key: cord-0004954-5budstbg authors: Takayama, N.; Kirn, A. title: An improved method for titration of mouse hepatitis virus type 3 in a mouse cell culture date: 1976 journal: Arch Virol DOI: 10.1007/bf01315624 sha: 8b0abe2cce5d59d104611f616f7f6e623bfcb648 doc_id: 4954 cord_uid: 5budstbg Plaque assay in DBT cells with DEAE-dextran and trypsin presents a titration system for MHV3 as sensitive as the LD(50) assay in mice. Plaque assay of mouse hepatitis virus type 3 (MttV3), a member of the genus coronavirus (7) , was carried out in mouse embryo cells (3) as well as mierofocus assay in mouse macrophages (5) . However these methods were either not so sensitive as the LD50 assay in mice or else not convenient for routine use. In this paper we describe an improved method for the titration of MHV3 in the same continuous eelI-line that makes the growth of MHV2 possible (4) . The cell-line SR-CDF1-DBT (DBT), originating from a mouse brain tumor, was kindly supplied by Dr. Hirano (Institute of Medical Sciences, University of Tokyo, Japan). DBT cells were grown in Eagle's minimum essential medium (MEM) supplemented with 10 per cent calf serum (CS) and 10 per cent tryptose phosphate broth (TPB). When DBT-monolayers were infected with MHV3 (American Type Collection Strain previously submitted to three cycles of plaque purification in our laboratory), the formation of s}mcytia became visible at 6 hours postinfeetion (p.i.) and increased in both size and number parallel to the virus titer. Plaque assays were performed on DBT-monolayers in 3.5 cm-diameter petri dishes, following the method described by HII~ANO et, al. (4) . In DBT cells overlaid with ME)/I containing 1 per cent Noble agar, 5 per cent CS, 10 per cent TPB, and 1 : 10,000 neutral red, MHV3-plaques of 0.6--1.4 mm (mean value = 0.9 ram) in diameter could be counted at 40 hours p.i. The plaques were clearly-defined and sometimes contained a deeply-stained area at their center. Comparative titrations showed that the plaque assay gave tigers lower by about half a log than did the LDs0 method in mice (Table 1) . To ira.prove the sensitivity of the plaque assay we examined the effect of diethylaminoethyl-dextran (DEAE-D) on the plaque formation and plaque diameter of MHV3, as BRADBUllNE and TYRREL]5 had reported that DEAE-D added to overlay agar increased the plaque number of human coronavirus (2). When DEAE-D was added to the overlay medium, no significant increase in the mean plaque number was produced compared with that of cultures overlaid with a DEAE-D-free medium. When DEAE-D was given along with the virus suspension to DBT cells, a clear-cut increase in the plaque number was found (Fig. 1) . The most remarkable increase was observed in those cells which received a. 50 ~zg/ml dose of DEAE-D. ~Vith a 25 tzg/mI dose of DEAE-D the mean plaque number was still 4 times greater than that of the control. With regard to the plaque diameter, no significant difference was found between the control group and a n y of the treated groups. The effect of trypsin on M H V 3 -p l a q u e formation in DBT-monolayers was also investigated. After infection, D B T cells were overlaid with MEM containing 1 per cent Noble agar and trypsin at a concentration of 5, 10 or 25 [zg/ml. The Dl3T-dishes were incubated at 37°C for 24 hours and plaques were stained with a second overlay medium consisting of MEM with 1 per cent Noble agar, 5 per cent CS, 10 per cent T P B and 1 : 10,000 neutral red at 37 ° C for 16--18 hours. Whereas an enhancing effect, on the plaque formation of myxoviruses has been reported (1, 6), no significant increase in the mean plaque n u m b e r of M H V 3 was observed. However, the plaques were larger (mean value --1.4 mm in diameter; range = 0.6--2.3 ram) and clearer t h a n those in the control group. The sensitivity of the plaque assay in DBT-monolayers was equal to t h a t of the LDs0 assay in mice when 50 ptg/ml of D E A E -D were added to the virus dilutions and the plaques could be counted without difficulty when the overlay m e d i u m contained 25 [zg/ml of trypsin (Table 1) . As D B T is an established cell-line, it is far less troublesome in handling t h a n mouse macrophages and, through the plaque assay on D B T cells, virus titers are determined in a shorter period of time t h a n through the LDs0 assay in mice which is widely used as a titration m e t h o d of M H V 3 today. Consequently, the assay method of M H V 3 described above can a d v a n t a g e o u s l y replace not only plaque assay on mouse embryo cells or mouse maerophages but also the LDs0 assay in mice. Plaque formation by influenza viruses in the presence of trypsin The propagation of "Coronaviruses" in tissue-culture Plaque formation by a mouse hepatitis virus 1%eplication and plaque formation of mouse hepatitis virus (MttV-2) in mouse cell line DBT culture Observations on the growth of mouse hepatitis virus (MItV-3) in mouse macrophages Genetie recombination for antigenic markers of antigenieally differen$ strains of influenza B virus