Biologicals (2001) 29, 67–73 doi:10.1006/biol.2001.0278, available online at http://www.idealibrary.com on Isolation and Replication of Rabies Virus in C6 Rat Glioma Cells (Clone CCL-107) J. Bordignon 1 , A. T. Piza 2 , M. Alvarez-Silva 3 , G. M. M. Caporale 4 , M. L. Carrieri 4 , I. Kotait 4 and C. R. Zanetti 1 1 Department of Microbiology, Immunology and Parasitology of the Federal University of Santa Catarina (MIP/CCB/UFSC), Campus Universita ´rio da Trindade, 88040-900 Floriano ´ polis, Brazil; 2 Valle ´e S.A.—Av. Eng. Luiz Carlos Berrini, 1253, Sa ˜ o Paulo, 04571-010, Brazil; 3 Department of Cell Biology, Embryology and Genetic of UFSC (BEG/CCB/UFSC), Campus Universita ´rio da Trindade, 88040-900 Floriano ´ polis, Brazil; 4 Pasteur Institute, Avenida Paulista, 393, Sa ˜ o Paulo, 01311-000, Brazil Abstract. The susceptibility of the C6 rat glioma cell line (ATCC; CCL-107) to rabies virus was character- ized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supernatants of infected cultures, by both BHK-21 cell infection and mice inoculation. C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new and useful system for rabies virus investigation. © 2001 The International Association for Biologicals Key words: rabies, C6 rat glioma cells, BHK-21 cell, NA cell, glycoprotein. Introduction Tissue culture techniques have long been used in studies related to rabies virus, and there are now a number of continuous cell lines used in research on pathogenesis, vaccine production and diagnosis of rabies (For reviews see refs 1 and 2). King 1 has listed 41 cell types (in primary cultures, diploid and heteroploid cell lines and leukocytes) from di#er- ent animal origins susceptible to rabies infection. BHK-21 3 and CER 4 were the first cell lines reported to be suitable for routine diagnosis of rabies. To the extent that rabies virus is highly neurotropic, cell lines of neuronal origin are now widely accepted as being the most sensitive systems. Human and mouse neuroblastoma cells are currently used in diagnostic tests. 5–8 However, the susceptibility of non-neuronal cells has also been extensively described. 1,9 In the present study the susceptibility of C6 rat glioma cells (ATCC; CCL-107) was tested for both fixed (PV) and wild (WRS) strains of rabies virus. This cell line was cloned from cerebral tumors induced by N-nitrosomethylurea. 10 The C6 glioma can present oligodendrocytic or astrocytic pheno- types, depending on culture conditions, and repre- sents a poorly di#erentiated lineage. 11–13 C6 was chosen because its susceptibility to vesicular stoma- titis virus, a member of the Rhabdoviridae family has been demonstrated. 14 In addition, the use of glial cells in rabies research has already been reported. 15 The susceptibility of C6 was demon- strated by infecting them with PV and WRS rabies virus strains. Fluorescent cytoplasmic inclusions were readily visible at 24 h post-infection (hpi) using an anti-ribonucleoprotein (RNP) rabies conju- gate. In addition, significant amounts of rabies *To whom correspondence should be addressed. E-mail: zanetti@ccb.ufsc.br 1045–1056/01/020067+07 $35.00/0  2001 The International Association for Biologicals