses following intrapleural administration of oncolytic SEPREHVIR in patients with malignant pleural mesothelioma. Journal for immunotherapy of cancer. 2015;3(Suppl 2):P335.

Nakamori M, Fu X, Rousseau R, Chen S-Y, Zhang X. Destruction of nonimmunogenic mammary tumor cells by a fusogenic oncolytic herpes simplex virus induces potent antitumor immunity. Molecular Therapy.9(5):658-65.

Toda M, Rabkin SD, Kojima H, Martuza RL. Herpes simplex virus as an in situ cancer vaccine for the induction of specific anti-tumor immunity. Human gene therapy. 1999;10(3):385-93.

Esaki S, Goshima F, Kimura H, Murakami S, Nishiyama Y. Enhanced antitumoral activity of oncolytic herpes simplex virus with gemcitabine using colorectal tumor models. International Journal of Cancer. 2013;132(7):1592-601.


  • There are currently no refbacks.

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.