Equine Encephalomyelitis Virus

HEYDRICK, FRED P. (Fort Detrick, Frederick, Md.), RALPH F. WACHTER, AND HENRY J. HEARN, JR. Host influence on the characteristics of Venezuelan equine encephalomyelitis virus. J. Bacteriol. 91:2343-2348. 1966.-Alterations in plaque size, virulence, and lipid content of Venezuelan equine encephalomyelitis (VEE) virus were examined for possible interrelationships among these properties during 10 serial passages in embryonated eggs, suckling mice, chick embryo fibroblasts, and L cells. The chick embryo host maintained the same large-plaque and virulence properties of the virus through 10 passages as seen in the original seed. Passage of virus in either L cells or chick fibroblasts rapidly produced populations that were, in the main, intermediate with respect to plaque size and virulence. Passage of virus in suckling mouse brain yielded populations that were intermediate with respect to plaque size only. The nature of the lipid of the virus, in terms of the ratio of petroleum ether-soluble to -insoluble lipid, changed after only one passage in all systems except in chick embryos. Nine additional serial passages failed to enhance these changes in viral lipid, suggesting that the decrease in the large-plaque and virulence properties was not directly associated with changes in lipid content. The properties of virulence and plaque formation of the equine encephalomyelitis group of viruses have been shown by a number of investigators to be influenced by passage of the virus in various host systems. For example, Marshall et al. (9) found that the large-plaque characteristic of naturally occurring strains of western equine encephalomyelitis virus was maintained consistently during passage in embryonated eggs, less consistently during passage in mouse brain, and was quickly replaced by virus that produced small plaques when passed in chick fibroblast cell culture. The literature contains several reports describing alterations in the properties of Venezuelan equine encephalomyelitis (VEE) virus after passage in cell culture (1, 5, 8, 10, 11). The work of Hardy and Hearn (4), Mussgay and Suarez (11), and Brown (2) indicated a correlation between loss of virulence resulting from passage in cell culture and loss in ability of the virus to form large plaques. In preliminary studies, Hearn and Soper (Bacteriol. Proc., p. 135, 1962) and Heydrick, Cree, and Wachter (Federation Proc. 23:400, 1964) emphasized that different host species varied in their ability to alter plaque size and virulence of VEE virus. An additional factor, the ratio of petroleum ether (PE)-soluble to PE-insoluble viral lipid also appeared to depend upon the host from which the virus was derived (Heydrick et al., Federation Proc. 23:400, 1964). It is the purpose of this paper to report the relationships and changes in plaque size, virulence, and lipid content of viral populations that occurred during 10 serial passages of a single strain of VEE virus, with the use of the chick embryo, suckling mouse brain, chick fibroblasts, and L cells as hosts. MATERIALS AND METHODS Virus seeds. The parent egg seed (PES) (5) of the Trinidad strain of VEE virus (10), which had received 13 passages in embryonated eggs, was used to initiate these studies. This seed possessed a titer of 109.2 mouse intracerebral (ic) LD50 per ml of 20% suspension. Embryo and yolk-sac seeds used to infect various hosts were prepared by inoculating 104.6 mouse ic LD50 of the PES strain into 10-day embryonated eggs via the allantoic route. After incubation for 24 hr, embryos and yolk sacs were harvested from moribund and dead eggs. These harvested tissues were homogenized separately, and prepared as 20% suspensions in Ringer-Locke solution. After low-speed centrifugation, the supernatant fluid was placed in ampoules and stored at -70 C. Passage of virus. The embryo and yolk-sac seeds, 2343 on N ovem er 3, 2017 by gest http/jb.asm .rg/ D ow nladed fom HEYDRICK, WACHTER, AND HEARN which titered 109 5 and 108.0 mouse ic LD50 per ml, respectively, were serially passed 10 times in 11-day embryonated eggs, suckling mice, and in monolayers of L cells and chick fibroblasts (CF). Prior to inoculation, cell monolayers for passage of virus were grown for 48 hr in either T-60 flasks or Roux bottles containing lactalbumin hydrolysate medium with 10% calf serum. The former were inoculated with 0.3 ml and the latter with 1.0 ml of a 1:10 dilution of either the embryo or yolk-sac seed. After an adsorption period of 20 min, the inoculum was removed and fresh growth medium was added. The viral suspensions were harvested after 36 hr of incubation at 37 C. Remaining serial passages were made with the use of harvested tissue culture supernatant fluids as inocula, each of which contained approximately 108 0 mouse ic LD5o. Serial passages in 1to 2-day-old suckling mice were initiated in a similar manner by intracerebral injection of 0.03 ml of a 1:100 dilution of either the embryo or yolk-sac seed. Infected brains were harvested after 24 hr, pooled, and stored as 10% suspensions in beef heart infusion broth (BHIB). Subsequent suckling mouse brain serial passages were carried out with approximately 106.0 mouse ic LD6o of the harvested brain suspensions. Passage of virus in chick embryo was begun with 0.25 ml of a 1:100 dilution of either the embryo or yolk-sac virus. Subsequent serial passages were made with approximately 107-0 mouse ic LD5o of the harvested embryo pools. All harvested material was stored at -70 C until tested. Virus assay and plaque size determination. Viral assays were performed by injecting 12to 14-g mice by the intracerebral (ic) and intraperitoneal (ip) routes with 10-fold dilutions of the preparation in BHIB. Viral titers were expressed as the mouse ic LDo0 and ip LD60 per ml. The CF monolayer plaque technique for intact virus, as described by Col6n and Idoine (3), was used to determine plaque size. Plaque diameters were measured to the nearest 0.25 mm. Counts and size determinations were made 2 and 3 days after inoculation; the longer incubation period, however, proved to give more accurate size differentiation. Three groups of plaque sizes were arbitrarily defined: large plaques (4 to 6 mm in diameter), intermediate plaques (2.0 to 3.5 mm), and small plaques (0.5 to 1.5 mm). Two dilutions of inoculum were employed in duplicate to provide between 20 and 120 plaques per plate. Fifthand tenth-passage preparations were tested simultaneously to minimize variation in plaque size resulting from influences other than those exerted by the host from which the virus was obtained. Purification ofvirus. The VEE virus from 20% chick embryo suspension, 10% suckling mouse brain suspension, or infected tissue culture fluid was purified by treatment with Celite, clarification with protamine sulfate, and concentration of the virus by high-speed centrifugation (59,000 X g) of the supematant fluid. The concentrated virus was placed on a continuous sucrose gradient, and centrifuged at 53,500 X g for 3 hr in a Spinco SW-25 swinging bucket rotor. The visible virus band, located at a density of 1.14, was removed by puncturing the side of the tube. Dialysis and lyophilization. Purified virus suspensions from a series of identical gradients were prepared for analysis by diluting the virus band of high sucrose content with 5% sucrose, and sedimenting the virus by high-speed centrifugation (105,000 X g for 2 hr). The resuspended virus pellet was dialyzed against distilled water, and lyophilized in small bottles. Samples were further dried to constant weight over phosphorus pentoxide before extraction of lipids. Lipid determination. Total lipid was determined by direct weighing of the extracted lipid. Lipid was removed with a microextraction apparatus similar to that described by Schaffer et al. (13), by use of the extraction procedure of Thomas (14).