Frank J. Jenkins¹, Alyson M. Donoghue² and John R. Martin³

¹ Department of Pathology and Infectious Diseases and Microbiology, University of Pittsburgh, and Division of Behavioral Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213
² Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD. 20814
³ Food and Drug Administration, Division of Antiviral Drug Products, Rockville, MD 20857.

Received 07/30/96; Accepted 08/27/96; On-line 10/04/96

METHODS

Cells and viruses.

Vero cells (American Type Culture Collection) were grown in Eagle's minimal essential medium (EMEM) supplemented with 10% Serum-Plus (SP; JRH, Rockville, MD) and 50 µg/ml gentamycin (USB, Inc. Cleveland, OH). LT-A^- cells were kindly provided by M.J. Tevethia (M.S. Hershey Medical Center, Pennsylvania State University, Hershey, PA) and grown in EMEM supplemented with 10% horse serum and gentamycin. Human TK- 143 cells were obtained from Bernard Roizman (University of Chicago, Chicago, IL) and grown in EMEM supplemented with 10% SP and gentamycin. Every fifth passage, the 143 cells were passaged in medium containing 50 µg/ml 5-bromodeoxyuridine (BUdR).

The properties of HSV-1(F), HSV-1(F) 305, and the recombinant viruses RBMu2, RBMu3 and REMu1 have been described elsewhere (4,5,9,10). Viral stocks were prepared and titered on Vero cells as previously described (9).

Viral growth curves

Confluent monolayers of Vero cells grown in six well dishes were infected with 1x10³ pfu and incubated at 37°C. At various times post infection, the cells were scraped into cell culture medium, frozen and thawed two times, and the viral titers determined on confluent Vero cell monolayers.

Mouse inoculations

Six week old male BALB/c mice (NCI, Bethesda, MD.) were inoculated intracerebrally (i.c.), intraperitoneally (i.p.), or onto scarified corneas with varying amounts of virus. All viral dilutions were made in cell culture medium as described above, with the control groups receiving cell culture medium alone.

Intracerebral and interperitoneal inoculations were performed on mice anesthetized by brief exposure to metofane followed by injection of 0.03 ml and 0.1 ml of virus sample, respectively. For corneal inoculations, the corneas of mice anesthetized by i.p. injections of 2 mg pentobarbital were scarified by 10 passes of a hypodermic needle, followed by the placement of 0.01 ml of virus sample on the scarified corneas. The virus samples were allowed to absorb for 30 minutes while the mice were under anesthesia. For neurovirulence studies, the inoculated mice were observed daily for three weeks, and the number of deaths recorded. PFU/LD50 ratios were determined by the procedure of Reed and Muench (11). To detect acute ocular infection and both acute and latent virus in the trigeminal ganglion, the tissues were removed aseptically from exsanguinated mice and processed as described below. All animal experiments met the standards for humane animal care and use as set by the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals.

Analysis of lytic and latent virus

For detection of lytic virus, the tissue samples were homogenized using a Tissumizer (Tekmar Corp., Cleveland, OH), subjected to three rounds of freeze/thaw and plated on confluent monolayers of Vero cells. For detection of latent virus, the tissues were first incubated for 5 days in 1.0 ml cell culture medium at 37°C in an atmosphere of 5% CO₂ in order to reactivate the latent virus. Following incubation, the tissue samples were homogenized as described above, alternately frozen and thawed three times and cocultivated with confluent monolayers of Vero cells. The Vero cells were monitored daily for 8 to 10 days for the appearance of HSV plaques.