Cellular alterations in the MCH line of Chinese hamster cells following infection with herpes simplex virus.

We previously reported the isolation of the aneuploid MCH line of Chinese hamster cells from a normal line originally derived by Dr. George Yerganian from female adult fibroblast derivatives which formed a short-term carrier state with herpes simplex virus.1 We also reported the occurrence of chromosomal aberrations during the first postinfection division with this virus, and subsequently the presence of persistent chromosomal aberrations in clonal sublines derived from infected cells.2 This paper is concerned with further studies of the effects of viral infection upon chromosomes of MCH cells. Materials and Methods.-Cell line: The MCH line and the composition of the medium employed were previously described.' All sera used were heated to 560 for 40 min before use. Virus: The SAE strain of herpes simplex virus used was previously described.1 Virus was harvested from cultures of HeLa cells inoculated 24 hr earlier with 108 TCID50 of virus. The cells were collected, washed 5-6 times with Hanks' balanced salt solution, and resuspended in 0.2 M P04 buffer containing 0.1% bovine serum albumin (Armour fraction V) at pH 7.0. The cells were then disrupted by 3 cycles of rapid freeze-thawing in a dry ice-alcohol mixture, followed by 4 min of sonication at maximum output in a 10 kc Raytheon sonic oscillator. Following disruption of cells, the suspension was centrifuged at 40, the supernatant collected, and then filtered through a millipore filter (MF type HA) in a Swinny hypodermic adaptor before use. Virus titrations were performed in six HeLa tubes per dilution, and the TCID50 calculated by the method of Reed and Muench.3 This strain of herpes simplex virus causes mainly a nonproliferative type of cytopathic effect in HeLa cells as described by Gray et al.4 Infection of cells: A stock bottle of MCH cells was trypsinized (0.25% Bactotrypsin-Difco) and centrifuged at room temperature. The resultant pellet was resuspended in medium, and the number of cells determined by direct count in a hemocytometer chamber. Three cc of a cell suspension containing 2 X 105 cells plus 0.1-0.2 ml of virus suspension or buffer was added to a 25 ml sterile plastic flask (Falcon Plastics). The cell-virus mixture was then placed on a rotating shaker and shaken (370 rpm) for 1 hr at 37"C. The suspension was either diluted directly for seeding of plates or first centrifuged and the cells washed to remove the bulk of residual virus. For cloning and plating efficiency studies, approximately 500 cells were seeded in Petri dishes. Within 24 hr the location of single attached cells was noted. These cells were observed during the next 7 days, and individual clones which were well isolated were removed with a capillary pipette and seeded in separate 25 ml plastic flasks. The flasks were incubated at 370 in an atmosphere of 5%Xc CO2 in air and refed every 2-3 days until a full cell sheet had formed. The cells were then trypsinized and passaged normally. Virus antiserum: Antiserum was prepared in rabbits by intravenous inoculation of 108 TCIJ)5 of virus for six consecutive weeks. Neutralizing antibody was assayed in HeLa cells by a constant virus-varying serum dilution method following incubation at room temperature for one-half hour. Serum from a single animal having a titer of 1:130 for 105 TCID50 virus was used throughout. Preinoculation bleeding from the same animal was used as control. All sera were heated to 560 for 40 min before use. Chromosome preparations: Approximately 2 X 105 cells in 3 ml medium were seeded in glass Petri dishes containing 2 coverslips. After 48-72 hr, colchicine was added to yield a final concentration of 0.5 pg/ml. Plates were reincubated for an additional 2-4 hr. The medium was made hypotonic by adding '/2 volume of prewarmed deionized water, and the plates were incubated