Veterinary World

     Open access and peer reviewed journal  

ISSN (Online): 2231-0916

ISSN (Print): 0972-8988


Home l Editorial board l Instructions for authors l Reviewer guideline l Open access policy l Archives l FAQ

Open Access

Copyright: The authors. This article is an open access article licensed under the terms of the Creative Commons Attribution License

( which permits unrestricted use, distribution and reproduction in any medium, provided the work is properly cited.

Research (Published online: 21-05-2015)

14. Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells - A. Sasikalaveni, K. G. Tirumurugaan, S. Manoharan, G. Dhinakar Raj and K. Kumanan

Veterinary World, 8(5): 636-639



   doi: 10.14202/vetworld.2015.636-639


A. Sasikalaveni: Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai - 600 007, Tamil Nadu, India;

K. G. Tirumurugaan: Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai - 600 007, Tamil Nadu, India;

S. Manoharan: Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai - 600 007, Tamil Nadu, India;

G. Dhinakar Raj: Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai - 600 007, Tamil Nadu, India;

K. Kumanan: Dean, Faculty of Basic Sciences, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai - 600 051, Tamil Nadu, India;


Received: 30-12-2014, Revised: 14-04-2015, Acceped: 19-04-2015, Published online: 21-05-2015


Corresponding author: K. G. Tirumurugaan, e-mail:

Citation: Sasikalaveni A, Tirumurugaan KG, Monoharan S, Dhinakar Raj G, Kumanan K (2015) Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells, Veterinary World 8(5):636-639.

Background: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA) cells, which enable quicker isolation of street rabies virus with minimum passages.

Objective: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells.

Materials and Methods: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test.

Results: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided.

Conclusion: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

Keywords: co-culture, isolation, fluorescent-antibody test, murine neuroblastoma, rapid tissue culture infection test.

1. Kaplan, M.M. (1973) An overview of laboratory techniques in the diagnosis and prevention of rabies and in rabies research. In: Kaplan, M.M. and Koprowski, H. editors. Laboratory Techniques in Rabies. 3rd ed. World Health Organization, Geneva. p21.
2. World Health Organisation. (1984) Report of the 2nd WHO Consultation on Monoclonal Antibodies for Rabies Diagnosis and Research. WHO, Germany.
3. Iwasaki, Y.I. and Clark, H.F. (1977) Rabies virus infection in mouse neuroblastoma cells. Lab. Invest., 36(6): 578-584.
4. Rudd, R.J. and Trimarchi, C.V. (1989) Development and evaluation of an in vitro virus isolation procedure as a replacement for the mouse inoculation test in rabies diagnosis. J. Clin. Microbiol., 27(11): 2522-2528.
PMid:2681254 PMCid:PMC267070
5. Hanlona, C.A., DeMattosa, C.A., DeMattosa, C.C., Niezgodaa, M., Hooperb, D.C., Koprowskib, H., Notkinsc, A. and Rupprechta, C.E. (2001) Experimental utility of rabies virus neutralizing human monoclonal antibodies in post exposure prophylaxis. Vaccine, 19(28-29): 3834-3842.
6. Goudsmit, J., Marissen, W.E., Weldon, W.C., Niezgoda, M., Hanlon, C.A., Rice, A.B., Kruif, J.d., Dietzschold, B., Bakker, A.B.H. and Rupprecht, C.E. (2006) Comparison of an Anti – Rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin. J. Infect. Dis., 193(6): 796-801.
7. Madusuadhana, S.N., Malavalli, B.V., Thankappan, U.P., Sundramoorthy, S., Belludi, A.Y., Pulagumbaly, S.B. and Sanyal, S. (2014) Development and evaluation of new immunohistochemistry based test for the detection of rabies virus neutralizing antibodies. Hum. Vaccin. Immunother., 10(5): 1359-1365.
8. Chander, V., Singh, R.P. and Verma, P.C. (2012) Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection. Indian J. Virol., 23(3): 317-325.
PMid:24293819 PMCid:PMC3550790
9. Mani, R.S. and Madusudana, S.N. (2013) Laboratory diagnosis of human rabies: Recent Advances. Sci. World J., 2013: 1-10.
PMid:24348170 PMCid:PMC3848253
10. Favi, M., Roos, O. and Yung, V. (1992) Evaluación de la técnica de cultivos cellular esfrente a la inoculación en ratoneslactantes en el diag¬nóstico de rabia Evaluation of the cell culture technique against inoculation in suckling mice in the diagnosis of rabies. Av. Cienc. Vet., 7(2): 172-179.
11. OIE – Organization for Animal Health. (20141) Manual for diagnostic test and vaccines for terrestrial animals. Available from: Last accessed on 08-04-2015.
12. Jayakumar, R., Thirumurugaan, K.G. and Raj, G.D. (2006) Phylogenetic characterization of rabies virus isolates from Chennai, India. Acta Virol., 50(4): 275-276.
13. Noguchi, H. (1913) Contribution to the cultivation of parasite of rabies. J. Exp. Med., 18(3): 314.
PMid:19867707 PMCid:PMC2125063
14. Levaditi, C. (1913) Virus rabiqueet culture des cellules in vitro. C R S Soc. Biol., 75: 505-509.
15. Larghi, O.P., Mebel, A.E., Lazara, L. and Savy, V.L. (1975) Sensitivity of BHK-21 cells supplemented with diethylaminoethyl-dextran for detection of street rabies virus in saliva samples. J. Clin. Microbiol., 3: 243-245.
16. Rudd, R.J. and Trimarchi, C.V. (1987) Comparison of sensitivity of BHK-21 and murine neuroblastoma cells in the isolation of a street strain rabies virus. J. Clin. Microbiol., 25(8): 1456-1458.
PMid:3305560 PMCid:PMC269247
17. Chabra, M., Mittal, V., Jaiswal, R., Malik, S. and Gupta, M. (2007) Development and evaluation of an in vitro isolation of street rabies virus in mouse neuroblastoma cells as compared to conventional tests used for diagnosis of rabies. Indian J. Med. Microbiol., 25(3): 263-266.
18. Umoh, J.U. and Blenden, D.C. (1983) Comparison of primary skunk brain and kidney and racoon kidney cells with established cell lines for isolation and propagation of street rabies virus. Infect. Immunol., 41(3): 1370-1372.
PMid:6885165 PMCid:PMC264649
19. Webster, W.A. and Casey, G.A. (1988) Diagnosis of rabies infection. In: Campbell, J.B. and Charlton, K.M., editor. Rabies. Kluwer Academic Publishers, Boston. p201-222.
20. Fontana, D., Kratje, R., Etcheverrigaray, M. and Priet, C. (2014) Rabies virus-like particles expressed in HEK293 cells. Vaccine, 32(24): 2799-2804.
21. Yamada, K., Noguchi, K. and Nishizono, A. (2014) Characterization of street rabies virus variants with an additional N-glycan at position 247 in the glycoprotein. Arch. Virol., 159(2): 207-216.
22. Wang, F.X., Zhang, S.Q., Zhu, H.W., Yang, Y., Sun, N., Tan, B., Li, Z.G., Cheng, S.P., Fu, Z.F. and Wen, Y.J. (2014) Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge. Vet. Microbiol., 174(3-4): 362-71.
23. Dastkhosh, M., Rahimi, P., Haghighat, S., Biglari, P., Howaizi, N., Saghiri, R. and Roohandeh, A. (2014) Cell culture extraction and purification of rabies virus nucleoprotein. Jundishapur. J. Microbiol., 7(9): e11734.
24. Matsumoto, T., Yamada, K. and Noguchi, K. (2010) Isolation and characterization of novel human monoclonal antibodies possessing neutralizing ability against rabies virus. Microbiol. Immunol., 54(11): 673-683.
25. Thirumeni Nagarajan., Wilfred E Marissen. and Charles E. Rupprecht. (2014) Monoclonal antibodies for the prevention of rabies: theory and clinical practice. Antibody Tech. J., 2014(4): 1-12.
26. Appel, M.J., Pearce-Kelling, S. and Summers, B.A. (1992) Dog lymphocyte cultures facilitate the isolation and growth of virulent Canine distemper virus. J. Vet. Diag. Invest., 4(3): 258-263.
27. Sharma, U.K., Song, H.F., Willingham, F.F., Hannig, J., Flexner, C., Farzadegan, H., Nicolau, C. and Schwartz, D.H. (1997) Diagnosis of human immunodeficiency virus infection using citrated whole blood. Clin. Diagn. Lab. Immunol., 4(3): 261-263.
PMid:9144360 PMCid:PMC170515
28. Osterhaus, A.D.M., Broeders, H.W.J., Visser, I.K.G., Teppema, J.S. and Vedder, E.J. (1990) Isolations of an orthopoxvirus from pox-like lesions of grey seal (Halichoerus Grypus). Vet. Rec., 127(4): 91-92.
29. Sapkal, G.N., Wairagkar, N.S., Ayachit, V.M., Bondre, V.P. and Gore, M.M. (2007) Detection and isolation of Japanese encephalitis virus from blood clots collected during the acute phase of infection. Am. J. Trop. Med. Hyg., 77(6): 1139-1145.